Nobel Week

It's been an interesting week so far, with the vesicle transport, Higgs boson, and now multiscale modeling being recognized.   While I clearly missed the ball this year in contrast to last year, I suppose it still leaves a potential Prize for molecular dynamics to be awarded in Chemistry, or perhaps Physics without the Higgs juggernaut lying in wait.

Of course, I am tremendously entertained to see the "those three do a lot of biological applications" comments already cropping up elsewhere.  Heh.   Read more!

Still alive....

You can cue the "Portal" song here - trying to not fall entirely away from this blog, especially as the most entertaining time of the year approaches for the chemical blogosphere.

Here's something for the quantum mechanics & photosynthesis fans - Excitation Energy Transfer in a Classical Analogue of Photosynthetic Antennae from J. Phys. Chem. B.  

I may have more to say about this and other things in the future.  In other news, I suppose we'll all be really careful with our supplemental information sections in the future........ Read more!

A neat bit of work.

From the people who brought you a structure of an unengineered full-length GPCR in phospholipid bilayers comes a structure of an unengineered full-length mercury transporter in phospholipid bilayers.  Gotta say that this is pretty neat.....

While new technical difficulties are otherwise occupying my efforts, I have enough things in the air to keep me busy.  Speaking of which, back to it! Read more!

A bit overdue, I suppose.

It seems that I have neglected you, dear blog.  I would express my regret, but, well, I've been busy.  I do hope that any occasional visitors have been having themselves a healthy, enjoyable, and productive 2013.

In any case, research is progressing to the point to where critical experiments will be run within the next few months, and presuming that my calculations are not too far off, we should obtain a reasonable amount of interesting data that will be published not long thereafter.   Which should help with the entire "what to do next?" conundrum.

However, before I meander to other topics, an observation - the one thing that keeps biochemistry challenging is that your samples are never entirely happy.  You can purify it to some vague approximation of homogenity, put it in a buffer it likes, add cryoprotectant, and flash freeze in countless little aliquots for storage at -80 degrees Celsius.  And when someone takes out an untouched aliquot 3 years later to check its viabiity, they find it's.....reluctantly alive.  You compare it to a sample made of protein you prepared a week ago, it's clearly working nowhere near its potential.  Now, I had the occasion to stop by my old grad school lab the other year, and the one student working on my old project is still using the small molecule ligands that was synthesized by myself and another student ~ 10 years ago. And that sample was not aliquoted out in tiny volumes, each one to only be used once, rest assured. 

Onto said other topics......

- I have to say I am extremely tickled and intrigued by the idea of approaching quantum mechanics as a certain generalization of probability theory.  It's probably a good thing I have no intentions of being a faculty member anywhere, as I'd probably try to pull off a presentation like this to students. 

- I sort of postponed my entire relativistic quantum chemistry self-study program.  I will get back to it.  One of these days.

- I am finding myself in the position where I need to start doing some serious practical and theoretical NMR teaching to a very small audience, though.   The whiteboards in lab are going to get messy, I suspect.

- Should probably start microblogging more often, to boot. 

Anyway, going to try and chime in more regularly here over the upcoming weeks and months. 




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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!

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.




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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!

Chiming in with a #ChemCoach entry

Here's my contribution to the ChemCoach Carnival, for what it's worth.


Your current job.

I am a postdoc in an actual chemistry department on the East Coast (USA) doing a mix of physical chemistry, biochemistry, and spectroscopy, along with dashes of molecular biology, computation, and misery. 

What you do in a standard "work day."

No such thing as a standard work day for me -  I'm presently slogging through heaps of molecular biology in order to validate a protocol for producing uniformly 13C/15N labeled protein (mostly since my PI isn't yet comfortable with how we do things in the 21st century).  Earlier this year, for example, I was doing everything from NMR on inorganic solids (after effecting some probe repairs on my own) as well as some p. chem. of lipid mixtures, in addition to protein NMR (solution and solid state). 

What kind of schooling / training / experience helped you get there?

 I was a biochem/chem undergrad (did my undergrad research in a biophysics lab - lasers and magnets, what more could one wish for?) and did my Ph.D. in an honest-to-Buddha chemistry department, doing biophysical chemistry.  I was briefly diverted into a year at a cancer research institute doing biophysics/soft matter-oriented work (that was a trip, let me tell you), but returned to my chemical roots. 

How does chemistry inform your work?

Just the other day I was trying to get this horrendous mix of inorganic chemicals to go into and stay in solution, actually - and people say there is no chemistry in biochemistry!  Heh.   My perspective on many of my scientific interests is rooted in my chemical background - for example, signal transduction could be translated as essentially controlling the rates of chemical reactions across interfaces, after all.  It's a rather essential component of what I do on a daily basis.

Finally, a unique, interesting, or funny anecdote about your career

I was at a conference where I spoke to the colleague of this scientist who had written a paper I had read and digested multiple times, as it was extremely germane to my interests.  He noted that said scientist had never followed up on this particular aspect since they found that obtaining reproducible data was far too tricky.  I should have realized then that my project might have been a bit ambitious, but I persevered and did manage to get reproducible data.  It just took another 2.5 years on top of the 2 years I had spent working on said project.....  Read more!

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.


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Prize ponderings

There have been some particularly interesting and worthwhile points made on the blogosphere over the last two days in the wake of the Nobel announcements.

1.) The success of small/investigator-driven/table-top science.  Actually, this applies to more than just the Chemistry Prize - see here for some comments regarding this year's Physics Prize. 

2.) Are we doing ourselves a disservice by discussing and debating the Nobel ad infinitum?  Is trying to find one to three people to recognize for a certain (set of) accomplishments really the best option?  How much of this is a holdover from how science used to be conducted pre-1900?  I happen to especially like Paul's Chemical Hall of Fame idea, and am willing to participate.  (I may want to nominate a physicist or two, though.*)

3.) Chemjobber brings up the interesting point as to whether the "mix" of chemistry that gets highlighted due to the Prize announcements is the one the community wants to present to the public. 

4.) There seems to be a sense that we need to circle the wagons a bit before the central science withers away.  I can't entirely disagree with this one.  I've been told multiple times that some of the questions I've stumbled over while doing biological chemistry regarding underlying questions of (mostly) physical and inorganic chemistry aren't really fundable, at least relative to the biological question with which I'm engaged.  You can only try and spin questions into applications for so long and so far before it gets tedious.


5.) I would read this post over at Everyday Scientist if you haven't already.  I'm in the same sort of boat as a bio/physical chemist.  I look at the Physiology/Medicine Prize and see work like in vitro fertilization and the H. pylori work recognized, and vast amounts of cell biology and systems physiology in their ranks (immunology, olfaction, neurobiology, and so on).  I view biochemistry as something which is securely rooted within the realm of chemistry.  Of course, this makes me wonder - while the issue of communicating chemistry to the public has been a discussion topic in various contexts over the years, maybe we also need to open up the lines of communications between chemists.  I'm not entirely sure how to go about doing this right at the moment, but I am definitely open to ideas.  

Of course, perhaps this is all just colored by my spectroscopic tendencies - if I can fit it into a coil,  cuvette, or beamline, consider my interest piqued.  Biological, chemical, geological, material, or physical. 

As always, the comment section is open. 

*: Erwin Hahn and Albert Overhauser.  Spin echoes and the Overhauser effect.  You know you want to agree with me! Read more!

A perhaps idiosyncratic view.

So, as it turned out, I called this year's Nobel Prize for Chemistry.  While I am now curious if I have any Elven ancestry (Elrond had that gift of foresight, right?), I feel I should note the following for those feeling that biochemistry isn't really chemistry.

I like biological chemistry since, in my view, it allows me to explore so many areas of chemistry.  I've synthesized isotopically enriched small molecules, I've purified proteins, I've been able to work with multiple spectroscopic and physical measurement methods in detail, I've examined paramagnetic inorganic solids with both success and failure, I've spectroscopically dissected multicomponent organic solids, I've had to explore certain types of self-assembling systems, I've cranked through theoretical treatments of metalloenzyme catalysis as well as polymer chemistry to make sense of my data over the years.....it's been a good time.

Extending from that, I like chemistry since it is perfectly situated to go hopping over boundaries.  I don't see why I shouldn't be able to figure out signal transduction pathways just because I'm a chemist.  I don't see why I should humbly hand over fundamental questions about the theoretical basis for spectroscopic methods to the physicists.   

Although right now biochemistry is giving me a headache.  Speaking of which, back to it.....

P.S. - A hearty congratulations to both Serge Haroche & David Wineland as well as, of course, Robert Lefkowitz & Brian Kobilka! Read more!

Nobel Notes

I am not even going to try and offer up any predictions or suggestions given last year's surprise.  I will, however, make a few comments, some of which I've mentioned in the past.


Molecular Dynamics

Martin Karplus is usually mentioned here.  Chembark has this folded into a more general computational chemistry prize with other scientists who have contributed greatly in other areas of comp. chem., as has the listing over at the Curious Wavefunction.  Not to take anything away from Prof. Karplus and his extraordinary career, but as was brought up last year, this position betrays a lack of appreciation for the development of MD.  I'm not sure how one could reward Karplus without snubbing the early accomplishments under the rug that showed the power of computational methods to ask questions and provide answers to physical problems.  Of course, it's also entirely possible that an MD prize would be one in Physics, which would be entirely acceptable as well.

GPCRs

While some have noted that a Prize for the recent structural accomplishments might seem premature, I can envision a slightly different scenario.  Kobilka's former mentor, Robert Lefkowitz, did receive the National Medal of Science a few years ago for his pioneering work in GPCRs, and has earned various other accolades over the years.  It's possible that there might be some sort of Prize that involves more on the biochemical side of things, but with an eye (and inclusion of) the structural work.

Magnetic Resonance

Not even going to try since I'll just keep babbling on for a while.

The Kavli Question

Will we ever see a dual Nobel/Kavli Prize laureate?  Or is the work that the Kavli Prizes recognize too interdisciplinary to make it through the Nobel process?  (Cue the whining of chemists who want to eliminate biochemistry from eligibility for the Nobel Prize.  Heh.)

P.S. - Really kind of hoping a physician wins the Nobel for chemistry, a chemist wins the Nobel for physics, and a physicist wins the Nobel for physiology/medicine.  It would be a tremendously hilarious week.









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Generality and Contingency.

There's an interesting post over at In the Pipeline regarding the nature of how enzymes work, and it has inspired excellent comments (as is par for the course at ItP).  One notion comes up that if so-called generalized mechanisms can't be devised, it's a failure of sorts for chemists.

My question - the photosynthetic reaction center is an enzyme (oxidoreductase).  Dinky soluble proteins that do isomerizations are enzymes.  Metalloproteins that rip apart chemical bonds are enzymes.  Some proteins float around in the cytoplasm.  Others are embedded are in a membrane of some sort or another.  Others exist at a membrane/solution interface.  Others are extruded from the cell to go do their thing in the cell's environment.  So, how general can a mechanism be for an enzyme? 

One of the things that is often brought up is that there's an element of historical contingency to consider when examining the history of life on this planet.   While we obviously can't just throw up our hands in defeat to try and understand the mechanistic details of biological chemistry, neither can we truck along without acknowledging that context and history are important aspects of the grander scheme.

If that is still not comforting, perhaps one can find some solace in Kornberg's reminder to trust in the universality of biochemistry.  Consider it a tradeoff - while your one enzyme might not shed light on all enzymes, it can shed light on the same enzyme (or similar ones) across many organisms, from the modestly-scaled unicellular beasties to the gargantuan eukaryotes that now populate the Earth.  Read more!

The Eternal Struggles

I managed to wrap up some experiments for a paper (sent back the revised version just the other day), While doing all of that over the last two weeks and change, I was stimulated to contemplate some long-standing issues off and on.  I figured they might be of mild interest.

The first - I'd like insight and numbers.

There's a fairly famous quote attributed to the late theoretical chemist Charles Coulson on obtaining insight versus just numbers.  My question - why can't we have both?  My  purely anecdotal experiences have suggested that chemists tend to be really ambivalent on this topic - on the one hand, we tend to be annoyed if we can't intuit everything from just a glance at the periodic table and a smattering of semiclassical physics (as I once vaguely alluded to recently), but on the other hand, we're quite quick to complain about things not being rigorous and how it's all just a model.  Other fields tend to be a bit less gripey about this sort of thing in my experience - they've either learned to deal with the indeterminacy or uncertainty, and/or come to grips with the ups and downs of toy models. 

The second - the perpetual translation that goes on in the head of anyone working at an interface.

A long time ago, I had gotten myself into a little back-and-forth because, in short, I was reading with my physics filter on when I should have been reading it with my chemistry filter.  This is hardly new, and it's certainly happened since then, for that matter.  It usually manifests in turns of phrase or underlying assumptions that - for example - aren't anything unusual in one setting but might be a bit odd or worse in another setting.  I'm not sure how to resolve this recurring situation, except to try and be more careful.   Suggestions would be welcomed.     

The third - is biochemistry really just "applied organic chemistry," as I was once informed as an undergraduate and have heard off and on since then?

Please.  One is only fooling the innocent undergraduates with that pompous bit of nonsense.  There's a reason it's called biochemistry - one needs to appreciate and understand how to navigate through the entirety of chemistry.  Once one casts aside the self-completing fantasies of some deluded chemists, it's rather straightforward to see interesting chemistry of all stripes manifest in biological systems.  There are incredible metalloenzymes that can fix nitrogen (nitrogenase), we have a chromophore bound to a membrane protein which experiences a photochemically induced conformational change (bacteriorhodopsin), and of course there's all of the multiple feedback and regulatory pathways that all seem to tie into one another in ever-increasingly labyrinthe but beautiful ways that seem to be well-attacked (to some extent, at least) with the mathematical formalisms of physical chemistry.  And all of that is just the tip of the iceberg.  Read more!

Danger!

Chiming in late on the Toxic Carnival, but I can't help but mention one of the most historically significant chemical threats to life on this planet.

My chosen chemical is arguably responsible for one of the greatest environmental catastrophes in history, whose effect was global in its reach and certainly changed the biological face of the planet.  Said chemical is still produced in vast amounts to this very day, and no one seems inclined to do anything about it.

The culprit, of course, is dioxygen (O₂).

Billions of years ago (a bit over 2 billion of them, in fact), the Earth's atmosphere became significantly more oxygenated due to the increasing extent of oxygenic photosynthesis. I can't even begin to fathom how many otherwise innocent anaerobic bacterial species must have been driven to extinction. Of course, it was perhaps just a matter of time once oxygenic photosynthesis evolved from its anoxygenic roots. 

The worst part is that oxygen still wreaks havoc among organisms to this very day.  Exposure to higher-than-normal partial pressures of oxygen can be toxic, and - in fact - many animals have elaborate mechanisms of oxygen transport that serve to protect the organism from unfettered oxidative damage, including specific "oxygen chaperones" (in essentially all vertebrates, this role is filled by hemoglobin).   Of course, anaerobic organisms are still susceptible to the threat of dioxygen in their environment to this very day.  The reasons for this can range from insufficient amounts of enzymes capable of metabolizing reactive oxygen species (catalase, peroxidase, and others) to oxygen poisoning their (frequently novel) catalysts  - err, metalloenzymes - that are specific for anaerobic metabolism. 

At a more molecular level, dioxygen is critical for the function of cytochromes P450, which has been termed "nature's blowtorch."  That doesn't sound very soothing, now does it?  One of the oxygen atoms is doubly reduced and scoots off as water, leaving behind a vicious biological oxidant which will insert the remaining oxygen atom even into fairly unreactive C-H bonds.  Reactive oxygen species such as superoxide and peroxide are produced as a result of oxidative phosphorylation, due to incomplete reduction of dioxygen by the cytochrome c oxidase complex.

When you take a deep oxygen-rich breath one of these days, think of the poor anaerobes who can't. You should feel a twinge of guilt. Read more!

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!

Black boxes and rigor.

There was a thought-provoking post over at the Curious Wavefunction regarding a Nature op/ed piece on the increasing "black boxification" of modern biological research.  While it is both concerning and makes for an easy bit of mockery, I have to sometimes wonder where one can draw a line.  For example, it's been fairly typical (in my experience) for some specific physical/spectroscopic method to be introduced in a manner consistent with one's expected minimum physical chemistry background.  It's not uncommon for there to be a step or two which is essentially "and we take this result from classical mechanics/electrodynamics" or "this is actually a result of a certain mathematical theorem/relation" in such an introduction.   Some might claim that there's a huge difference between not knowing a technique relies upon a particular mechanism versus (for example) not having worked out a laborious series expansion for a particular term that yields the desired form in that case.  But I would view it as a caution - what happens if you stumble across a case where, in fact, you need to go back and rederive the expression for a term since some parameter or limiting case has changed?  Naturally, you find that if you end up relying upon that method in your research to any significant extent, you are going to dig in deeper.  You will figure out what the limiting cases are, and where any approximations are likely to break down. 

Of course, here I'm reminded that there is a difference between being able to contend with the formalisms of an argument and being able to develop a more physically rooted intuition for said argument.  I suspect many of us have encountered the "it's not rigorous enough" student somewhere along the line - they're the ones who find the experimental nature of scientific research a bit troubling and are worried that we're not careful enough with our mathematics.  We don't want to go in that direction either, of course.  Well, those of us who are scientists and not just frustrated mathematicians, at least.  I'd like to think that there can be a fruitful synergy between the two - when one is able to invoke physical intuition is a good time to develop one's mathematical skills and understanding, and then later on apply that mathematical expertise to a new problem where intuition is lacking at the start. 

I have more stuff to blather about, as it distracts me from extremely unfortunate technical difficulties in my own research.  Stay tuned. Read more!

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!

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!

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!