The best seat in the house for watching HIV entry

One of my major reasons for attending BPS this year was to expand my knowledge in a field that isn’t very important at all for the work that I do in my day to day.  My work involves designing molecules that can alter protein function and hopefully “drug” an interaction or protein conformation that is useful therapeutically.  The readouts for whether we are successful are pragmatic ones — we look at cell viability, downstream effects, preservation or desolation of certain cellular pathways as needed.  What we generally don’t concern ourself with is confirming with mechanistic insight how exactly the molecules we make do what they do.  So I decided to go learn more about biophysical techniques for looking at protein dynamics and allostery — the best place to do that was BPS.

Well I’ve learned a lot, and have a lot more to learn from all the papers and techniques that others have suggested I look into.  One of the most fascinating examples of a study hoping to shed light on protein dynamics of therapeutic importance was presented yesterday by James Munro.  Professor Munro used single molecule FRET to monitor conformational changes in the HIV envelope protein gp120 as it interacted with receptors on the host cell surface.  Only one envelope protein on each HIV virion was dually labeled, with a FRET donor at one relatively “fixed” location, and an acceptor at one of three locations on nearby loops of gp120.  FRET is a very powerful technique, and smFRET is even better since it gives conformational trajectories that can give valuable information about the kinetics being observed. However with two labels, the FRET readout is one dimensional — only one coordinate is generated, with points along a line of FRET efficiency indicating the distance between two points on a protein surface.  Can something as complex as HIV envelope binding and entry be observed usefully along a single coordinate?

The answer, as published in Science last year, is yes.  With the choice of a coordinate indicating the distance between the V1/V1 loop region and the V5 loop in the outer domain, Munro and his coworkers were able to observe three distinct conformations accessed by the envelope protein: a highly occupied low-FRET “ground” state indicating prefusion envelope protein, a high-FRET state indicating the envelope protein bound to its receptor CD4, and an intermediate-FRET state indicating binding to both CD4 and the HIV coreceptor.  Many experiments with both laboratory and clinically-derived HIV strains with a variety of ligands confirmed this result.  The smFRET kinetics also supported this view, as fitting the traces to a three-state Markov model showed many transitions from unbound to CD4-bound, and transitions from CD4-bound to CD4+Coreceptor-bound, but very rarely transitions from unbound directly to the doubly-bound conformation.

This choice of coordinate was not a lucky guess, it was guided by existing low-resolution structures of the envelope protein during membrane fusion, and even so, likely was the result of many grad student/postdoc-years of trial and error.  What this study does show is that even complicated and dynamic processes like HIV membrane fusion can often be monitored and deep information gleaned from a very clever choice of one coordinate.

I’ve often spend time choosing a coordinate to succinctly show the transitions in a molecular dynamics system of interest, and seeing someone not only choose the right coordinate, but get a working smFRET experiment working along it for such a cool system was a lot of fun.

It’s good to be single in 2015

Single molecule work has really stood out to me at this meeting, as researchers are developing all sorts of interesting methods to study molecules both in vitro and in live cells. As someone who doesn’t do single molecule work, I found the Biophysics 101: Super-Resolution Microscopy session on Monday tremendously insightful. I think for the first time I truly understood what goes into improving resolution for optical microscopy techniques. In particular, I found Dr. Keith Lidke’s presentation very thorough and easy to understand, as he went through theory and then stepped through each and every acronym (fPALM, STORM, etc) and what was different about each one. I understand that the presentations will be posted online somewhere; does anyone have a link to this? Please share it in the comments or send it and I will be happy to post it here as well.

Having absorbed some basic background knowledge, I wandered over to the Single-Molecule Platform session on Tuesday morning. In particular, I was curious about a presentation on the development of single-molecule electron paramagnetic resonance (EPR) by Richelle M. Teeling-Smith from Ohio State. I use EPR spectroscopy in my own work to study protein-protein interactions and conformational changes. There are a variety of EPR methods, but the one I utilize employs the placement of thiol-reactive spin labels to introduce paramagnetic centers into proteins. The technique relies on the sensitivity of the label to its environment, and is a useful probe for protein dynamics on timescales that cannot be achieved by optical microscopy.

In the traditional continuous-wave EPR experiment, this label (or the corresponding protein it is attached to) needs to be enriched to a high enough concentration (typically 50-200 uM) to achieve a robust signal in the experiment. This is definitely a few more than a single molecule, and this the measurement is heavily averaged. So, I was quite curious about how this group had managed to develop a strong enough signal from one molecule to make a measurement.

Teeling-Smith and colleagues employed the covalent tethering of a nanodiamond label, which I’d never heard of previously but apparently is enriched with paramagnetic centers, to a single molecule of DNA. On its own, the nanodiamond achieves a robust signal that is sensitive to whether it is free in solution or attached to something bigger (much like a nitroxide spin label). It’s of course much larger than a nitroxide label (which is about the size of an amino acid side-chain), which I suspect makes it impractical for work such as my own. However, it can clearly be a sensitive reporter in larger systems, and may offer information at a level of detail that is not possible with single-molecule optical techniques. I found the work very creative and fascinating, and I will be curious to see how it progresses in the coming years.

Challenges and Opportunities in NIGMS reforms

Grant writing seems to become the central part of all PIs’ daily task, with multiple deadlines coming up all year around, and even a graduate student like me can feel the decline of the funding climate. While Biophysics Society meeting serves not only an opportunity to share and inform new biophysical advances,  also as an chance for people to voice and address concerns. Today at the “Conversation with NIGMS Director
Jon Lorsch” session, Dr. Lorsch introduced to us his vision and reform plan for the funding agencies. Of all, Dr. Lorsch specifically explained the new pilot program, “Maximizing Investigators’ Research Award (MIRA)”. MIRA is an innovative program that grants the investigator more flexibility and sustained support compared to the normal R01 grants. The longer time duration, larger MIRA grant is designed to reduce the number of the grants that one PI has to write for application, decrease the total number of the grants application, and reduce the valuable time that academic faculty members spend on grant reviewing. MIRA appears to be quite bold reform aiming to streamline and increase efficiency for grant application processes. However, the question still remains, “Which PIs are more likely to get a grant like MIRA?” There are growing concerns over the increasing averaged age for PIs to get their first R01.  The national averaged increased from 35 yrs to 45 yrs in the past 30 years. The support for early career investigators appear to be the concern of most of the attendees. Although the MIRA also promises to boost the selection of young investigators, the challenges and competitions that the young assistant professors face on a daily basis still seems like a great barrier for the career development.

In a way that science grant is like trust fund: the money is there, but you need to wait until a certain age to use them.

The Biophysical Society Logo: Random or Profound?

Today was my last day at the Biophysical Society Meeting and I have to say that I truly enjoyed my first experience at this conference! I’m used to the American Chemical Society National Meetings, which are significantly larger, so this was a nice change of pace. Blogging was definitely a new facet to a scientific conference for me, so I hope you’ve enjoyed reading these posts as much as I’ve enjoyed writing them!

I wanted to bring up an amusing question that someone mentioned to me earlier this week and see if it boggles your minds as much as it did mine! I attended the New Member Coffee break on Monday since I just recently joined the Biophysical Society and a relatively new PI at my table brought up a question he hadn’t managed to find an answer to…

What does the Biophysical Society logo represent?

We’ve all seen it – horizontal lines sandwiching the name of the society – and it’s even in the banner for this blog at the top of the page. Does anyone actual know the origin of the logo, though? Is there a significance behind the design or an interesting history that explains its look? I tried Google-ing it and came up with nothing!

Since I had absolutely no idea what the logo meant, I decided to take up the task of unofficial investigator and I promised the initial question poser that I would blog about it so his question could reach a larger audience. For the last two days, I have been asking people at random what they think the logo represents and I got an interesting array of responses! The most amusing thing I noted was that everyone had their own unique interpretation – I kid you not, I did not have a single idea given to me more than once! Below is a list of some of the answers I got from new and old members alike:

1. An hourglass energy landscape
2. Artistically enhanced error bars
3. Noise
4. A poorly drawn Jablonski Diagram
5. A rotational-vibrational spectrum
6. Representation of the number of subgroups you can join
7. Loop and beta-sheet structure representations
8. A free induction decay
9. Single molecule FRET data
10. Random scribbles the society founder drew when drunk
11. “Old school DNA sequencing thingy”
12. Absolutely nothing

I hate to disappoint, but I never found someone who actually knew the answer! That’s kind of wonderful if you think about it. Just take a look at those responses… you can see the expertise of some people poking through. We have some physical chemists, NMR spectroscopists, structural biophysicists, single-molecule experts, and a class of folks with a great sense of humor. The interpretations I received seemed to be biased, in a sense, by the way each person viewed science. Whatever “lens” we use to see the world of biophysics trains us to approach problems in a particular way and view the results with unique perspective. Maybe that’s the point of an ambiguous logo. It’s nothing and everything all at once depending on who happens to be looking at it. Isn’t that a lot like this particular field we all love so much? You might view a certain biological phenomena one way and I might disagree, but that’s the point. That’s how discoveries continue and our knowledge evolves. We come up with answers, but there has to be someone out there who remains skeptical. It keeps us honest and makes it possible for us to rewrite textbooks when old ideas turn out to be wrong or incomplete.

…or maybe the logo really is just a set of horizontal lines that looked neat. That’s a bit anti-climactic, though, so I think I’ll stick with my idealized view of it representing the beautiful, multi-faceted nature of biophysics. I think that meshes quite nicely with the enormous diversity of research that culminated at this conference and the vast array of viewpoints I’m sure many of you heard with regards to your own research at your poster session or talk.

Feel free to prove me wrong in the comments if you have an idea about the logo’s meaning or actually know the answer!

The foodie speaks

The last couple of days have been crazy, in every sense of the word. Most of the time I found myself running… catch a platform session, to meet someone I planned on meeting, to the poster sessions, to lunch. It’s amazing how much have been fit into a single day, and I am equally surprised by my ability to cope with it all. I had my own poster on Monday, and it was a great experience presenting one for the first time.

I did, however, manage time to venture out of the Convention Center. Sunday night, for example, I was at Pratt Street Ale House with my colleagues. It’s less than a five-minute walk from the Convention Center. I am not much into beer (I know! I apologize!!!), but I would say that the Bishops Breakfast was well worth it. So was the Breckenridge-Vanilla Porter. The atmosphere at the Ale House is pretty great too!

Monday night we tried out Luna Del Sea, a seafood restaurant also quite close to the Convention Center. It’s expensive (one meal is a minimum of $30), but the ambiance combined with the delicious shrimps and scallops definitely makes up for it.

And oh, did I mention I have finally managed to have Maryland Crab Cake? I have been waiting to try one ever since I heard about it. I will take this opportunity to thank Ellen Weiss, the Director of Policy and Communications of the Biophysical Society, for informing me about this particular item. It is nothing like anything I have had before, and would surely have it again before I leave Baltimore tomorrow (and when I am back, this will be the first thing I have!).

That will be all for now. Will be back soon with more.

“Yes! Yes! But why? But why?”

This was what Klaus Schulten heard from an experimental colleague after he had told him what he’d found and it neatly highlights one the key advantages of molecular simulation; you can see in extraordinary detail what is happening or, as Klaus put it in his National Lecture last night,

It doesn’t only agree with the experiment, it tells you more.

Implicit in that statement is that the simulation must first agree with experiment, which is pretty obvious, but still worth saying! The best way of achieving this, in my experience, is that you work closely with experimentalists, preferably from the beginning of a project. Klaus did not explicitly spell this out it but it was clear from his lecture that this is what he always tries to do.

He also showed several very nice examples from ankyrin repeats to cadherins to aquaporins where simulations from his group have made predictions that were subsequently confirmed by experiment. Predictions of this kind are, I think, the possible the best demonstration of the utility and power of computer simulation in molecular biophysics. Another key theme was choosing the appropriate level of description for any simulation, or as Klaus put it,

Chemical detail is important.

This is true; we must resist the urge to discard detail to make our simulations simpler and faster at the expense of biological accuracy and instead chose the most appropriate description for the system at hand.

Given the audience was mostly experimentalists it was striking how long Klaus spent talking about the tools i.e. the software, or to be specific the molecular dynamics (NAMD) and visualisation (VMD) codes that his group have developed and now supported for around twenty years. But then again these are important and both NAMD and VMD are very widely used so why not talk about them?

What is unusual is that both codes have a full-time developer which allows them to make regular releases, have good documentation and also nurture a community of academic contributors. I think the question this raises should be why don’t more academic codes have this degree of support? And, in tandem with that, why don’t we as a community use more software engineering tools, as encouraged by Software Carpentry and institutes like the Software Sustainability Institute in the UK.

That Klaus ended with an advertisement for a training course is especially poignant for me; I attended the first NAMD summer school back in 2003 and this really kick-started my PhD (I was at the end of my first year). As an aside I enjoyed the anecdotes and stories during the lecture, like the one I started with, and so I thought I’d end with one of my own. During the summer school in 2003 I have a vivid memory of Klaus addressing us all and booming out “if you use molecular dynamics to calculate free energies you are braindead!”. So when I saw those free energy profiles last night I smiled. I’m glad that even distinguished scientists can change their minds.

DNA Methylation at single molecule level

Just heard this talk….Nanomechanics of DNA Methylation!!! Always drew my interest as it is one of the major epigenetic modifications leading to gene silencing. Gene silencing occurs when a cell develops or exercises the ability to prevent or reduce the expression of a certain gene. These modifications do not alter the sequence of the DNA but they affect how the genes are read by the cell. Every tumor without any exceptions, has varied frequencies of DNA methylation pattern, indicating an altered genome. No doubt, it has been studied to be a cause of breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, aging, Rett Syndrome and many more. Already epigenetic therapies towards drug development involving cancer is driven by DNA methylation inhibitors (FDA approved) like 5-azacytidine (Vidaza), 5 aza-2/-deoxycytidine, Decitabine (Dacogen) and there are many more in various phases of clinical trials. Henceforth, seeing this at a single molecule level becomes even more exciting. There have been recent papers studying these, but the one which almost resembled the work done by Csaba I. Pongor was by Kaur et al “Hydrophobicity of methylated DNA as a possible mechanism for gene silencing.” Physical biology 9, no. 6 (2012): 065001. While both were able to observe a shorter contour length in the methylated form of the DNA, and an increase in persistence length in the methylated form, but the interpretations seemed quite different, Csaba’s work had in additional a lowering of stress modulus on methylation. I would have certainly liked to observe a titration study on the degree of methylation and observed the effects, likewise done by kaur et al, but still beautiful work!! Well it is indeed fascinatinig how just adding a single methyl group to the DNA can give such huge variations to it. Surely more work needs to be done at single molecule level to understand this mechanism further and I would be looking forward to it.