Venkatramanan Krishnamani

Optimization of an Elastic Network Augmented Coarse Grained Model to Study CCMV Capsid Deformation

The major protective coat of most viruses is a highly symmetric protein capsid that forms spontaneously from many copies of identical proteins. Structural and mechanical properties of such capsids, as well as their self-assembly process, have been studied experimentally and theoretically, including modeling efforts by computer simulations on various scales. Atomistic models include specific details of local protein binding but are limited in system size and accessible time, while coarse grained (CG) models do get access to longer time and length scales but often lack the specific local interactions. Multi-scale models aim at bridging this gap by systematically connecting different levels of resolution. Here, a CG model for CCMV (Cowpea Chlorotic Mottle Virus), a virus with an icosahedral shell of 180 identical protein monomers, is developed, where parameters are derived from atomistic simulations of capsid protein dimers in aqueous solution. In particular, a new method is introduced to combine the MARTINI CG model with a supportive elastic network based on structural fluctuations of individual monomers. In the parametrization process, both network connectivity and strength are optimized. This elastic-network optimized CG model, which solely relies on atomistic data of small units (dimers), is able to correctly predict inter-protein conformational flexibility and properties of larger capsid fragments of 20 and more subunits. Furthermore, it is shown that this CG model reproduces experimental (Atomic Force Microscopy) indentation measurements of the entire viral capsid. Thus it is shown that one obvious goal for hierarchical modeling, namely predicting mechanical properties of larger protein complexes from models that are carefully parametrized on elastic properties of smaller units, is achievable.

The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes. DOI: http://dx.doi.org/10.7554/eLife.17438.001

Cellular encoding of Cy dyes for single-molecule imaging

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment. DOI: http://dx.doi.org/10.7554/eLife.19088.001

CMPyMOL: A Tool for Protein Contact-Map Analysis

Contact-maps are reduced 2D representation of the 3D spatial configuration of a protein. Many valuable structural features like secondary structures, inter and intra-protein interactions, interacting domains, etc., can be readily identified from these maps. However, it is not straightforward and intuitive to reckon the spatial organization of the contact regions from reduced representation. The CMPyMOL software attempts to bridge this gap as an interactive graphical tool for protein contact-maps that interfaces with PyMOL for 3D visualization. Importantly, CMPyMOL helps understand the functional importance of contacts by providing visual overlays of various structural and biochemical properties of a protein on top of its contact-map.Contact–maps are reduced 2D representation of the 3D spatial configuration of a protein. Many valuable structural features like secondary structures, inter- and intra-protein interactions,interacting domains, etc., can be readily identified from these maps. However, it is not straightforward and intuitive to reckon the spatial organization of the contact regions from reduced representation. The CMPyMOL extention for molecular visualization software PyMOL attempts to bridge this gap as an interactive graphical tool for protein contact-maps that interfaces with PyMOL for 3D visualization. Specifically, CMPyMOL helps understand the functional importance of contacts by providing visual overlays of various structural and biochemical properties of a protein on top of its contact-map.

DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions

We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiquitin-binding proteins, including interactors that we verify biochemically. To demonstrate the specificity of DEEPN, we show that DEEPN allows simultaneous comparison of candidate interactors across multiple bait proteins, allowing differential interactions to be identified. This feature was used to identify interactors that distinguish between GTP- and GDP-bound conformations of Rab5.

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Measuring Large Membrane Protein Dimerization in Lipid Bilayers by Forster Resonance Energy Transfer

Our understanding of the thermodynamics forces in membrane protein assembly in a lipid bilayer is limited, because there are not many equilibrium models for studying protein association in lipid bilayers. We have developed a new model system based on the homodimeric CLC-ec1 Cl−/H+ antiporter (WT) that offers an opportunity to investigate this. A single tryptophan substitution (I422W) in WT yields a monomer/dimer mixture in detergent. We quantitatively labeled CLC-ec1 with Cy3 donor and/or Cy5 acceptor to measure Forster resonance energy transfer (FRET) changes upon dimerization. By single-molecule TIRF microscopy we measured the molecular FRET efficiency of as 0.7 ± 0.1 (n=3). We then measured the population FRET efficiency of I422W in large membranes while varying the protein/lipid fraction (χ) to determine a Kd of dimerization. Emission spectra obtained from the fluorometer is fit using an in-house software Fytt to decompose contributions from the sensitized emission of donor, acceptor, FRET and background and to quantify FRET efficiency (E). We determined E for the positive dimer control by co-labelling with Cy3/Cy5 across a range of χ. Measuring I422W-Cy3 + I422W-Cy5 in membranes shows low E at low χ that increases and saturates to all-dimer E at high χ, indicating a dimerization reaction in membranes. Furthermore, we observe reversibility of the reaction when going from a high χ to low χ by diluting all-dimer I422W membranes with empty lipid bilayers, and the appearance of FRET upon fusion of separately labelled I422W-Cy3 with I422W-Cy5 membranes, indicating subunit exchange. We compared 2:1 POPE/POPG vs. POPC lipid compositions, and while both show a similar reaction, the POPC data fits well to a dimerization isotherm with Kd(I422W) = 4 x 10−6 protein/lipid, suggesting that we are measuring equilibrium dimerization reaction in membranes.