Thematic Meeting, Berlin 2017: Session III

Welcome back to my post-conference note. Let’s jump right into the third session on “Interpreting Experiments Through Molecular Simulations“ including talks by Ceclilia Clementi, Michael Feig and Arianna Fornili on Saturday afternoon and with Shang-Te Danny Hsu, Jana Selent and Massimiliano Bonomi on Sunday morning.

The challenges of computational biophysics aiming to bridge molecular and cellular studies is among others due to the characterization of macromolecular systems by sets of different timescales, separated by large gaps. Celilia Clementi tackles this challenge by combining coherent state analysis and Markov State Modeling. Furthermore, she introduced a theoretical framework for the optimal combination of simulation and experiments in the definition of simplified coarse-grained Hamiltonian protein models. As those simplified models loose information, the combination with experimental data makes them more realistic and provides larger time-scales. She further illustrated the method and mentioned as application the coarse-graine model of FIP35.

Next, Michael Feig investigated protein dynamics and stability with crowders in simulations and experiments. He states protein destabilization in Villin due to crowding, in order to explain the differing results from NMR vs. MD. With Villin under dilute conditions, he observed oligomer formation in MD, whereas transient oligomers form on timescales longer than rotational translation diffusion. Rotational diffusion is slowed down by additional factors. In addition to his study on Villin, he could identify transitions between conformations connecting all states of Bacterial genomic DNA, based on targeted MD.

The last talk of this Saturday was given by Arianna Fornili about identification of rescue sites for protein function. As rescue mutations can be mimicked by drugs, their locations is of interest for drug design wherefore she developed a method. Double Force Scanning (DFS) mimics mutations by external forces, using an elastic network model to represent protein dynamics. In oder to model structural perturbations, linear response theory is used. In detail, she used the Fibonacci lattice for uniform distribution for force vectors. The performance of DFS was tested on p53, predicting 80% non-rescue sides correctly, and on an evolutionary dataset, where 79% of evolutionary rescue sites where predicted correctly.

Subsequently after Ariannas talk and a short coffee break, the first poster session was started. Although it was very crowed (which might have been a live experiments from the organizers, fitting to the last session), posters of high interests have been presented, leaving the presenters no break for a small sip of water. Afterwards, the program was open for all those aiming to explore the city of Berlin. On Sunday morning, the third session was continued with talks from Shang-Te Danny Hsu, Jana Selent and Massimiliano Bonomi.

The first session of this Sunday started with a rather biological topic on the structural basis of substrate recognition and chaperone activity of ribosome-associated trigger factor (TF) regulated by monomer-dimer-equilibrium from Shang-Te Danny Hsu. As the structure contains highly dynamic regions, those parts could not be easily resolved. This was also confirmed by solid state NMR studies, showing ribosome binding-induced conformational change in the ribosome binding domain of TF (TF-RBD). Shang-Te revealed TF substrate specificity by peptide array analysis, originating from recognition of averaged property rather than an exact sequence. Produced SAXS data showed a misfit between those and the crystal structure of TF. Further studies using pulse dipolar ESR spectroscopy revealed multiple dimer configurations of TF, which could be identified by chemical cross-linking. Furthermore, he modeled a dynamical system using several different techniques such as crystallography, NMR, SAXS. Comment: good luck for your PhD student 😉

Next, Jana Selent presented her work on the functional dynamics of the distal C-tail of arrestin. As introduced by her, phosphorylation of the GPCR C-tail triggers the arrestin pre-complex, including a partial C-tail displacement of arrestin. To investigate the role of the distal C-tail, she performed all-atom MD simulations and site-directed mutagenesis studies. She described its conformational space with preference to bind to positively charged residues. Tryptophan-induced dynamic quenching was increased for some residues. Furthermore, she investigated the mechanism of IP6-induced displacement, where IP6 displaces residue 393 to 400 but not further down upon binding to GPCR. As second topic, she investigated the C-edge loop of arrestin. By MD simulations, she showed that C-edge loop of pre-activated arrestin was able to penetrate the membrane, in contrast to activated arrestin. This was confirmed by quenching mutagenesis experiments. Finally, she postulates a step-wise binding process from unbound GPCR over an arrestin-GPCR pre-complex including the displacement of the distal C-tail and the penetration of the C-edge into the membrane, followed by the high affinity complex.

The last talk for this session was hold by Massimiliano Bonomi on integrative structural and dynamical biology with PLUMED-ISDB. As computational and experimental technique have their challenges or errors, a hybrid or integrative method could provide a more realistic view. By providing the module PLUMED-ISDB, he presents a way to investigate heterogeneous systems by including experimental data with a priori information. It uses a Bayesian inference method which accounts for data noise and averaged ensembles. He applied this metainference approach to cryo-EM data, able to explain the data better with a lower resolution as a mixture of dynamics and noise.

At last, he addresses challenges to the community, to those I could not agree more and therefore will end my post with his wishes: More distribution of ensembles of the community like structural models, model populations and protocols; the establishment of robust methods for ensemble comparison and validation; and a way to facilitate comparison of different ensemble modeling approaches by sharing methodologies.

Cheers,

Johanna

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Thematic Meeting, Berlin 2017: Session I & Session II

Just a blink and this fantastic conference was over. Equally my time for writing some posts passed by, which preparation I fairly underestimated. So, now I would like to share my notes with you. Hope you enjoy reading my post-conference posts, to remember the great talks or get an impression, what you missed! Let’s start with session I & II!

After planning this meeting thoroughly together with Helen Berman, Andrea Cavalli and Gerhard Hummer for several years, Kresten Lindorff-Larsen addressed the opening remarks emphasizing what they aimed for this program and how much they looked forward to tackle it. And they were not alone! Finally, the first session on “Disordered Protein Ensembles” started, including talks by Adriaan Bax, Tanja Mittag, Teresa Head-Gordon and Paul Robustelli on Friday and Martin Blackledge and Birthe Kragelund on Saturday morning.
For me it was the first time I heard such comprehensive collection of talks about (intrinsically) disordered proteins (IDP) and this session had a great mixture of experimental methods to computational strategies and the benefit of their conjunction.

Starting with Adriaan Bax, he observed protein folding and misfolding by pressure jump NMR with special focus on HIV-1 Protease. The challenge in there is indeed the pressure but by managing this, he observed pressure-induced fully reversible unfolding including slow exchange time scale. Additionally, monomer exchange faster than stable dimer. He further looked into single residue cis-trans isomerisation. By investigating hydrogen bonding network stability, he sees a significant shift towards low pressure. For Abeta(1-40) fibril formation, real time NMR signals disappear after dropping pressure from 2.4 kbar to 1 bar, which is then heterogeneous, but rapidly reappears after jumping to high pressure. For his third system (Ubiquitin), the showed the influence of pressure by revealing an intermediate state, leaving open that this might also be due to mutations.

Next Tanja Mittag studied the effect of multi-site phosphorylation on conformations of intrinsically disordered proteins, exemplary S. cerevisiae transcription factor Ash1. Ash1 is expected to be collapsed and to expand upon multi-site phosphorylation but SAXS shows only little differences. By following an ensemble optimization method, she identified preferences for expanded conformations to be insensitive to screening of long-range electrostatic interactions, but reacting to the presence of weak local structural preferences. She identifies sequence features, mainly a relationship between proline and charged amino acids, deriving intrinsic sequence code expansion. She argues against chain compaction upon multi-site phosphorylation due to proline isomerization or presence of pSer/Arg or pThr/Arg salt bridges. Additionally, all-atom simulations could reproduce the experimental observations. Finally, she states that a large enough concentration of Prolines distributed along a sequence can buffer changes in net charge per residue while changes in local conformation occur.

After a refreshing coffee break, Teresa Head-Gordon presented new methods for generating and evaluating conformational ensembles. She looked at secondary structure propensities for Abeta42 and Abeta43 and observed differences in N-terminus and central hydrophobic core with REMD simulation, indicating either bad force-fields, poor sampling or both. There she introduced Temperature Cool-Waking (TCW) using annealed importance sampling and could indicate poor sampling. She states that TCW and polarizable forcefields work for folded proteins and are robust for IDPs, whereas Boltzmann priors may be questionable for IDPs. Monte-Carlo side chain ensemble (MC-SCE) has implemented a sophisticated energy function to distinguish different side chain packings based on Boltzmann factor. She concluded that MC-SCE and entropy expansions are informative about improved catalysis.

For the last talk of the first day, Raul Robustelli from D.E. Shaw Research gave insights in ongoing force field development, focusing on ordered and disordered protein states. The problem of current force fields for IDPs is that they tend to being structurally too compact relative to experiments. In this context, he stated the importance of an improved water model, as water dispersion energies have been systematically underestimated. Therefore, he introduced the TIP4P-D water model. Enhanced hydrogen bond potential and ‘fractional’ charges showed improved fits of non-bonded parameters to quantum data. As those analysis fit nicely, still further improvements have to be conducted, e.g. to challenge the over-representation of beta sheets.

With Paul Robustelli, an interesting first day at the “Conformational Ensembles from Experimental Data and Computer Simulations” conference ended and everybody went over to the welcome reception to enjoy a beautiful evening on the terrace outside. Perfect time for some networking. You can find some impression on twitter (@BiophysicalSoc).

Day two started with Martin Blackledges talk about “Large-scale Protein Conformational Dynamics from NMR and Molecular Simulation. From Fundamental Biophysics to Biological Function”. He aims to understand large-scale domain motion in influenza, where temperature is a key factor. Problematically is that its crystal conformation cannot bind to importin alpha. For NMR, its 2 domains do not interact with each other, rather having an open and a closed form. He finds that highly conserved salt bridges are involved in stabilizing the closed formation. He uses chemical exchange saturation transfer (CEST) to measure interconversion rate and population of substates. Using integrated structurally dynamics (NMR, smFRET, SAXS) CEST analyses are supported and dynamic interconversion between open and closed form of H5Na influenza 627 are revealed .
To explain how IDPs interact with their physiological partners, Martin introduced Asteroids, a selection tool of ensemble descriptions of intrinsically disordered systems, which was tested using target ensembles. In order to understand the mechanism of highly dynamic IDPs interactions, first their intrinsic dynamics have to be understood and therefore he developed a physical framework by comparing conformational sampling between 274 -298 K.
Using the ABSURD (average block selection using relaxation data) procedure, he could reproduce the experimental data, overcoming current limitations of MD simulations of IDPs. It identifies ensembles of trajectories of IDPs and can thereby map extent of inter-residue dynamic correlation.

The last talk for this session was held by Birthe Kragelund on dynamics and disorder in class 1 Cytokine receptors. She states that disorder is an important and integral part of membrane proteins as intrinsically disordered regions (IDRs) co-structure them as regulatory platforms with the need of structural information on bound state. Still, they are often not recognized. For membrane proteins, other players come into account. Disordered lipid binding motifs still need to be identified and understood, especially short linear motifs. In general, she combined experimental and computational techniques, including NMR, X-ray scattering, mass spectrometry, simulation and molecular modeling, solving the prolactin receptor monomeric structure. As the transmembrane domain is a weak dimer with highly specific interactions, its study is more challenging. She could identify two conformations, providing more insights into its dynamical function, e.g. that specificity can be modified by one single methyl group. Finally, she describes the multiple transmembrane domain states which are influenced by lipid composition, kinase binding and dimerization.

After the coffee break, the second session started on “Integrative and Hybrid Methods” with Andrej Sali, Alexandre Bonvin and Ji-Joon Song started.

Andrej Sali introduced us his research on integrative structure determination, where he uses experiments, physical theory and statistical inference to maximize accuracy, resolutions, completeness and efficiency. He presented 4 general steps of his integrative modeling platform (IMP), first naming gathering of information by experimental data, statistical inference and physical principles, second designing system representation and scoring, third followed by sampling and finally analysis and validation. Following this workflow for the Spindle Pole Body, he obtained a validated structure including insights into functional implications of the model.

Next, Alexandre Bonvin presented his integrative modeling platform HADDock (high ambiguity driven docking). It incorporates ambiguous and low-resolution data to aid the docking of up to 6 molecules and has a powerful algorithm to handle flexibility at the interface including refinement in explicit solvent. Using RNA-polymerase II, he introduced DisVis for explorative modeling and consistency quantification of information content of distance restraints solely based on geometric considerations. It can provide information about possible interfaces and calculable information to guide modeling but does not account for conformational changes and energetics.

Ji-Joon Song focused with his talk on the human importin4_histone H3/H4 Asfla complex, which is a perfect example for a successful integrative structural approach, as he was able to solve the whole complex by combining several techniques, such as x-ray crystallography, SAXS, EM, Mass spectrometry, biological application and modeling. He also stated that C-importin4 directly interacts with histon H3 peptide via a highly acid patch. The validation of the complex via single particle electron microscopy, small angle x-ray scattering and cross-linking mass spectrometry confirmed conformational flexibility.

Closing his talk, many discussions were extended during a manifold but time-wise rather tight lunch outside at the venue. Enjoying the sun we were waiting for the next session, curious what surprises there might be… You want to know what happened? Check out my next post on the third session about “Interpreting Experiments through Molecular Simulations”!

Cheers,

Johanna

Berlin calling – let’s get thrilled!

Hey folks! I am Johanna Tiemann and as you can read I got the honor to post some comments and personal perspectives of this Biophysical Society thematic meeting, ‘Conformational Ensembles from Experimental Data and Computer Simulations’ that will be held in Berlin, Germany from the 25th to the 29th of August 2017.

Originally from Munich, Germany, I studied Bioinformatics in Berlin and joined Peter Hildebrand’s lab at Charité Medical University already during my Bachelor and Master thesis. The cluster of knowledge within the lab and institute (e.g. Klaus Peter Hofmann and others) stimulated my fascination about G-protein coupled receptors (GPCR), modeling, simulating and coding. So here I am in my second year PhD that I started in Berlin and now continue in Leipzig. First time I saw the announcement for this meeting I got excited as the Harnack house is located at the green and cozy campus of my Alma Mater, the Free University of Berlin. Although it is rather off the beaten track, it is great to get out of the hectic city center of Berlin and focus on science. As a venue for a conference it is pleasant as you will not be that much distracted by the busy city life but rather can get in touch intensively with each other. If you still have energy to spare after a day of very interesting talks and posters, the night life of Berlin is definitely worth to visit, especially during the weekend (plus there is public transport during the whole night). If you need some advice what to see in Berlin – I am happy to share my experience ;-).

During my time as student assistant and PhD student, I already attended several interesting conferences and workshops but this will be my first BPS meeting and the first time as a blogger at such an event. Of course, I got excited when I noticed the organizing committee, as I already had the pleasure of meeting Andrea Cavalli and Kresten Lindorff-Larsen at a CECAM meeting in Lausanne 2016, leaving no doubt that this conference will be full of highlights with a great mixture of computational and experimental topics.

So what else is there to say about me? I am an enthusiastic, always smiling structural biologist. Within the lab of Peter Hildebrand and with great collaborations, I develop tools for protein modeling (especially loop prediction) and the placement of internal water molecules. Oh, and I like moving pictures, so I put GPCRs and their binding partners in a box and simulate them. To enhance understanding of complex dynamic processes and promote scientific transparency, I want to make analysis and especially visualization of those dynamics with our tools more accessible to others. When I am not driving water molecules in my simulations crazy, I try to see the world while hopping from one conference to another or I can be found abroad with friends, cycling, climbing or trying some new sports. I also like taking pictures, so be warned – you might end up in one with me 😉

Coming to the end, I am looking forward to attending this very interesting BPS meeting and see you all later!

Cheers, Johanna Tiemann

The Science Behind the Image Contest Winners: Light Trails of Receptor Tyrosine Kinase EphA2

The BPS Art of Science Image Contest took place again this year, during the 60th Annual Meeting in Los Angeles. The image that won second place was submitted by Thomas Newport, a PhD student at the University of Oxford. His image shows simulated dynamics of the ectodomain of the receptor tyrosine kinase EphA2 (shown as glowing lines) as well as different conformations that highlight the possible movement of this receptor relative to the membrane surface. Newport writes here about the image and the science it represents, as well as his experience as a first-time BPS Annual Meeting attendee.

Newport Thomas-57-84474

The Biophysical Society’s 2016 meeting was unlike any other conference I have attended in my admittedly fairly short academic career. Thousands of posters, talks, and exhibits clamour for attention over a packed six day schedule. Distinguished professors haggle over conclusions with terrified grad students, while suited salespeople draw in new customers with the promise of free bags, USB drives, and flat-pack microscopes.

Probably the highlight of this, my first Biophysical Society meeting, was winning second prize in the Art of Science image competition, or as my supervisor helpfully emailed me a few minutes later, “congratulations, you’ve won second prize in a beauty contest, collect $10.” The competition: ten gorgeous images, some computer generated, some from microscopes, telling a selection of stories from across the vast field of biophysics.

My image was developed in collaboration with Matthieu Chavent, and shows simulated motions of a key signalling protein as it interacts with the cell membrane. His paper explains more of the scientific background and is well worth a read. Matthieu had already visualised the protein using VMD, a popular open source visualisation tool for molecular dynamics simulations, and traced the paths followed by several atoms as the protein moved between two states. By this point my enthusiasm for digital art and 3D visualisation was fairly well known so we met up to discuss turning this data into art.

Blender3D has been my tool of choice for 3D digital art since I was in high school. It’s open source, has great developer and user communities, and can be used for anything from game development to movie production. Working on the data in Blender gave me complete control of the scene, letting me get the composition, lighting and materials just right. I drew inspiration from long-exposure “light painting” photography, where movement can be captured using trails of light. The light trails were probably the most difficult part to get right – transparent and glowing enough to look ethereal but clear enough to be easy to follow.

It seems to have been a hit – the image has been used as the cover of Structure journal and even appeared on the 2015 Biochemistry Department Christmas card (Matthieu even photoshopped some festive hats onto the proteins, although sadly they didn’t make it to the final card). I’ve run a couple of courses teaching Blender to structural biologists, and it definitely seems like a few people were put off by easily fixable data compatibility issues. Once I’ve got a moment free I’d really like to improve the way Blender handles structural biology data to take some of the data wrangling out of developing scientific art. If you’d like to help, you can find me on GitHub or Twitter (@tnewport).

Concepts in biophysics and structural biology are often most effectively communicated through images, from the iconic double helix of DNA to Jane Richardson’s now ubiquitous protein ribbon diagrams. New technologies are creating new ways for us to tell scientific stories in visual media, as still images, videos, and interactivities. This year’s BPS Art of Science competition showcased some amazing works of art that have come out of these advances, and I’m looking forward to an even tougher competition next year.

Biophysics on World AIDS Day

December 1 is World AIDS Day. More than 35 million people worldwide are living with HIV, with 66% of new infections occurring in sub-Saharan African countries. Many biophysicists study HIV/AIDS, and we recently spoke with BPS member Bo Chen, University of Central Florida, and biophysicist Lesley Earl, a member of the Laboratory of Cell Biology, Biophysics Section, NIH/NCI/CCR, led by BPS member Sriram Subramaniam, about the research their labs have conducted relating to the HIV virus, and how biophysics research contributes to our understanding of HIV and potential therapies. 


 

Bo Chen, University of Central Florida

What is the connection between your research and HIV/AIDS?

My research is to understand the structure and formation of a protein shell called “capsid” that encloses the viral genome materials. Our results can potentially shed light to design antiviral drugs against HIV/AIDS. Even better, once we completely understand the formation of this protein shell, we can use it as a template to design customized nanoparticles with designated assembly morphology, stability, and affinity for biotechnologies in applications such as biosensors, drug delivery vehicles.

Why is your research important to those concerned about HIV/AIDS?

The integrity of the capsid is critical to the infectivity and function of the HIV particle.  Specifically, the capsid is formed by the self-assembly of hundreds of copies of the capsid proteins. When a virus infects a host cell, the capsid needs to disassociate at the right time and location to release the viral genome materials to initiate the replication process by hijacking the host cell machineries. Molecular biology and biochemistry experiments have shown that the capsid is more than just a protective armor. It is actively involved in multiple steps of the viral life cycles, including the reverse transcription of the viral RNA to DNA and subsequent importation of the DNA into the host nucleus. Afterwards, as new viral materials are produced by the host cells, hundreds of copies of these capsid proteins need to come together and assemble into the capsid in the same morphology, encapsulating the right amount of the viral genome materials. The infectivity of the HIV particle will be lost or attenuated if the capsid deviates from the normal morphology or stability. Therefore, the HIV capsid is considered as a promising antiviral drug targets, and elucidation of the capsid structure and assembly mechanism will help find new therapeutic strategies against the AIDS. Yet the structure and formation mechanism of the capsid is still not well-understood.

How did you get into this area of research and how long have you been working on it?

I started to characterize the HIV capsid assembly structure by solid state NMR when I was a postdoc in Dr. Robert Tycko’s group at NIDDK, NIH. We were looking for some large proteins for solid state NMR, to push the capability of the structural biology technique.  Two years into the project, I had a wild idea to simulate the self-assembly process in addition to my experimental efforts. It was inspired by the pioneering work of Dr. Gregory Voth, Dr. Michael Hagan, and Dr. David Chandler. With my passion for Mathematics and geometry, I came up with a novel model to simulate the self-assembly of the HIV capsid. Since then, it has been about eight years.

Do you receive public funding for this work? If so, from what agency?

I was awarded the 2013 Young Investigator Award by the Air Force Office of Scientific Research. I was so obliged to their support, without which our research could not be where it is today.

Have you had any surprise findings thus far?

We have two very interesting findings. Firstly, we enhanced our simulation model such that it not only captures the shape of individual capsid protein, but also provides sufficient simulation speed to simulate the self-assembly of the capsid proteins in 3D. In that sense, it is very unique. It enables us to accurately incorporate the experimental high resolution structure and dynamics information of the capsid protein into the simulation. We demonstrated that the capsid protein prefers to assemble into hexameric lattice with only mild curvatures in the majority of its time in the solution, where it samples an ensemble of different conformations, as assessed by solution NMR work from Dr. Marius Clore’s group. The conical tips with sharp curvatures are assembled by the capsid proteins in a highly selective conformation state, along a distinct assembly pathway.  Due to the ability to capture the protein structure at high resolution, we further probed the importance of the variations at the dimerization interface of the capsid protein. We showed that subtle variations of the crossing angle between helix 9 from the monomers in the dimer will not affect the curvature or morphology of the assembly,but dramatically change the assembly pathway. We are really excited with our results, published in BBA General Subjects, 1850,p2353-2367, and we are anxious to see if they can be confirmed by experiments and alternative theoretical work, as you know, a theory is only a theory until it is proven correct.

BoChenImage

Simulations with our novel coarse-grained model show that the tip of the conical HIV capsid can be formed by the capsid proteins in pentameric like conformation. The final assembled structure, exhibits co-existence of quasi-equivalent pentamers and hexamers as shown above. Figure from Xin, Q. et al. (2015) BBA-Gen Subjects 1850,2353-2367.

What is particularly interesting about the work from the perspective of other researchers?

To our knowledge, our work was the first 3D simulation of the HIV capsid assembly by a high resolution coarse-grained model that can capture accurately the protein structure. Our results therefore, can be directly compared with experimental observation. If it is correct, its knowledge will provide rational guidance to anti-HIV drug design targeting the capsid protein. As I mentioned above, our work brings us one step closer to constructing nano-architecture derived from the HIV capsid protein for a wide-range of applications in bio-nanotechnology. On an even broader scale, our model can be generalized to simulate other large biomolecular assembly, which is a very challenging topic. Such systems involve a large number of molecules, and the assembly process extends a time range that is too slow for simulation with all atom molecular dynamics but too fast for direct experimental analysis. So the protein molecular structure has to be coarse-grained in simulations to enable the simulation of such large systems. As you are probably aware, the function of the protein directly ties with its structure. So the unique strength of our model as we demonstrated on HIV capsid assembly is that we can retain the structural information quite well, without a prohibiting computational cost. We hope our work will help and inspire others with similar interests, just like my model was partly inspired by the pioneers in this field.

What is particularly interesting about the work from the perspective of the public?

Well, they can see their tax money is well-spent :). Specific to the public concerns about health related issues, our results, if verified by other scientists, can be highly valuable for rational design of anti-HIV drugs and help fight AIDS. In fact, the capsid shell is a ubiquitous component for all viruses, whose formation mechanism is still debated. There is an abundance of viruses as pathogens that cause many other terrible diseases, including Hepatitis virus, Dengue virus, to name a few, and the list can go on and on. More broadly, many of the enzymes and proteins perform their duty not alone, but in cohort assemblies. Our model may be applied to investigate such systems of interest.


 

Lesley Earl, Laboratory of Cell Biology, Biophysics Section, NIH/NCI/CCR

What is the connection between your research and HIV/AIDS?

Our laboratory is generally focused on studying the 3D structure of cells, viruses, and proteins by electron microscopy. A primary focus for the lab over the last decade or so has been uncovering how HIV invades target cells. We have studied a number of aspects of this process, including structural analyses of the HIV envelope glycoprotein (the viral surface protein that binds to the target cell and allows the virus to enter), the formation of the HIV core (the protein structure that forms around and protects the virus’ genetic material), and the cellular structures of the HIV virological synapse (which allows much more efficient viral transfer between cells).

Why is your research important to those concerned about HIV/AIDS?

One of the most frustrating things about the many years of research into HIV has been our inability to design a vaccine that is effective against a broad range of HIV strains. In our structural studies we have been looking at what makes certain types of antibodies effective in neutralizing the virus and preventing infection. While we haven’t found a silver bullet yet for designing vaccine immunogens, we have uncovered some really interesting findings, such as the way in which certain neutralizing antibodies from patients can actually stop the envelope glycoprotein from opening up and binding to target cells. This type of information is critical for understanding how HIV functions, and for designing new therapies.

How did you get into this area of research and how long have you been working on it?

For the last 15 or so years, our group has been working on developing the ability to determine high resolution structures of proteins by cryo-electron microscopy. In 2006 or so, we were hunting around for something really interesting and important to work on, something where structure could really make a difference, and which we could also use as a platform to continue developing our technologies. We decided that working on the HIV envelope glycoprotein was just the thing – it’s a highly dynamic protein present in high concentration on the surface of the virus, and at the time, there were no structures at all of the native envelope glycoprotein. We published the first structure of the native unliganded envelope glycoprotein trimer in 2008, and have followed that with in-depth studies of the structural mechanism of HIV binding and entry into target cells.

Do you receive public funding for this work? If so, from what agency?

As a lab within the NIH Intramural Program, we primarily receive funding from the National Cancer Institute, as well as from the Intramural AIDS Targeted Antiviral Program.

Have you had any surprise findings thus far?

LesleyEarlImage

An uninfected T cell (blue) reaching around and attaching to an HIV-infected T cell (green), which has HIV virions (glowing yellow) on the surface. Image credit: Donald Bliss, National Library of Medicine.*

 

Truthfully, HIV continues to surprise us, no matter how long we work on it. Early on, we saw for the first time what seemed to be a twisting opening motion of the HIV envelope glycoprotein; in 2014, we found that even though the top portion of the trimer twists open, the base seems to stay still, suggesting that the interface between these two sections of the trimer must adjust as the trimer engages with receptors on the surface of the cell. Another surprising finding came from our study of the HIV core – we were looking at images of HIV virions from an infected culture, and realized that the generally accepted model for core formation, which suggests that the protein core subunits nucleate and build up from one end inside a virus particle, didn’t align with what we were seeing. Instead, we could see sheets of core material rolling off the membrane into the classic cone-like core shape. And finally, in our cellular studies, we were extremely surprised to see that uninfected cells seem to send out finger-like membrane extensions towards infected cells, apparently facilitating their own infection.

What is particularly interesting about the work from the perspective of other researchers?

I think the most interesting piece of our work in terms of what is of use to other researchers has been our cryo-electron tomographic studies of antibody-mediated neutralization of the envelope glycoprotein. By understanding what allows an antibody to prevent the virus from infecting cells, researchers working on designing an HIV vaccine can design novel protein variants what will elicit the right types of antibodies from patients, which is a key step in rational vaccine design.

What is particularly interesting about the work from the perspective of the public?

One of the great things about 3D electron microscopy techniques is that they give us the ability to actually see what’s going on at the cellular or even atomic level. The types of images being produced from our lab – whether they’re 3D models of proteins from single particle analysis, or models of viruses from cryo-electron tomography, or models of cells from focused ion beam scanning electron microscopy – really bring the biology of HIV to light.

 

*The data from which this image was derived is from the following publication: Do T, Murphy G, Earl LA, Del Prete GQ, Grandinetti G, Li G, Estes J, Rao P, Trubey C, Thomas J, Spector J, Bliss D, Nath A, Lifson JD, and Subramaniam S. 3D imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J Virol. 2014 Sep;88(18):10327-39. doi: 10.1128/JVI00788-14.

Biophysical Society Summer Research Program: A Novel Internship for the Science-Addicted

My name is Manuel Castro, I am a rising senior at Arizona State University, and my major is Biochemistry with a focus in medicinal chemistry. From a relatively young age, I knew that my love of science was considerably broad. I enjoy the fields of chemistry, biology, and physics; through undergraduate lab reseCastro,Manuel headshotarch opportunities, I became more familiar with the interdisciplinary concept of biophysics, and subsequently, the breadth and depth associated with this area of study. When my lab mentor told me about the Biophysical Society Summer Research Program, I enthusiastically applied.

At Arizona State, I work in an NMR lab that focuses on characterizing the structure and function of membrane proteins. Under Dr. Wade Van Horn, my work in his lab has helped direct me towards achieving a career within the large realm of biophysics; namely, structural biology. Upon receiving my acceptance letter to the BPS Summer Program, I began looking into various professors at UNC Chapel Hill that complemented my interests. I quickly found Dr. Matt Redinbo, a professor whose lab also focuses in the structural and chemical biology of proteins involved in human disease, but with X-ray diffraction instead.

Coming from an NMR lab, I entered Dr. Redinbo’s crystallography lab with the intention of exploring the structural biology spectrum more broadly. I really wanted to learn X-ray crystallography first hand to help me decide this coming year where to focus my applications for graduate school programs. I expected my work in Dr. Redinbo’s lab would be very general, including making buffers, cleaning dishes, etc. To my pleasant surprise, the same day I met Dr. Redinbo in person, he already had me setting up crystal trays. Within a few more weeks into the BPS course, I was shadowing graduate students using the x-ray source, which I consider my favorite part of the summer course thus far. In addition to the research, I have learned a lot about scientific communication. We give presentations which help train us for graduate-level coursework by having us present on what their research is about and the direction it is headed.

The program also offers classes that introduce important topics of physical chemistry, biochemistry, molecular biology and biophysics. For those who have taken those classes, the course serves as a wonderful review; for those that have not, it is a fantastic introduction to central themes of biophysical studies. These are formal courses with important feedback such graded assignments and quizzes; however, the courses are not for credit. This promotes a comfortable learning environment for students of all levels of education and disciplines.

Overall, I think that this summer has been one of the best of my life so far. The BPS Summer Program allowed me to travel across the country and make new friends from various fields and interests. I would strongly suggest this internship to anyone who is passionate about science, and I have no regrets when I reflect upon my stay at UNC Chapel Hill.

Bringing Together Biophysicists in the Hoosier State

The Biophysical Society recently sponsored a networking event for biophysicists in Indiana. The event, titled “The Hitchhiker’s Guide to the Protein Galaxy: A Mini-symposium on Integrating Structure, Function, and Interactions of the Protein Universe,” was held at Purdue University on May 13-14, 2015, and was organized by Satchal K. Erramilli, Duy P. Hua, Adriano Mendes, Phillip Rushton, Brendan Sullivan, Sakshi Tomar, all of Purdue University. Satchal Erramilli reports on the event – and explains its interesting title.

poster

Attendees mingle during the poster session.

You many wonder why we chose the “Hitchhiker’s Guide to the Galaxy” as the theme for a symposium on protein science and biophysical research. The classic novel by Douglas Adams, which we often wax nostalgic about, is an excellent work of fiction, but is also filled with concepts that can readily be applied to scientific inquiry. The book contains a long tangent on the validity of mice models, includes an excellent digression on “Somebody Else’s Problems”, and is peppered with discussions on evolution. Most significantly, the book’s most famous story – the meaning of life – is an allegory for asking the right questions, as important an exercise as any for a scientist.

And so we drew on themes from the book for our symposium, “The Hitchhiker’s Guide to the Protein Galaxy”. We were charmed to find many attendees and sponsors shared our enthusiasm for the novel, and some of the credit for the event’s success has to be ascribed to the theme. Indeed, we found many attendees were as excited to discuss the book as they were to discuss science.

The symposium was held at Purdue University on May 13th and 14th, 2015, with the goal of bringing together protein scientists and biophysical researchers from all across campus. Thanks to the generosity of our sponsors, we were able to expand the scope of the event to include attendees and presenters from nearby institutions and, in some cases, beyond. We had over 130 attendees, with nearly two dozen coming from nearby institutions in Indiana and Illinois, and some even as far off as Cornell, the N.I.H., and Texas Tech. This can only be described as apropos for a Hitchhiker-themed symposium.

We felt the event should reflect the depth and breadth of structural biology research here at Purdue, and thus it included aspects of protein science ranging from basic to applied, from individual proteins to whole cell studies. The presenters had backgrounds as diverse as their topics, and included several young faculty members, postdoctoral scholars, and senior graduate students. Topics ranged from protein structure and function to biophysical methods and high-resolution electron microscopy, and much more. Attendees clearly enjoyed being able to hitchhike around the Protein Galaxy during the two days of the symposium.

organizers

The Hitchhiker’s Guide to the Protein Galaxy organizers pose for a photo.

The presence and participation of our external attendees enriched the experience for all at this event, and their interactions with local researchers offered the potential for fruitful collaborations. In particular, we recognize our keynote speaker, Dr. Tony Kossiakoff, who was an excellent guest of honor for the event and drew an audience exceeding the venue’s capacity. As an unexpected addition, and entirely a product of the enthusiasm of several of our visitors, we were able to organize an impromptu career workshop, which our graduate students found tremendously useful. We hope to have even more external presenters next time.

We definitely plan to have this event again next year. We can only hope to again receive such tremendous support from our sponsors, who far exceeded our expectations with their willingness to sponsor the symposium, the awards, and contribute in many other ways. We were thrilled to get this networking grant from the Biophysical Society, which, besides providing us with money, also provided visibility for the event beyond what we could have hoped for. A big shout out goes to April Murphy, who made time not just to assist us and help market the symposium but also to visit us and take part in our event. It was a pleasure to work with her and everyone else who helped make this a success, and we hope to see her and many others at the next iteration of this symposium. See you again next year, fellow hitchhikers!