Yet another "quantum effects in photosynthesis" paper.
Waiting for the rampant speculation that can arise from this paper, as well.
Off to troubleshooting the remainder of my afternoon!
Read more!
1 day ago
Showing posts with label biophysics. Show all posts
Showing posts with label biophysics. Show all posts
Friday, June 21, 2013
Friday, December 21, 2012
Off the back burner.....
Meant to post about this a while back, but never quite got around to mentioning it.
Lipid Bilayers and Membrane Dynamics: Insight into Thickness Fluctuations.
I suspect anyone reading this post knows that membranes are far from static entities, ranging from lateral diffusion of individual lipids within the bilayer to collective motions of the membrane. Here, the authors report of a thickness fluctuation, which is exactly what it sounds like -
From here.
The authors utilized both small-angle neutron scattering and neutron spin echo spectroscopy on these samples, as neutrons offer remarkable versatility in terms of probing various length and energy scales, as is presented here -
In any case, I thought it was interesting. They are - based on what I've heard - looking at lipid bilayers with proteins, but I haven't seen it come out yet in the literature. I think as people become more interested in what is really going on in complex biological systems, we're going to need to look beyond the purely molecular length and energy scales to the mesoscopic regime (however one defines it).
Happy holidays and New Year to all! Read more!
Lipid Bilayers and Membrane Dynamics: Insight into Thickness Fluctuations.
I suspect anyone reading this post knows that membranes are far from static entities, ranging from lateral diffusion of individual lipids within the bilayer to collective motions of the membrane. Here, the authors report of a thickness fluctuation, which is exactly what it sounds like -
From here.
The authors utilized both small-angle neutron scattering and neutron spin echo spectroscopy on these samples, as neutrons offer remarkable versatility in terms of probing various length and energy scales, as is presented here -
In any case, I thought it was interesting. They are - based on what I've heard - looking at lipid bilayers with proteins, but I haven't seen it come out yet in the literature. I think as people become more interested in what is really going on in complex biological systems, we're going to need to look beyond the purely molecular length and energy scales to the mesoscopic regime (however one defines it).
Happy holidays and New Year to all! Read more!
Monday, November 19, 2012
Speak of the devil....
...and the devil, he shall appear.
A recent discussion at the Curious Wavefunction briefly touched upon the role of (macro)molecular crowding in biochemical studies. I am presently preparing to see whether a certain set of experiments are feasible, and in some of the potential tests, I am adding a fair amount of crowding agents to my samples.
The samples that have those crowding agents are presently the only ones that exhibit any enzymatic activity. The other samples are dead, biochemically speaking. One of the "dead" samples does exhibit complex formation in one of my alternate tests, but that's it. Further tests are needed, of course, and it's certainly possible that I can retain enzymatic activity in the other samples upon logically adjusting my current protocols. But perhaps there's a lesson here to be learned.
Read more!
A recent discussion at the Curious Wavefunction briefly touched upon the role of (macro)molecular crowding in biochemical studies. I am presently preparing to see whether a certain set of experiments are feasible, and in some of the potential tests, I am adding a fair amount of crowding agents to my samples.
The samples that have those crowding agents are presently the only ones that exhibit any enzymatic activity. The other samples are dead, biochemically speaking. One of the "dead" samples does exhibit complex formation in one of my alternate tests, but that's it. Further tests are needed, of course, and it's certainly possible that I can retain enzymatic activity in the other samples upon logically adjusting my current protocols. But perhaps there's a lesson here to be learned.
Read more!
Wednesday, October 31, 2012
Lit Links
So, in case any were wondering, my area on the East Coast was mostly spared the wrath of Hurricane to Post-Tropical Cyclone Sandy. Some rain, a bit more wind, but not many power outages in the immediate area. I am however fairly well prepared for any mystery zombie apocalypses that might arise (from the dead). I hope that all of you reading who were subject to its furor endured the storm as well as possible.
In any case, some bits of possible interest -
1.) PNAS has a special feature this week on "the Chemical Physics of Protein Folding." Sadly, it's behind a paywall for the time being.
1.5.) Related to this, I once mentioned a while back in a comment (I believe over at the Inquisitive Ket) about one of the less-important reasons Levinthal's paradox never really bothered me, namely, that proteins aren't really free to sample all possible conformations due to their interactions with other proteins (even indirectly due to crowding), the solvent, and with itself. In any case, it's always interesting to see people carefully examine these sorts of questions in the recent literature.
2.) Gaining structural insight occasionally takes a while. It also reminds me of the utility of neutron science for biochemistry - the ability to use contrast variation using selective deuteration make it possible to probe multicomponent systems. And let's not forget that one can also use neutrons for spectroscopic measurements.
Anyway, back to the actual science….. Read more!
In any case, some bits of possible interest -
1.) PNAS has a special feature this week on "the Chemical Physics of Protein Folding." Sadly, it's behind a paywall for the time being.
1.5.) Related to this, I once mentioned a while back in a comment (I believe over at the Inquisitive Ket) about one of the less-important reasons Levinthal's paradox never really bothered me, namely, that proteins aren't really free to sample all possible conformations due to their interactions with other proteins (even indirectly due to crowding), the solvent, and with itself. In any case, it's always interesting to see people carefully examine these sorts of questions in the recent literature.
2.) Gaining structural insight occasionally takes a while. It also reminds me of the utility of neutron science for biochemistry - the ability to use contrast variation using selective deuteration make it possible to probe multicomponent systems. And let's not forget that one can also use neutrons for spectroscopic measurements.
Anyway, back to the actual science….. Read more!
Tuesday, October 16, 2012
Some minor notes.
The reason they're making your son take chemistry? Because they're mean. Or perhaps because they hold to some archaic notion that education is about broadening one's horizons and tempering the intellect, not just about preparing one for any particular career path. I haven't decided.
Small-molecule synthetic chemistry in the NY Times? A pleasant surprise to see in the national newspaper of record, at least last I checked.
Microbial physiology via NMR. So cool. Also, kind of jealous. I really need to get some awesome stuff done sooner rather than later.
I think that will be all for the evening.
Read more!
Small-molecule synthetic chemistry in the NY Times? A pleasant surprise to see in the national newspaper of record, at least last I checked.
Microbial physiology via NMR. So cool. Also, kind of jealous. I really need to get some awesome stuff done sooner rather than later.
I think that will be all for the evening.
Read more!
Saturday, May 19, 2012
Quantum biology - A Rose By Any Other Name?
I believe I may have off-handedly mentioned some of this work somewhere in a blog comment semi-recently, but I suppose some further thoughts would not be out of place at this time. I bring it up because of this preprint and this article about said preprint.
Clearly, on one level, quantum mechanics - via chemistry - underlies biology. This is, I suspect, a fairly unoffensive statement. Chemical reactions are quantitatively studied in a quantum mechanical framework, and I don't see biochemical reactions being much different.
On another level, direct appeal to quantum mechanical behavior to explain biology can seem kind of silly. Biology is slow, wet, messy, and takes place at a whole bunch of time and length scales. I imagine many of us recall the exercise - likely done in an introductory general chemistry course, at least in my experience - where one calculates the de Broglie wavelength for an electron and then, say, a baseball.
Of course, when one looks at table 1 in the preprint, a light goes on. Long-range electron transfer in proteins? The role of tunneling in enzyme catalysis? Vision - which involves the photochemistry of a protein-immobilized chromophore? Photosynthesis? A proposed radical spin mechanism for avian magnetoreception?
This all reeks of physical(ly predisposed) chemists trying to get their dirty mitts onto a whole lot of funding. Not that there's anything wrong with that, mind you - I'm presently trying to work "quantum biology" into my CV/resume as we speak.
But for sake of argument, let's take a look at the Fenna-Matthews-Olsen (FMO) protein from a green sulfur bacterium where quantum effects were observed via 2D electronic spectroscopy.
Yep, definitely a protein. But what's all of that inside the protein?
Why, it looks like the protein is the wrapping for a photochemically delicious filling of chlorophyll molecules! I could envision that this is the sort of environment which would be conducive for maintaining some sort of quantum mechanical excitation.
But what about - for instance - microtubules, which some have suggested play a role in consciousness via quantum mechanical effects? Why, there's even GTP (GDP) known to associate with tubulin in the structure! Let's take a look -
Hmmm. Let's focus on the GTP and GDP, so how about….
Well, that was kind of anticlimactic. That's it? That is somehow supposed to sustain and nurture our very consciousness from the harsh decoherent world out there? I find myself skeptical.
I suppose that is as good a place to end as any. I may or may not have more to say in the future after I've had a chance to properly consume and digest the preprint. While I do find the idea of nature exploiting exciton transport, radical spin pair chemistry, proton tunneling, and so on incredibly exciting - I don't think taking that and wantonly speculating is the best route.
FYI - This was also mostly a chance to play around some more with UCSF Chimera. Just started using it a bit earlier this year, so if anyone has any tips or list of useful tricks, please share! The structures were generated with this program using PDB ID 3ENI (FMO protein) and 1JFF (tubulin). Read more!
Clearly, on one level, quantum mechanics - via chemistry - underlies biology. This is, I suspect, a fairly unoffensive statement. Chemical reactions are quantitatively studied in a quantum mechanical framework, and I don't see biochemical reactions being much different.
On another level, direct appeal to quantum mechanical behavior to explain biology can seem kind of silly. Biology is slow, wet, messy, and takes place at a whole bunch of time and length scales. I imagine many of us recall the exercise - likely done in an introductory general chemistry course, at least in my experience - where one calculates the de Broglie wavelength for an electron and then, say, a baseball.
Of course, when one looks at table 1 in the preprint, a light goes on. Long-range electron transfer in proteins? The role of tunneling in enzyme catalysis? Vision - which involves the photochemistry of a protein-immobilized chromophore? Photosynthesis? A proposed radical spin mechanism for avian magnetoreception?
This all reeks of physical(ly predisposed) chemists trying to get their dirty mitts onto a whole lot of funding. Not that there's anything wrong with that, mind you - I'm presently trying to work "quantum biology" into my CV/resume as we speak.
But for sake of argument, let's take a look at the Fenna-Matthews-Olsen (FMO) protein from a green sulfur bacterium where quantum effects were observed via 2D electronic spectroscopy.
Yep, definitely a protein. But what's all of that inside the protein?
Why, it looks like the protein is the wrapping for a photochemically delicious filling of chlorophyll molecules! I could envision that this is the sort of environment which would be conducive for maintaining some sort of quantum mechanical excitation.
But what about - for instance - microtubules, which some have suggested play a role in consciousness via quantum mechanical effects? Why, there's even GTP (GDP) known to associate with tubulin in the structure! Let's take a look -
Hmmm. Let's focus on the GTP and GDP, so how about….
Well, that was kind of anticlimactic. That's it? That is somehow supposed to sustain and nurture our very consciousness from the harsh decoherent world out there? I find myself skeptical.
I suppose that is as good a place to end as any. I may or may not have more to say in the future after I've had a chance to properly consume and digest the preprint. While I do find the idea of nature exploiting exciton transport, radical spin pair chemistry, proton tunneling, and so on incredibly exciting - I don't think taking that and wantonly speculating is the best route.
FYI - This was also mostly a chance to play around some more with UCSF Chimera. Just started using it a bit earlier this year, so if anyone has any tips or list of useful tricks, please share! The structures were generated with this program using PDB ID 3ENI (FMO protein) and 1JFF (tubulin). Read more!
Sunday, January 8, 2012
You Shall Pass!
I saw this paper, and it was just asking be blogged about here. I figured I’d give it a shot.
Disclaimer – Not my work, never met any of the authors (although I’m sure they’re all within six degrees of me scientifically). The paper is open access, which I think is a good policy for me to adhere to in any future efforts along these lines.
Citation: L.A. Clifton, et al. “Low Resolution Structure and Dynamics of a Colicin-Receptor Complex Determined by Neutron Scattering.” The Journal of Biological Chemistry. Vol. 287, No. 1, pp. 337-346; January 2, 2012.
Among the many things that bacteria can do, one of them is knocking off other bacteria. There are a number of ways to go about this critical task, not surprisingly, and one of them involves proteins known as bacteriocins. These are proteins that the bacterium uses to kill off potential competitors, as they typically go after closely related bacteria. In this paper, the authors are focusing on Colicin N (ColN), a bacteriocin produced by E. coli. ColN depolarizes the inner membrane of Gram-negative bacteria by forming pores in the inner membrane, resulting in cell death.
The question the authors address is a fundamental one – how does ColN get past the lipopolysaccarhide-decorated outer membrane of a bacterium? It is ~ 40 kDa in size - so, clearly, not going to be able to easily masquerade as an ion or small molecule and pass unhindered through a pore in the outer membrane. The authors note that past research on ColN demonstrated that it is dependent on the presence of an outer membrane protein, OmpF (or related porins), to be effective. Cells that are OmpF-deficient will not be killed off by ColN. I should note that OmpF is a trimeric porin that permits the passage of ions and small molecules through the outer membrane. It was suggested that ColN could pass through the OmpF pore, but would need to be completely unfolded to do so. So there is clearly something going on here that is interesting.
The paper describes a multipronged approach to this question – the authors integrate microscopy, neutron reflectivity, and small angle neutron scattering (SANS). The authors step through their case – they first present the thin film imaging (Brewster’s angle microscopy) and neutron reflection data for their model of the OmpF/phospholipid monolayer. The microscopy suggests similar stability for the OmpF/phospholipid monolayer, although different topography and compression behavior (the formation of domains appears less evenly distributed in the OmpF/phospholipid monolayer, and there are “kinks” in the isotherm for the phospholipid-only monolayer compared to the OmpF-containing one). The neutron reflection data also seems to support the existence of an OmpF/phospholipid bilayer, despite Fig. 3B being mislabeled by my eye. Normally the neutron “refractive index” - neutron scattering length density, aka nSLD – is plotted as a distance away from some reference (e.g., an easily determined interface or a metal layer on which your sample is ultimately deposited). It seems that is what they intended to write (the x-axis seems to be labeled as such) but is mislabeled with the “Q/A-1” tag.
In any case, much of biologically-oriented neutron scattering is dependent on the existence of contrast variation in the nSLD. You can purchase deuterated compounds (such as lipids), prepare buffers in deuterium oxide, and even express & purify deuterated proteins. You then mix and match your deuterated and protonated components to see what each component looks like when in complex with everything else. It is a low-resolution means of doing so, but the benefits can outweigh the disadvantages.
The authors move onto the ColN portion of their work, showing the microscopy and neutron reflection data for ColN interacting with the OmpF/lipid monolayer. The time-lapse microscopy of ColN with the pure lipid monolayer and the OmpF/lipid monolayer shows increased image intensity, but appears to “smear” homogeneously with the pure lipid monolayer while forming larger, brighter spots with the OmpF/lipid monolayer. Their analysis of the neutron reflectivity data indicates the presence of the ColN in the same layer with the OmpF, and not just interacting with its surface, as they see in the ColN + pure lipid monolayer sample. Given the contrast variation matching, they state that they are able to see ColN extend as it inserts into the lipid region, suggesting that it is unfolding to some extent. The increase in surface pressure would suggest that it is not going through the OmpF pore but is, instead, inserting into the lipid region next to the OmpF. If it was inserting through the pore channel, the surface pressure might be expected to level off and not keep increasing.
The SANS data round out the story – they’re looking at the ColN/OmpF complex in detergent. (I know, I know.) Anyway, their data-derived model has one of the ColN domains slithering down between the cleft between OmpF monomers, while the remainder of ColN remains protruding outward. If you look at Fig. 6C, the blue distance distribution (where you are only looking at scattering from ColN) has two peaks, one that overlaps with the red trace (where one is only looking at OmpF) and a separate peak. So this at least makes sense. They do discuss the potential for translocation via the pore, and some recent literature on that possibility.
Mostly, I thought that this was a really interesting bit of research – while there is the obligatory mention of potential application to antibiotic development, it’s pretty obvious that the fundamental scientific question of “how does a largish protein get across a cell membrane where the cell has no interest in letting it inside?” I think that the experiments were reasonable, were carefully done, and did not set off too many massive alarms in my brain while reading. I would like to think that you could use something like nanodiscs or bicelles for the SANS studies so you could at least approximate a native membrane environment – clearly, sample homogeneity is a concern, as scattering methods can be notoriously sensitive. (Did I ever tell you about the time I spent a good afternoon into evening washing banjo cells for SANS experiments since said cells were just disgusting?) I haven’t worked with nanodiscs – although I’ve heard and read more than I can shake a stick at - and my experience with bicelles hasn’t been quite so detail-oriented, so maybe it would require sublime experimental mastery beyond the typical.*
Anyway. That was kind of fun. Also, how many of you saw Ohm’s Law Survives to the Atomic Scale? I imagine people will want to confirm this, as it is definitely seems really cool. Clearly, it was custom-made by “hand” (well, scanning tunneling microscope), so no immediate applications to large-scale mass production any week soon, but that isn’t why we do science.
Now, off to think about thermodynamics for a while. I need to come up with a reasonable explanation of some data today….. Read more!
Disclaimer – Not my work, never met any of the authors (although I’m sure they’re all within six degrees of me scientifically). The paper is open access, which I think is a good policy for me to adhere to in any future efforts along these lines.
Citation: L.A. Clifton, et al. “Low Resolution Structure and Dynamics of a Colicin-Receptor Complex Determined by Neutron Scattering.” The Journal of Biological Chemistry. Vol. 287, No. 1, pp. 337-346; January 2, 2012.
Among the many things that bacteria can do, one of them is knocking off other bacteria. There are a number of ways to go about this critical task, not surprisingly, and one of them involves proteins known as bacteriocins. These are proteins that the bacterium uses to kill off potential competitors, as they typically go after closely related bacteria. In this paper, the authors are focusing on Colicin N (ColN), a bacteriocin produced by E. coli. ColN depolarizes the inner membrane of Gram-negative bacteria by forming pores in the inner membrane, resulting in cell death.
The question the authors address is a fundamental one – how does ColN get past the lipopolysaccarhide-decorated outer membrane of a bacterium? It is ~ 40 kDa in size - so, clearly, not going to be able to easily masquerade as an ion or small molecule and pass unhindered through a pore in the outer membrane. The authors note that past research on ColN demonstrated that it is dependent on the presence of an outer membrane protein, OmpF (or related porins), to be effective. Cells that are OmpF-deficient will not be killed off by ColN. I should note that OmpF is a trimeric porin that permits the passage of ions and small molecules through the outer membrane. It was suggested that ColN could pass through the OmpF pore, but would need to be completely unfolded to do so. So there is clearly something going on here that is interesting.
The paper describes a multipronged approach to this question – the authors integrate microscopy, neutron reflectivity, and small angle neutron scattering (SANS). The authors step through their case – they first present the thin film imaging (Brewster’s angle microscopy) and neutron reflection data for their model of the OmpF/phospholipid monolayer. The microscopy suggests similar stability for the OmpF/phospholipid monolayer, although different topography and compression behavior (the formation of domains appears less evenly distributed in the OmpF/phospholipid monolayer, and there are “kinks” in the isotherm for the phospholipid-only monolayer compared to the OmpF-containing one). The neutron reflection data also seems to support the existence of an OmpF/phospholipid bilayer, despite Fig. 3B being mislabeled by my eye. Normally the neutron “refractive index” - neutron scattering length density, aka nSLD – is plotted as a distance away from some reference (e.g., an easily determined interface or a metal layer on which your sample is ultimately deposited). It seems that is what they intended to write (the x-axis seems to be labeled as such) but is mislabeled with the “Q/A-1” tag.
In any case, much of biologically-oriented neutron scattering is dependent on the existence of contrast variation in the nSLD. You can purchase deuterated compounds (such as lipids), prepare buffers in deuterium oxide, and even express & purify deuterated proteins. You then mix and match your deuterated and protonated components to see what each component looks like when in complex with everything else. It is a low-resolution means of doing so, but the benefits can outweigh the disadvantages.
The authors move onto the ColN portion of their work, showing the microscopy and neutron reflection data for ColN interacting with the OmpF/lipid monolayer. The time-lapse microscopy of ColN with the pure lipid monolayer and the OmpF/lipid monolayer shows increased image intensity, but appears to “smear” homogeneously with the pure lipid monolayer while forming larger, brighter spots with the OmpF/lipid monolayer. Their analysis of the neutron reflectivity data indicates the presence of the ColN in the same layer with the OmpF, and not just interacting with its surface, as they see in the ColN + pure lipid monolayer sample. Given the contrast variation matching, they state that they are able to see ColN extend as it inserts into the lipid region, suggesting that it is unfolding to some extent. The increase in surface pressure would suggest that it is not going through the OmpF pore but is, instead, inserting into the lipid region next to the OmpF. If it was inserting through the pore channel, the surface pressure might be expected to level off and not keep increasing.
The SANS data round out the story – they’re looking at the ColN/OmpF complex in detergent. (I know, I know.) Anyway, their data-derived model has one of the ColN domains slithering down between the cleft between OmpF monomers, while the remainder of ColN remains protruding outward. If you look at Fig. 6C, the blue distance distribution (where you are only looking at scattering from ColN) has two peaks, one that overlaps with the red trace (where one is only looking at OmpF) and a separate peak. So this at least makes sense. They do discuss the potential for translocation via the pore, and some recent literature on that possibility.
Mostly, I thought that this was a really interesting bit of research – while there is the obligatory mention of potential application to antibiotic development, it’s pretty obvious that the fundamental scientific question of “how does a largish protein get across a cell membrane where the cell has no interest in letting it inside?” I think that the experiments were reasonable, were carefully done, and did not set off too many massive alarms in my brain while reading. I would like to think that you could use something like nanodiscs or bicelles for the SANS studies so you could at least approximate a native membrane environment – clearly, sample homogeneity is a concern, as scattering methods can be notoriously sensitive. (Did I ever tell you about the time I spent a good afternoon into evening washing banjo cells for SANS experiments since said cells were just disgusting?) I haven’t worked with nanodiscs – although I’ve heard and read more than I can shake a stick at - and my experience with bicelles hasn’t been quite so detail-oriented, so maybe it would require sublime experimental mastery beyond the typical.*
Anyway. That was kind of fun. Also, how many of you saw Ohm’s Law Survives to the Atomic Scale? I imagine people will want to confirm this, as it is definitely seems really cool. Clearly, it was custom-made by “hand” (well, scanning tunneling microscope), so no immediate applications to large-scale mass production any week soon, but that isn’t why we do science.
Now, off to think about thermodynamics for a while. I need to come up with a reasonable explanation of some data today….. Read more!
Thursday, December 22, 2011
Trust but verify.
The question of how much to trust computational methods is brought up here at Chemiotics II. My answer is that it depends on what one is looking for in the first place.
If one is looking for some sort of completely accurate and precise way to have all biological phenomenona fall out of "first principles," well, I wouldn't hold my breath. Of course, I don't think anyone is really waiting for that. At least I hope not. I believe my feelings on these sorts of issues are best described by personal experiences I've had with computational methods.
In grad school, I had an interest in this one mid-sized protein (somewhere between 40 to 60 kDa) that was known to bind this particular ligand. There was a crystal structure of the protein with and without ligand, although of course it was hardly the entire story (which is why it was the subject of my research attentions). In any case, collaborators did some MD simulations, and it was consistent with what we had found and was known. In their next bit of work, they mentioned that they found something new regarding the mechanism of ligand binding. This was going on the same time as I was doing some work, and as it turned out, my data did not rule it out. And so new research was inspired for those who took up the project after I left.
Currently, I am embroiled in a sordid and complex tale of transmembrane signaling involving the receptor and varying amounts of soluble cytoplasmic proteins that propagate that signal. There was a fairly recent paper detailing MD studies of the signaling process. Well, part of it, I suppose - huge chunks on either end of the transmembrane receptor were not included, and none of the cytoplasmic proteins that bind and are modified by the receptor were included in the study. Certainly a daring attempt, but it's hard to get too worked up over it when it doesn't resemble anything that I actually work with on a daily basis.
In short....I think properly used, it can be a useful way to bridge what is measured experimentally with the metaphors we use to describe processes. (For example - people love using descriptions involving simple machines, but what is actually measured are thermodynamic or spectroscopic quantities. Of course, "force spectroscopy" looks to change this, but when you yank apart a protein, you are no longer just gently playing around at kT or sub-kT conditions to see what kinds of deformations you get naturally or as a response to some stimulus. Anyway....) Certainly, for small enough systems, I am inclined to give them a proper reading, and in cases where the system might be larger but is somewhat well characterized, the same applies. In giant systems where they toss out a number of critical components or oversimplify to the point of absurdity, I am generally far more skeptical.
Merry Christmas to those who celebrate, Happy Hanukkah to those who celebrate, and a delightful winter holiday season to the rest. Read more!
If one is looking for some sort of completely accurate and precise way to have all biological phenomenona fall out of "first principles," well, I wouldn't hold my breath. Of course, I don't think anyone is really waiting for that. At least I hope not. I believe my feelings on these sorts of issues are best described by personal experiences I've had with computational methods.
In grad school, I had an interest in this one mid-sized protein (somewhere between 40 to 60 kDa) that was known to bind this particular ligand. There was a crystal structure of the protein with and without ligand, although of course it was hardly the entire story (which is why it was the subject of my research attentions). In any case, collaborators did some MD simulations, and it was consistent with what we had found and was known. In their next bit of work, they mentioned that they found something new regarding the mechanism of ligand binding. This was going on the same time as I was doing some work, and as it turned out, my data did not rule it out. And so new research was inspired for those who took up the project after I left.
Currently, I am embroiled in a sordid and complex tale of transmembrane signaling involving the receptor and varying amounts of soluble cytoplasmic proteins that propagate that signal. There was a fairly recent paper detailing MD studies of the signaling process. Well, part of it, I suppose - huge chunks on either end of the transmembrane receptor were not included, and none of the cytoplasmic proteins that bind and are modified by the receptor were included in the study. Certainly a daring attempt, but it's hard to get too worked up over it when it doesn't resemble anything that I actually work with on a daily basis.
In short....I think properly used, it can be a useful way to bridge what is measured experimentally with the metaphors we use to describe processes. (For example - people love using descriptions involving simple machines, but what is actually measured are thermodynamic or spectroscopic quantities. Of course, "force spectroscopy" looks to change this, but when you yank apart a protein, you are no longer just gently playing around at kT or sub-kT conditions to see what kinds of deformations you get naturally or as a response to some stimulus. Anyway....) Certainly, for small enough systems, I am inclined to give them a proper reading, and in cases where the system might be larger but is somewhat well characterized, the same applies. In giant systems where they toss out a number of critical components or oversimplify to the point of absurdity, I am generally far more skeptical.
Merry Christmas to those who celebrate, Happy Hanukkah to those who celebrate, and a delightful winter holiday season to the rest. Read more!
Tuesday, September 27, 2011
Surely you jest!
So, I strongly recommend everyone checks out this paper -
Accessing protein conformational ensembles using room-temperature X-ray crystallography
- which was just published in PNAS this week. The paper cites a 2004 paper by Bertil Halle (which I mentioned a while back) on the potential consequences of flash freezing and cryocrystallography.
Enjoy! Read more!
Accessing protein conformational ensembles using room-temperature X-ray crystallography
- which was just published in PNAS this week. The paper cites a 2004 paper by Bertil Halle (which I mentioned a while back) on the potential consequences of flash freezing and cryocrystallography.
Enjoy! Read more!
Tuesday, September 20, 2011
No, I am not going to talk about the recent paper on the success of Foldit. Mostly since if you can even get a crystal structure for something, it's probably not agonizingly painful enough for me to work on - as I've said before, give me your disordered, your poorly soluble, your aggregated masses yearning to be analyzed.
Anyway, I wanted to mention this interesting-looking paper:
Binding Leverage as a Molecular Basis for Allosteric Regulation. I haven't had a chance to really dig into the paper, but the idea itself is simple enough - ligand binding can couple to various collective motions in proteins to varying extents, due to which we observe allosteric modulation of enzyme function. There are obvious oversights (one example that they mention in the paper - the lack of attention paid to proteins that aren't enzymes such as signaling proteins of various types), and I'd want to pore through which structures they used in the PDB (e.g., how did they deal with the family of structures that are generated by NMR if applicable). Then again, I usually consider thought-provoking ideas worth the publication, even if a judiciously skeptical outlook may make them seem a little less lustrous. Read more!
Anyway, I wanted to mention this interesting-looking paper:
Binding Leverage as a Molecular Basis for Allosteric Regulation. I haven't had a chance to really dig into the paper, but the idea itself is simple enough - ligand binding can couple to various collective motions in proteins to varying extents, due to which we observe allosteric modulation of enzyme function. There are obvious oversights (one example that they mention in the paper - the lack of attention paid to proteins that aren't enzymes such as signaling proteins of various types), and I'd want to pore through which structures they used in the PDB (e.g., how did they deal with the family of structures that are generated by NMR if applicable). Then again, I usually consider thought-provoking ideas worth the publication, even if a judiciously skeptical outlook may make them seem a little less lustrous. Read more!
Thursday, July 7, 2011
As has been often noted about a number of topics, one’s biases will always skew one’s perspective.
On the one hand, we have this very interesting paper where the authors suggest correlated motions in ubiquitin over a distance of ~ 15 Angstroms (1.5 nm) based on further squeezing information from previously acquired NMR data with the help of computational methods. On the other hand, there is this other very intriguing paper where the authors put forth using gadolinium tags as a way to obtain structural constraints in proteins on the order of ~ 6 nm (60 Angstroms) via ESR/EPR techniques.
In the former, we’re looking at proposed long-distance correlations based on a bunch of relatively weak, short-range interactions (NOEs and RDCs), while in the latter we have nanometer-scale distance constraints being derived from a technique that is well matched to determining distances at the nanoscale. I figure the astute reader can figure where I stand on each given my tone.
Suffice it to say, it’s the reason why I’ve recently developed an interest in 19F NMR (oh, to work with a nucleus that has a decent gyromagnetic ratio and isn’t as common as protons in biological materials!), for one, as well as metal binding tags for paramagnetic relaxation enhancement studies.
In other news, my resolution for the second half of 2011 is to always try and work in a mention of the Helmholtz free energy into each discussion I am involved in that touches upon thermodynamics, as I think far too many chemists have gotten comfortable in their Gibbsian-oriented world.
I suppose this is one of those “it was bound to happen” things – one of the summer undergraduates who is in my lab at my current institution mentioned that the undergrad biochem lab uses a hexahistidine-tagged protein for overexpression & purification. I of course remember having to prep and subsequently grind up giant amounts of animal muscle to extract a protein in my undergrad biochem lab. I feel slightly dusty.
With that observation, I will call this blog post to an end. Read more!
On the one hand, we have this very interesting paper where the authors suggest correlated motions in ubiquitin over a distance of ~ 15 Angstroms (1.5 nm) based on further squeezing information from previously acquired NMR data with the help of computational methods. On the other hand, there is this other very intriguing paper where the authors put forth using gadolinium tags as a way to obtain structural constraints in proteins on the order of ~ 6 nm (60 Angstroms) via ESR/EPR techniques.
In the former, we’re looking at proposed long-distance correlations based on a bunch of relatively weak, short-range interactions (NOEs and RDCs), while in the latter we have nanometer-scale distance constraints being derived from a technique that is well matched to determining distances at the nanoscale. I figure the astute reader can figure where I stand on each given my tone.
Suffice it to say, it’s the reason why I’ve recently developed an interest in 19F NMR (oh, to work with a nucleus that has a decent gyromagnetic ratio and isn’t as common as protons in biological materials!), for one, as well as metal binding tags for paramagnetic relaxation enhancement studies.
In other news, my resolution for the second half of 2011 is to always try and work in a mention of the Helmholtz free energy into each discussion I am involved in that touches upon thermodynamics, as I think far too many chemists have gotten comfortable in their Gibbsian-oriented world.
I suppose this is one of those “it was bound to happen” things – one of the summer undergraduates who is in my lab at my current institution mentioned that the undergrad biochem lab uses a hexahistidine-tagged protein for overexpression & purification. I of course remember having to prep and subsequently grind up giant amounts of animal muscle to extract a protein in my undergrad biochem lab. I feel slightly dusty.
With that observation, I will call this blog post to an end. Read more!
Sunday, February 8, 2009
Some random thoughts....
Or, Structures, Struggles, and Teaching People How to Make Nuclei Dance.
So, a few issues of interest and/or importance to yours truly. This is going to be short.
1.) Vaults! I've suddenly become very entranced with both their structural (39-fold dihedral symmetry for the win!) and functional (no one really knows what they do!) beauty. The structure of rat liver vault at 3.5 Angstroms was recently published (see here) – they were able to clearly assign the major vault protein (MVP) to the electron density, while the other proteins and ribonucleic acid is still up in the air. The internal dimensions of approximately 620 Angstroms in length and 400 Angstroms in diameter is enough to encapsulate most entities within the cell. There's some mention that they might interact with lipid rafts, given some sequence homology considerations. Amusingly, since the number of coordinates and structure factors exceeded what could be put in a PDB file, it's been deposited under three separate accession codes.
2.) I mentioned not too long ago that I'm having some issues with our resident BIAcore (surface plasmon resonance) system. Let me describe to you a representative issue with the current chip and setup: The baseline for the control surface after being coated with buffer and blocking agent is significantly lower than the original baseline. It is possible we have a bad chip, or a bad instrument (some sort of drift in the optical components), or something else. So while troubleshooting and such is going on, it brings to mind a major difference between my current work and former work in terms of nature and aspect. With the SPR setup, it's a unit that is, for lack of a better phrase, a single entity. Sure, we can remove and insert sensor chips, and it's controlled by the adjacent computer, but it's basically a large self-contained box. I can't really go peeking inside, checking things out for myself since it's a shared facility instrument. Back when I was boldly going where no one had gone before in the world of NMR, it was all modular and accessible. I had a oscilloscope checking on the forward and reverse power going to and coming from the probe (remember, in ssNMR, one works with higher RF powers than in solution, typically), I could break out the network analyzer to check the performance of the probe or an amplifier/frequency generator, I could open up the probe to see what chaos might have befallen us, I could always quickly toss in a standard sample (adamantane, KBr, glycine) to quickly assess how far off-course we might have strayed.... I could change filters/attenuators in an appropriate fashion to see where misery was striking at the heart of my experiment. And there was a sense of general, simple assessibility – I could use any network analyzer that I could borrow from someone in the department to check what I might see with my lab's. I could always repack a chemical standard with new material from scratch without it being a big deal or expenditure of time. I could examine the raw data to check and see if the FID was starting at a maximum or minimum (there's a way to check the angle in the SPR software, but it doesn't seem to be something most people do), for instance.
I suppose it's a matter of personal experience – my graduate experience was analogous to the laboratory where I worked as an undergraduate (where we did primarily EPR and time-resolved optical spectroscopy). I – as a general rule of thumb – like to know what's going on at a certain foundational level. I am not a fan of being told, “Yeah, you need to call the company and ask.” I expect with time I will get more used to such commercially available instrumental setups, but until then, I shall lament this state of affairs.
3.) I have a wild bug up my nose about the issue of how to best teach NMR to people. I think most people familiar with magnetic resonance first become acquainted with it via the module in introductory organic chemistry classes, with maybe a mention of it in the introductory physics sequence. Now, I don't really think that there's anything wrong with this – when I was taking organic chemistry, I had the inspired thought, “Hey, protons are spin-1/2 particles, but so are electrons....what about coupling those spin-1/2 particles with nuclei that are spin-1/2 particles? Could we correlate nuclei to electrons? This could be useful for metal-containing systems, since you've got unpaired electrons!” I did some math, and then I found out who George Feher was, and then my moment of insight became bittersweet. I had a few extra drinks that weekend to get over the disappointment, and now that I've told my “how I independently rediscovered ENDOR” story, I won't be tempted to tell it again for a while. However, back to the question - what about the next step? It seems that most people get a “structural determination via spectroscopic/physical methods” sort of class, where they also get into additional methods such as IR, UV/Vis, mass spec, and the like. But, let's say that I become science czar, and I mandate that NMR be removed from this class and be included in a one semester (or equivalent) course in NMR. What else should we teach in this course? How broadband should its audience be – should we let biochemists who have a thing for structural biology expect to get something out of it as well? What about physics majors who are plotting on becoming chemical/molecular physicists and possibly develop the next generation of NMR experiments and applications? How much “death by operator algebra” should we have in this class? What kinds of experiments should be include in the lab component? I have my own ill-formed opinions at this point in time, but it's been fun to mull over on my own and I thought I should share.
And just like that, I'm gone....
Read more!
So, a few issues of interest and/or importance to yours truly. This is going to be short.
1.) Vaults! I've suddenly become very entranced with both their structural (39-fold dihedral symmetry for the win!) and functional (no one really knows what they do!) beauty. The structure of rat liver vault at 3.5 Angstroms was recently published (see here) – they were able to clearly assign the major vault protein (MVP) to the electron density, while the other proteins and ribonucleic acid is still up in the air. The internal dimensions of approximately 620 Angstroms in length and 400 Angstroms in diameter is enough to encapsulate most entities within the cell. There's some mention that they might interact with lipid rafts, given some sequence homology considerations. Amusingly, since the number of coordinates and structure factors exceeded what could be put in a PDB file, it's been deposited under three separate accession codes.
2.) I mentioned not too long ago that I'm having some issues with our resident BIAcore (surface plasmon resonance) system. Let me describe to you a representative issue with the current chip and setup: The baseline for the control surface after being coated with buffer and blocking agent is significantly lower than the original baseline. It is possible we have a bad chip, or a bad instrument (some sort of drift in the optical components), or something else. So while troubleshooting and such is going on, it brings to mind a major difference between my current work and former work in terms of nature and aspect. With the SPR setup, it's a unit that is, for lack of a better phrase, a single entity. Sure, we can remove and insert sensor chips, and it's controlled by the adjacent computer, but it's basically a large self-contained box. I can't really go peeking inside, checking things out for myself since it's a shared facility instrument. Back when I was boldly going where no one had gone before in the world of NMR, it was all modular and accessible. I had a oscilloscope checking on the forward and reverse power going to and coming from the probe (remember, in ssNMR, one works with higher RF powers than in solution, typically), I could break out the network analyzer to check the performance of the probe or an amplifier/frequency generator, I could open up the probe to see what chaos might have befallen us, I could always quickly toss in a standard sample (adamantane, KBr, glycine) to quickly assess how far off-course we might have strayed.... I could change filters/attenuators in an appropriate fashion to see where misery was striking at the heart of my experiment. And there was a sense of general, simple assessibility – I could use any network analyzer that I could borrow from someone in the department to check what I might see with my lab's. I could always repack a chemical standard with new material from scratch without it being a big deal or expenditure of time. I could examine the raw data to check and see if the FID was starting at a maximum or minimum (there's a way to check the angle in the SPR software, but it doesn't seem to be something most people do), for instance.
I suppose it's a matter of personal experience – my graduate experience was analogous to the laboratory where I worked as an undergraduate (where we did primarily EPR and time-resolved optical spectroscopy). I – as a general rule of thumb – like to know what's going on at a certain foundational level. I am not a fan of being told, “Yeah, you need to call the company and ask.” I expect with time I will get more used to such commercially available instrumental setups, but until then, I shall lament this state of affairs.
3.) I have a wild bug up my nose about the issue of how to best teach NMR to people. I think most people familiar with magnetic resonance first become acquainted with it via the module in introductory organic chemistry classes, with maybe a mention of it in the introductory physics sequence. Now, I don't really think that there's anything wrong with this – when I was taking organic chemistry, I had the inspired thought, “Hey, protons are spin-1/2 particles, but so are electrons....what about coupling those spin-1/2 particles with nuclei that are spin-1/2 particles? Could we correlate nuclei to electrons? This could be useful for metal-containing systems, since you've got unpaired electrons!” I did some math, and then I found out who George Feher was, and then my moment of insight became bittersweet. I had a few extra drinks that weekend to get over the disappointment, and now that I've told my “how I independently rediscovered ENDOR” story, I won't be tempted to tell it again for a while. However, back to the question - what about the next step? It seems that most people get a “structural determination via spectroscopic/physical methods” sort of class, where they also get into additional methods such as IR, UV/Vis, mass spec, and the like. But, let's say that I become science czar, and I mandate that NMR be removed from this class and be included in a one semester (or equivalent) course in NMR. What else should we teach in this course? How broadband should its audience be – should we let biochemists who have a thing for structural biology expect to get something out of it as well? What about physics majors who are plotting on becoming chemical/molecular physicists and possibly develop the next generation of NMR experiments and applications? How much “death by operator algebra” should we have in this class? What kinds of experiments should be include in the lab component? I have my own ill-formed opinions at this point in time, but it's been fun to mull over on my own and I thought I should share.
And just like that, I'm gone....
Read more!
Wednesday, January 28, 2009
It's Cold In Here
Or, One More Reason to Take Protein-Ligand Crystal Structures with A Grain of Salt
This is going to be a really short post. Sorry, folks.
Ashutosh pointed out the other week to be wary of ligands in the Protein Data Bank (PDB) – see here. This is of course good advice, and everyone should read that post on the off chance they haven't already done so. Done? Good!
One other thing that I think is underappreciated by those who aren't protein crystallographers/structural biologists/biophysical BAMFs is that, for the most part, modern (synchrotron) protein crystallography is done under cryogenic conditions. Now, this typically is a bit warmer than the boiling point of liquid nitrogen, so you've got a protein crystal wallowing around somewhere above 90 K but not too far above 150 K. I'm not going to say that this is an entirely bad thing – it minimizes radiation damage, allowing for the higher-flux radiation sources to do their job. This of course can lead to higher-resolution structures and most of us generally appreciate that. However, here's the question – does it accurately represent the protein-ligand complex under physiological conditions?
A really interesting analysis of this question was addressed a few years back in 2004 by Bertil Halle from Lund University. I would recommend reading the paper, but if you're interested in the abbreviated conclusions, to wit:
1.)Flash-cooling of protein crystals – and subsequent cryocrystallography – is capable of retaining the general backbone fold and positioning of the protein, but
2.)The quenching of the solvent/ligand/ion degrees of freedom are not necessarily an accurate representation of the complex under physiological conditions and are probably more indicative of the system at the glass transition.
So the next time you look at a crystal structure and wonder, “How in Hades did that ligand end up there?” think about this. For a macroscopic analogy, consider the following structure:
Now, if it were 50 degrees colder, while the overall positioning would be similar, there's no reason to expect that every finger, strand of hair, and toe would be in the same position.
Expect more thoughtful posting one of these days, but don't hold your breath....
Read more!
This is going to be a really short post. Sorry, folks.
Ashutosh pointed out the other week to be wary of ligands in the Protein Data Bank (PDB) – see here. This is of course good advice, and everyone should read that post on the off chance they haven't already done so. Done? Good!
One other thing that I think is underappreciated by those who aren't protein crystallographers/structural biologists/biophysical BAMFs is that, for the most part, modern (synchrotron) protein crystallography is done under cryogenic conditions. Now, this typically is a bit warmer than the boiling point of liquid nitrogen, so you've got a protein crystal wallowing around somewhere above 90 K but not too far above 150 K. I'm not going to say that this is an entirely bad thing – it minimizes radiation damage, allowing for the higher-flux radiation sources to do their job. This of course can lead to higher-resolution structures and most of us generally appreciate that. However, here's the question – does it accurately represent the protein-ligand complex under physiological conditions?
A really interesting analysis of this question was addressed a few years back in 2004 by Bertil Halle from Lund University. I would recommend reading the paper, but if you're interested in the abbreviated conclusions, to wit:
1.)Flash-cooling of protein crystals – and subsequent cryocrystallography – is capable of retaining the general backbone fold and positioning of the protein, but
2.)The quenching of the solvent/ligand/ion degrees of freedom are not necessarily an accurate representation of the complex under physiological conditions and are probably more indicative of the system at the glass transition.
So the next time you look at a crystal structure and wonder, “How in Hades did that ligand end up there?” think about this. For a macroscopic analogy, consider the following structure:
Now, if it were 50 degrees colder, while the overall positioning would be similar, there's no reason to expect that every finger, strand of hair, and toe would be in the same position.
Expect more thoughtful posting one of these days, but don't hold your breath....
Read more!
Wednesday, January 21, 2009
Clusters of Spins (AKA the Promised NMR Lit Post)
So, here's the citation-heavy post about some interesting work done on systems of primarily – but not exclusively – biological interest via magnetic resonance spectroscopy since the turn of the century (I've always wanted to say that).
I mentioned CERM at the University of Florence in my last post for good reason – it's something of a place where, sooner or later, all paths seem to cross there in one way or another. It's either having read one of the texts that's come out of the faculty there, collaboration, or you just find inspiration in what they're doing. Because I have a certain unavoidable bias towards ssNMR out of (most likely) residual Stockholm Syndrome, I will mention this paper as an example of extending the work done at Florence in using NMR to understand paramagnetic metalloproteins in solution to the solid state.
Lyndon Emsley and coworkers at ENS-Lyon are pursuing a number of really interesting projects in magnetic resonance, both of basic importance to NMR (see, for instance, this paper) as well as to problems in chemistry, metabonomics, biology, and paramagnetic systems.
Paul Ellis & coworkers at PNNL have done some really stunning work with Zn-67 ssNMR and Mg-25 ssNMR. Zinc is one of the most common metal cofactors found in biology, and they've managed to do direct measurements of zinc in proteins via solid state NMR (see here and here for examples). Given that it's a quadrupolar nucleus, it's even more awesome. (While comments noting that they do this sort of work at really low temperature are accurate, it does not diminish its luster.) Magnesium-25 is another nucleus that this group has focused on as of late. See this paper for some Mg-25 ssNMR. The applications to half-integer quadrupolar nuclei in general, as well as their efforts in developing low-temperature ssNMR methods, are of interest in general and not just for biological systems.
Dieter Suter and his group at Dortmund have done some really neat things with optically-detected magnetic resonance, including metalloprotein research and their work on NMR of quantum wells. You can read more at their webpage here Fortunately for all of us, you can find a number of publications here. Suter was also involved in the Pines' group work on geometric phases in the late 1980s, so if you love it when interesting theory and experiment comes together, it should bring a smile to your face.
The Jaroniec group at Ohio State has been doing a variety of interesting things in solid state NMR, but I will mention their work with spin-labeled proteins here since it most tickles my fancy. You can read about it here, where they used spin-labeled proteins to obtain long-range structural constraints. Another advantage of spin-labeling is in facilitating assignment of congested NMR spectra – since you know where you introduce your spin label, you could potentially “blank out” certain residues that crop up in these congested regions to simplify assignments.
This is by no means a comprehensive list or thorough assessment of the field, more just intended to whet one's appetite. I would also like to point out the lecture notes from a solid state NMR workshop for graduate students and postdocs here. It may be a bit much to take in all at once, but some of you may find it makes for interesting reading despite not having a chance to hear the actual lecture. (FYI – no, I did not attend this workshop.) The list of speakers is a lineup of some of the major players in (biological) solid state NMR in the United States. If one's curiosity is still rampant, and you wish to expand your geographical purview, here is a list of lecture notes from a European ssNMR school (which, in case you're trying to pin me down, I did not attend) taught by a number of the major names in ssNMR in Europe. While I've long since downloaded these files and saved them, I can't guarantee that all lecture notes are still functional.
FYI - If I didn't mention you or your advisor or past advisor, I really didn't mean to do so. I would be pretty sure you've done/are doing awesome work, it's just that this is not a detailed journal club-style post. I will take recommendations, though, for any possible "journal club"-style posts, where the focus is on a single article or group of related articles. Tell me what totally BAMF science you've done, I'd love to hear it!
Read more!
I mentioned CERM at the University of Florence in my last post for good reason – it's something of a place where, sooner or later, all paths seem to cross there in one way or another. It's either having read one of the texts that's come out of the faculty there, collaboration, or you just find inspiration in what they're doing. Because I have a certain unavoidable bias towards ssNMR out of (most likely) residual Stockholm Syndrome, I will mention this paper as an example of extending the work done at Florence in using NMR to understand paramagnetic metalloproteins in solution to the solid state.
Lyndon Emsley and coworkers at ENS-Lyon are pursuing a number of really interesting projects in magnetic resonance, both of basic importance to NMR (see, for instance, this paper) as well as to problems in chemistry, metabonomics, biology, and paramagnetic systems.
Paul Ellis & coworkers at PNNL have done some really stunning work with Zn-67 ssNMR and Mg-25 ssNMR. Zinc is one of the most common metal cofactors found in biology, and they've managed to do direct measurements of zinc in proteins via solid state NMR (see here and here for examples). Given that it's a quadrupolar nucleus, it's even more awesome. (While comments noting that they do this sort of work at really low temperature are accurate, it does not diminish its luster.) Magnesium-25 is another nucleus that this group has focused on as of late. See this paper for some Mg-25 ssNMR. The applications to half-integer quadrupolar nuclei in general, as well as their efforts in developing low-temperature ssNMR methods, are of interest in general and not just for biological systems.
Dieter Suter and his group at Dortmund have done some really neat things with optically-detected magnetic resonance, including metalloprotein research and their work on NMR of quantum wells. You can read more at their webpage here Fortunately for all of us, you can find a number of publications here. Suter was also involved in the Pines' group work on geometric phases in the late 1980s, so if you love it when interesting theory and experiment comes together, it should bring a smile to your face.
The Jaroniec group at Ohio State has been doing a variety of interesting things in solid state NMR, but I will mention their work with spin-labeled proteins here since it most tickles my fancy. You can read about it here, where they used spin-labeled proteins to obtain long-range structural constraints. Another advantage of spin-labeling is in facilitating assignment of congested NMR spectra – since you know where you introduce your spin label, you could potentially “blank out” certain residues that crop up in these congested regions to simplify assignments.
This is by no means a comprehensive list or thorough assessment of the field, more just intended to whet one's appetite. I would also like to point out the lecture notes from a solid state NMR workshop for graduate students and postdocs here. It may be a bit much to take in all at once, but some of you may find it makes for interesting reading despite not having a chance to hear the actual lecture. (FYI – no, I did not attend this workshop.) The list of speakers is a lineup of some of the major players in (biological) solid state NMR in the United States. If one's curiosity is still rampant, and you wish to expand your geographical purview, here is a list of lecture notes from a European ssNMR school (which, in case you're trying to pin me down, I did not attend) taught by a number of the major names in ssNMR in Europe. While I've long since downloaded these files and saved them, I can't guarantee that all lecture notes are still functional.
FYI - If I didn't mention you or your advisor or past advisor, I really didn't mean to do so. I would be pretty sure you've done/are doing awesome work, it's just that this is not a detailed journal club-style post. I will take recommendations, though, for any possible "journal club"-style posts, where the focus is on a single article or group of related articles. Tell me what totally BAMF science you've done, I'd love to hear it!
Read more!
Saturday, January 10, 2009
Summations
Another week, another post. So, in my last post, there was that interesting looking Hhyperfine term that describes the interaction between the nuclear spins and any unpaired electrons in the environment. I will make a few points about paramagnetic NMR and something I've been reading up on as of late in the biochemistry/biophysics field. And a random comment or two, of course, at the end.
NMR of paramagnetic species, while a topic of growing interest and fruitful research over the past couple of decades, still tends to get people not involved in the field to look at you and think, “But what about the enhanced relaxation, broadened lines, and paramagnetic shifts? It just sounds really hard!” To anyone who might be thinking that as they read this, I would like to note that a number of the metals involved in biological systems are, in fact, quadrupolar nuclei. Also, something like one-third of all elements can exhibit paramagnetism. So, you can pick one challenge or another, it appears. Life may not be easy, but think about it like this. Since there hasn't been a whole lot of work done compared to other areas, it's a growth field with plenty of opportunity. Besides, as the bioinorganic chemistry folks like to remind us, the number of metalloproteins in nature probably hovers around at least 20 to 25%, if not a bit higher. I hardly think neglecting all of the proteins out there is a good idea just because you're terrified of some hard work. To say nothing of potential applications in materials science and chemistry!
One thing to keep in mind is that the effects aren't necessarily the same in every direction. If you have a paramagnetic center which isn't isotropic, and there's a distinct anisotropy, one could have two marker peaks that are the same distance from the metal but only detect one because of the anisotropy. If you've ever done any EPR, or have any familiarity with it, one can – in principle – resolve the components of the g-tensor. (Determining the angles between the g-tensor and the molecular frame is far more challenging from my understanding, though, and not something I've ever done.) Sometimes there will be axial symmetry (where two of the components are equal to one another), other times all three components of the g-tensor will be different. In any case, while there is practically always the averaging of the electron magnetic moment, distance matters when dealing with paramagnetic systems. If you're close up, you'll not only experience more of the effect, but it's more sensitive to any anisotropy. If you're further out, less effect, and it looks more isotropic.
So, experimentally speaking, one can decompose an observed chemical shift from a paramagnetic sample, δ obs, as follows:
δ obs = δdiamagnetic + δcontact + δ dipolar.
The nomenclature here can get a bit confused, so step-by-step, and hopefully not too confusing, here we go. δdiamagnetic is the “base” diamagnetic chemical shift, what you would get if there was no metal there. δcontact is the so-called contact shift (or Fermi contact shift) – this is what one gets from the average delocalized electron density on the nucleus interacting with the nucleus's nuclear spin magnetic moment. δ dipolar is also known as the pseudocontact or dipolar shift – this is what you probably think it is, just a through-space dipolar interaction between the nuclear spin and the electron spin. Now, there are all sorts of neat computer programs that can incorporate paramagnetic restraints for structure calculations/refinements, determine magnetic susceptibility tensors from NMR data, and other related pursuits.
If any of this sounds interesting to you, please let me know. I can pump out a lit post with a number of citations without much difficulty during the week, it would be my pleasure. Or if you'd rather receive an email, let me know. However, for a starting point, I would strongly recommend a look at the work done by Ivano Bertini, Claudio Luchinat, and coworkers at the University of Florence here. That should keep you busy for a while.....
I've gotten interested in the entire “intrinsically disordered proteins” topic as of late. It's interesting for a couple of reasons, some related to my current research, others simply since it's a neat topic. It makes one more careful in one's thinking that proteins just fold to some structure and stay there – proteins actually have functions, which are connected to other macromolecules and their functions. A protein can bind a ligand/substrate, where it may adopt a restricted ensemble of conformations. That protein's function may be modulated by interacting with another ligand or with another protein (for instance, an electron transfer protein), which could alter its structure. It may finally need to adopt another conformation to release the ligand or product. So whenever someone says, “Well, I've determined the structure of a protein,” what you should really be thinking is, “That person has determined the structure of a protein at a particular point along its functional pathway.” It also serves as a handy thing to bring up in the inevitable protein folding discussions that crop up every so often offline and online. Not every protein comes away from translation and adopts its active conformation right away, after all. As a short and hopefully accessible example, if we'll remember our blood clotting biochemistry, there are a whole bunch of zymogens (or proenzymes, if you will) involved in the process. They're activated upon trauma in healthy individuals – there's no need for them to be active when you're not bleeding, after all. There's a somewhat recent paper I'm still digesting here on a model involving intrinsic disorder with coupling allostery between binding sites. It's an interesting topic, mostly since I've been focused on shorter-range interactions earlier on in my education/research, and am now becoming more intrigued on what I will facetiously call “structural systems biology” - what is the structural and dynamic underpinnings of all these neat things that happen in cell biology and physiology from a physical chemistry/biophysics perspective?
I'm always intrigued by the interplay between solution and solid state NMR, and how one tends to delve into the other in order to get information that is typically denied to them in their home territory. You have the solution NMR folk introducing residual dipolar couplings, you have the solid state NMR people trying to minimize the effect of the geometric/angular effects in measuring certain parameters, it's all kinds of fun. I will probably comment on this here and there in the future, I think it's endlessly fascinating despite no longer actively in the magnetic resonance community.
There was an interesting post on equipment (the abundance or lack thereof, and of varying quality) at In the Pipeline the other day here. I've since moved onto a postdoc in an honest-to-Buddha biochemistry/biophysics lab since my graduate school days in a (biphysical/NMR) chemistry lab, and have had use of pre-made kits on occasion. I think it depends on one's perspective and background – it can be a crutch to use them, but I don't plan on doing a great deal of molecular biology while I'm here. Is it really the best use of my time or the lab's money to pour my own gels (as I did in grad school) or prepare my own DNA plasmid purification materials, especially if I'm not going to be doing it week in and week out for the next 2 to 3 years?
The one thing I did miss in graduate school, compared to my undergraduate institution, was the presence of a professional research-grade electronics shop. I especially missed it in my last two years of graduate school, when I seemed to get problems with instrumentation about every three to four months. While we managed to get a good amount of support from our department's NMR facility, it's not quite the same as having a facility capable of not only repairing instruments but also helping one devise potential work-arounds or new solutions. Of course, it is a character-building process, taking apart an NMR probe, diagnosing the problem, and then fixing it (at least until it fluxes up again).
In any case, a substantial enough second post, I think. Best wishes to all!
Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it. Read more!
NMR of paramagnetic species, while a topic of growing interest and fruitful research over the past couple of decades, still tends to get people not involved in the field to look at you and think, “But what about the enhanced relaxation, broadened lines, and paramagnetic shifts? It just sounds really hard!” To anyone who might be thinking that as they read this, I would like to note that a number of the metals involved in biological systems are, in fact, quadrupolar nuclei. Also, something like one-third of all elements can exhibit paramagnetism. So, you can pick one challenge or another, it appears. Life may not be easy, but think about it like this. Since there hasn't been a whole lot of work done compared to other areas, it's a growth field with plenty of opportunity. Besides, as the bioinorganic chemistry folks like to remind us, the number of metalloproteins in nature probably hovers around at least 20 to 25%, if not a bit higher. I hardly think neglecting all of the proteins out there is a good idea just because you're terrified of some hard work. To say nothing of potential applications in materials science and chemistry!
One thing to keep in mind is that the effects aren't necessarily the same in every direction. If you have a paramagnetic center which isn't isotropic, and there's a distinct anisotropy, one could have two marker peaks that are the same distance from the metal but only detect one because of the anisotropy. If you've ever done any EPR, or have any familiarity with it, one can – in principle – resolve the components of the g-tensor. (Determining the angles between the g-tensor and the molecular frame is far more challenging from my understanding, though, and not something I've ever done.) Sometimes there will be axial symmetry (where two of the components are equal to one another), other times all three components of the g-tensor will be different. In any case, while there is practically always the averaging of the electron magnetic moment, distance matters when dealing with paramagnetic systems. If you're close up, you'll not only experience more of the effect, but it's more sensitive to any anisotropy. If you're further out, less effect, and it looks more isotropic.
So, experimentally speaking, one can decompose an observed chemical shift from a paramagnetic sample, δ obs, as follows:
δ obs = δdiamagnetic + δcontact + δ dipolar.
The nomenclature here can get a bit confused, so step-by-step, and hopefully not too confusing, here we go. δdiamagnetic is the “base” diamagnetic chemical shift, what you would get if there was no metal there. δcontact is the so-called contact shift (or Fermi contact shift) – this is what one gets from the average delocalized electron density on the nucleus interacting with the nucleus's nuclear spin magnetic moment. δ dipolar is also known as the pseudocontact or dipolar shift – this is what you probably think it is, just a through-space dipolar interaction between the nuclear spin and the electron spin. Now, there are all sorts of neat computer programs that can incorporate paramagnetic restraints for structure calculations/refinements, determine magnetic susceptibility tensors from NMR data, and other related pursuits.
If any of this sounds interesting to you, please let me know. I can pump out a lit post with a number of citations without much difficulty during the week, it would be my pleasure. Or if you'd rather receive an email, let me know. However, for a starting point, I would strongly recommend a look at the work done by Ivano Bertini, Claudio Luchinat, and coworkers at the University of Florence here. That should keep you busy for a while.....
I've gotten interested in the entire “intrinsically disordered proteins” topic as of late. It's interesting for a couple of reasons, some related to my current research, others simply since it's a neat topic. It makes one more careful in one's thinking that proteins just fold to some structure and stay there – proteins actually have functions, which are connected to other macromolecules and their functions. A protein can bind a ligand/substrate, where it may adopt a restricted ensemble of conformations. That protein's function may be modulated by interacting with another ligand or with another protein (for instance, an electron transfer protein), which could alter its structure. It may finally need to adopt another conformation to release the ligand or product. So whenever someone says, “Well, I've determined the structure of a protein,” what you should really be thinking is, “That person has determined the structure of a protein at a particular point along its functional pathway.” It also serves as a handy thing to bring up in the inevitable protein folding discussions that crop up every so often offline and online. Not every protein comes away from translation and adopts its active conformation right away, after all. As a short and hopefully accessible example, if we'll remember our blood clotting biochemistry, there are a whole bunch of zymogens (or proenzymes, if you will) involved in the process. They're activated upon trauma in healthy individuals – there's no need for them to be active when you're not bleeding, after all. There's a somewhat recent paper I'm still digesting here on a model involving intrinsic disorder with coupling allostery between binding sites. It's an interesting topic, mostly since I've been focused on shorter-range interactions earlier on in my education/research, and am now becoming more intrigued on what I will facetiously call “structural systems biology” - what is the structural and dynamic underpinnings of all these neat things that happen in cell biology and physiology from a physical chemistry/biophysics perspective?
I'm always intrigued by the interplay between solution and solid state NMR, and how one tends to delve into the other in order to get information that is typically denied to them in their home territory. You have the solution NMR folk introducing residual dipolar couplings, you have the solid state NMR people trying to minimize the effect of the geometric/angular effects in measuring certain parameters, it's all kinds of fun. I will probably comment on this here and there in the future, I think it's endlessly fascinating despite no longer actively in the magnetic resonance community.
There was an interesting post on equipment (the abundance or lack thereof, and of varying quality) at In the Pipeline the other day here. I've since moved onto a postdoc in an honest-to-Buddha biochemistry/biophysics lab since my graduate school days in a (biphysical/NMR) chemistry lab, and have had use of pre-made kits on occasion. I think it depends on one's perspective and background – it can be a crutch to use them, but I don't plan on doing a great deal of molecular biology while I'm here. Is it really the best use of my time or the lab's money to pour my own gels (as I did in grad school) or prepare my own DNA plasmid purification materials, especially if I'm not going to be doing it week in and week out for the next 2 to 3 years?
The one thing I did miss in graduate school, compared to my undergraduate institution, was the presence of a professional research-grade electronics shop. I especially missed it in my last two years of graduate school, when I seemed to get problems with instrumentation about every three to four months. While we managed to get a good amount of support from our department's NMR facility, it's not quite the same as having a facility capable of not only repairing instruments but also helping one devise potential work-arounds or new solutions. Of course, it is a character-building process, taking apart an NMR probe, diagnosing the problem, and then fixing it (at least until it fluxes up again).
In any case, a substantial enough second post, I think. Best wishes to all!
Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it. Read more!
Thursday, January 1, 2009
Perspectives on Interfaces
I remember being asked once in graduate school by an undergraduate if I had instant recall of all of the detailed information on proton chemical shifts for all of the various chemical environments out there. While I didn't laugh – to be honest, while I remembered the general trends, there was no way I could rattle anything else off at the drop of a hat0 – it reminded me of the disconnect that I occasionally felt while in graduate school for chemistry. I never felt like much of a chemist – the aspect to chemistry that really distinguishes it from the other natural sciences1, synthesis, was something I did sparingly once I finished my undergraduate organic chemistry sequence. Grad school was focused on the area of solid state nuclear magnetic resonance (NMR) spectroscopy – a topic which gets minimal mention, if any, in most undergraduate curricula in the natural sciences. Worst of all2, I worked on proteins while in grad school – given the occasional bit of heat that can get generated come October depending on who's gotten the call from Stockholm, having any connection to biochemistry can make one become the token biochemist who tries to humbly explain why the Nobel went to someone who may actually have done some pretty decent work and is not, in fact, an unwashed lump of pirate scum.
However, being firmly ensconced between biology, chemistry, and physics is actually a really great place to be in my books. The biggest advantage3 is that I can lament the biological understanding of chemists and physicists, the physics blindness that chemists and biologists exhibit, and the appalling lack of chemical intuition that biologists and physicists demonstrate. However, I try not to do this too often, as I find it obnoxious and mostly only of use while at happy hour tweaking people's noses about this sort of thing. I of course hear the “jack of all trades, master of none” refrain, so I figure it all balances out in the end. While in grad school, in addition to playing with a couple of proteins via solid state NMR, I also had the opportunity to dip my toes into the uses of solid state NMR in investigating polymorphism in organic compounds, coordination chemistry complexes, a few discussions on possibly utilizing it for various materials, and the standard hijinks with cryogenic liquids that are part and parcel of every magnetic resonance laboratory (LN2 chilled vodka has a certain ambience to it).
So, given that NMR is a wonderfully interdisciplinary technique, and because I feel like rambling on about certain applications thereof given my past research interests (temporarily on hold while I get accustomed to things in the new laboratory where I make my home), I feel compelled to start what I suspect will be an infrequent but persistent habit of discussing various aspects of NMR dear to my heart. Now, when I think of NMR, I think of many things4, but among the first is the Hamiltonian which describes the plethora of interactions possible during an NMR experiment:
Htotal = HZeeman + HRF + Hdipolar + Hchemical shift + Hquadrupolar + Hhyperfine + Hscalar
The first term, HZeeman, is the interaction between the nuclear spins and the static applied magnetic field of the magnet iself, while the second term, HRF, is the interaction between the nuclear spins and the varying applied magnetic fields – aka the radiofrequency pulses – that compose the NMR experiment. The next term, Hdipolar, is composed of the various dipolar interactions that are at play in between the nuclear spins. This can be divided into the homonuclear and heteronuclear dipolar interactions. The chemical shift component, Hchemical shift, appears next, describing the shielding the nuclear spin experiences from its local chemical environment. The quadrupolar interaction, Hquadrupolar, crops up at this point, describing the interaction of the nuclear spins with the local electric field gradient for nuclei whose spins are equal to or greater than 1. The following term is the hyperfine interactions, Hhyperfine, where the nuclear spin is interacting with an unpaired electron. The last term is the scalar coupling (or J-coupling), Hscalar, which is the indirect through-bond interaction between two nuclear spins. Now, one can rewrite and manipulate this Hamiltonian's form as one sees fit or finds convenient – one could include the scalar couplings in the dipolar interaction term, for instance. Here, the off-diagonal matrix elements of the tensor describing the dipolar interaction would account for the scalar coupling and other effects.
Depending on what one does, these interactions may be nonexistent, attenuated, or averaged out. Sometimes you want to reintroduce these interactions (the excellent work on residual dipolar couplings is an example of this), other times you want to quench them (for instance, the interest in developing heteronuclear decoupling methods in solid state NMR of organic solids, including proteins and other biological samples). Obviously, if one never works with paramagnetic samples, fretting about the hyperfine interaction term is likely not the best use of one's time. And, well, if you do MRI, you may not have to think too much about chemical shifts in certain cases.5
Anyway, now that I've bored everyone with some elementary ramblings on NMR in anticipation of more interesting posts later on, I think I'll end this section. I do intend to keep writing about things of general scientific interest, my occasional thoughts about other things that cross my mind in related veins, and hopefully tweaking a few noses. I will eventually get the blog list and layout figured out in due time, but don't hold your breath. Any recommendations or suggestions - whether it be blogging technicalities or about the content - will be appreciated (although not necessarily followed up on). Hope everyone who's reading this had a delightful holiday season and New Year's!
Notes:
0.) I do have pretty good recall when it comes to the 13C chemical shifts of interest for amino acids, and a lesser level of recall when it comes to the 15N chemical shifts. Of course, that's from years of interpreting NMR spectra, not because I made any specific effort to stash them in my memory.
1.) At least in my opinion. Your perspective may differ on this, of course. I've made the point elsewhere that chemistry is all about structure, reactivity, and synthesis (see here ) and that instead of having grad students synthesize something interesting, biochemistry has model organisms synthesize the sample of interest (GFP, for instance) to allow the biochemist to focus on something more than just preparing the sample. This is a far longer, more subtle, and more complicated argument than I am giving justice to here – perhaps I will bring it up in the future.
2.) Just kidding.
3.) There are also challenges to being stuck in the middle.
4.) Not least the numerous NMR jokes. Here's one I'm sure you've heard - “How many NMR spectroscopists does it take to prepare a sample?” “Zero – they get it from a collaborator.” For those of you who are only familiar with solution state NMR, the tradition of coming up with inventive names for pulse sequences also exists in solid state NMR – HORROR, MELODRAMA, CRAMPS, and WISE, among others.
5.) This comment is based on a postdoc candidate talk I gave at an MRI lab - one of the students remarked that he hadn't thought much about chemical shifts once he finished up his magnetic resonance class, as his dissertation research primarily focused on relaxation phenomenona.
Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it. Read more!
However, being firmly ensconced between biology, chemistry, and physics is actually a really great place to be in my books. The biggest advantage3 is that I can lament the biological understanding of chemists and physicists, the physics blindness that chemists and biologists exhibit, and the appalling lack of chemical intuition that biologists and physicists demonstrate. However, I try not to do this too often, as I find it obnoxious and mostly only of use while at happy hour tweaking people's noses about this sort of thing. I of course hear the “jack of all trades, master of none” refrain, so I figure it all balances out in the end. While in grad school, in addition to playing with a couple of proteins via solid state NMR, I also had the opportunity to dip my toes into the uses of solid state NMR in investigating polymorphism in organic compounds, coordination chemistry complexes, a few discussions on possibly utilizing it for various materials, and the standard hijinks with cryogenic liquids that are part and parcel of every magnetic resonance laboratory (LN2 chilled vodka has a certain ambience to it).
So, given that NMR is a wonderfully interdisciplinary technique, and because I feel like rambling on about certain applications thereof given my past research interests (temporarily on hold while I get accustomed to things in the new laboratory where I make my home), I feel compelled to start what I suspect will be an infrequent but persistent habit of discussing various aspects of NMR dear to my heart. Now, when I think of NMR, I think of many things4, but among the first is the Hamiltonian which describes the plethora of interactions possible during an NMR experiment:
Htotal = HZeeman + HRF + Hdipolar + Hchemical shift + Hquadrupolar + Hhyperfine + Hscalar
The first term, HZeeman, is the interaction between the nuclear spins and the static applied magnetic field of the magnet iself, while the second term, HRF, is the interaction between the nuclear spins and the varying applied magnetic fields – aka the radiofrequency pulses – that compose the NMR experiment. The next term, Hdipolar, is composed of the various dipolar interactions that are at play in between the nuclear spins. This can be divided into the homonuclear and heteronuclear dipolar interactions. The chemical shift component, Hchemical shift, appears next, describing the shielding the nuclear spin experiences from its local chemical environment. The quadrupolar interaction, Hquadrupolar, crops up at this point, describing the interaction of the nuclear spins with the local electric field gradient for nuclei whose spins are equal to or greater than 1. The following term is the hyperfine interactions, Hhyperfine, where the nuclear spin is interacting with an unpaired electron. The last term is the scalar coupling (or J-coupling), Hscalar, which is the indirect through-bond interaction between two nuclear spins. Now, one can rewrite and manipulate this Hamiltonian's form as one sees fit or finds convenient – one could include the scalar couplings in the dipolar interaction term, for instance. Here, the off-diagonal matrix elements of the tensor describing the dipolar interaction would account for the scalar coupling and other effects.
Depending on what one does, these interactions may be nonexistent, attenuated, or averaged out. Sometimes you want to reintroduce these interactions (the excellent work on residual dipolar couplings is an example of this), other times you want to quench them (for instance, the interest in developing heteronuclear decoupling methods in solid state NMR of organic solids, including proteins and other biological samples). Obviously, if one never works with paramagnetic samples, fretting about the hyperfine interaction term is likely not the best use of one's time. And, well, if you do MRI, you may not have to think too much about chemical shifts in certain cases.5
Anyway, now that I've bored everyone with some elementary ramblings on NMR in anticipation of more interesting posts later on, I think I'll end this section. I do intend to keep writing about things of general scientific interest, my occasional thoughts about other things that cross my mind in related veins, and hopefully tweaking a few noses. I will eventually get the blog list and layout figured out in due time, but don't hold your breath. Any recommendations or suggestions - whether it be blogging technicalities or about the content - will be appreciated (although not necessarily followed up on). Hope everyone who's reading this had a delightful holiday season and New Year's!
Notes:
0.) I do have pretty good recall when it comes to the 13C chemical shifts of interest for amino acids, and a lesser level of recall when it comes to the 15N chemical shifts. Of course, that's from years of interpreting NMR spectra, not because I made any specific effort to stash them in my memory.
1.) At least in my opinion. Your perspective may differ on this, of course. I've made the point elsewhere that chemistry is all about structure, reactivity, and synthesis (see here ) and that instead of having grad students synthesize something interesting, biochemistry has model organisms synthesize the sample of interest (GFP, for instance) to allow the biochemist to focus on something more than just preparing the sample. This is a far longer, more subtle, and more complicated argument than I am giving justice to here – perhaps I will bring it up in the future.
2.) Just kidding.
3.) There are also challenges to being stuck in the middle.
4.) Not least the numerous NMR jokes. Here's one I'm sure you've heard - “How many NMR spectroscopists does it take to prepare a sample?” “Zero – they get it from a collaborator.” For those of you who are only familiar with solution state NMR, the tradition of coming up with inventive names for pulse sequences also exists in solid state NMR – HORROR, MELODRAMA, CRAMPS, and WISE, among others.
5.) This comment is based on a postdoc candidate talk I gave at an MRI lab - one of the students remarked that he hadn't thought much about chemical shifts once he finished up his magnetic resonance class, as his dissertation research primarily focused on relaxation phenomenona.
Disclaimer - Any arguable/unrigorous point in the above post is 99.9% me being lazy about it. Read more!
Subscribe to:
Posts (Atom)
