Session P40: Protein Fluctuations and Conformation Changes

Atomistic simulations of the MS2 coat protein conformational transition

During the replication of many viruses, hundreds to thousands of proteins self-assemble to form a protective protein coat, called a capsid, around the viral nucleic acid. Often these proteins have identical amino acid sequences with slightly different, or quasi-equivalent, conformations, which join in precise spatial arrangements. Although the structure of completed capsids is known to atomic resolution, little is known about the assembly intermediates and how protein conformations are selected during assembly. In this talk, we will use all-atom simulations to investigate how protein-RNA interactions guide conformational transitions of capsid proteins from the single-stranded RNA bacteriophage MS2. Since conformational changes occur on timescales which are not accessible to all-atom simulations, we use enhanced sampling methods to sample probable transition pathways and corresponding free energy profiles. Specifically, we will present free energy profiles associated with the MS2 capsid protein conformation in the presence and absence of RNA.   

Conformational changes of surface immobilized proteins studied by combined Atomic Force Microscopy and Fluorescence Spectroscopy

Atomic Force Microscopy (AFM) and Fluorescence Spectroscopy techniques have provided unique methods for characterizing conformational changes in proteins. Here we are using a technique called nanografting to immobilize proteins at well defined locations on atomically flat surfaces. In nanografting the AFM tip is used to shave alkanethiol molecules from a prescribed patch on a surface coated with an alkanethiol monolayer. Thiol-linked proteins in the surrounding solution are then able to self assemble on the newly exposed surface patch in a highly ordered array of the order of 100nm. Stable and meta-stable conformations of fluorescently tagged proteins and other molecules assembled in this manner can then be characterized using a combination of AFM and Fluorescent Resonance Energy Transfer (FRET). Due to the high spatial, temporal and force resolution provided by both AFM and FRET, a free energy landscape of the protein may be determined using this technique.   

The Glassy State of Crambin and the THz Time Scale Fluctuations Related to Protein Function

THz experiments have been used to characterize the picosecond time scale fluctuations taking place in the model protein crambin. Using both hydration and temperature as an experimental parameter, we have successfully identified the collective fluctuations ($\le $ 200 cm$^{-1})$ in the protein (Figure 1). Observation of the transition of the protein dynamics in the THz spectrum from both below and above the glass transition temperature (T$_{g})$ provides unique insight into microscopic interactions and modes that allow the solvent to couple with the protein dynamics (Figure 2). Our findings suggest that the solvent dynamics on the picosecond time scale not only contribute to the flexibility of the protein but also provides a dynamical parameter that allows the protein to modulate local regions of its structure that are distinct from the protein whole. These distinct dynamical regions of the protein may be important for energy transport and hence, linked with protein function.   

Stabilization of peptide helices by length and vibrational free energies: \emph{Ab initio} case study of polyalanine

Helices are one of the most abundant secondary structure ``building blocks" of polypeptides and proteins. Here, we explore helix stabilization as a function of peptide length and temperature [harmonic approximation to the vibrational free energy (FE)], for the alanine-based peptide, Ac-Ala$_n$-LysH$^+$ $n$=4-15, in the gas phase. For $n$=4-8, we predict the lowest energy structures in density-functional theory, using the van der Waals (vdW) corrected[1] PBE exchange-correlation potential. $\alpha$-helices become the lowest energy structures at $n\approx$7-8 on the potential energy surface, but only barely and if including vdW interactions. At finite temperatures, the helices are further stabilized over compact conformers. While the vibrational entropy is the leading stabilizing term at 300 K, also the zero-point-energies favor the helical structures. For $n\geq$8, the $\alpha$-helix should be the only accessible conformer in the FE surface at 300 K, in agreement with experiment[2] and with our own comparison[3] of calculated \emph{ab initio} anharmonic IR spectra to experimental IR multiple photon dissociation data for $n$=5, 10, and 15. [1] Tkatchenko and Scheffler, PRL 102, 073055 (2009); [2] Kohtani and Jarrold, JACS 108, 8454 (2004); [3] Rossi et al., JPCL 1, 3465 (2010).   

A Physics-based Approach of Coarse-graining the cytoplasm of E. coli

We have investigated protein stability in an environment of E. coli cytoplasm using coarse-grained computer simulations. To coarse-grain a small slide of E. coli's cytoplasm consisting of over 16 million atoms, we developed a self-assembled clustering algorithm (CGCYTO). CGCYTO uses a tunable resolution parameter ($\lambda $, ranging from 0 to 1) to justify the resolution of a cytoplasm, depending on the size of a test protein for the computation of covolumes and the volume of a macromolecule in the cytoplasm. We compared the results from a polydisperse cytoplasm model (PD model) from CGCTYO with two other coarse-grained hard-sphere cytoplasm models: (1) F70 model, macromolecules in the cytoplasm were modeled by homogeneous hard spheres with a radius of 55{\AA} and (2) HS model, each macromolecule in the cytoplasm is modeled by hard spheres of various sizes. It was found that the folding temperature Tf of a test protein (apoazurin) is $\sim $5 degrees higher in a PD model than that in a F70 model. In addition, there is a deviation of 1.7 degrees on Tf when an apoazurin is randomly placed at different voids formed by particle fluctuations in a PD model, 0.7 degrees higher than that in a HS model.   

Investigation of flexibility in Myosin V using a new 3D mechanical model

This paper presents the development of a three dimensional rigid multibody model for the simulation and analysis of motor protein locomotion. The interesting aspect of this model is that it retains the mass properties, in contrast to the commonly used models which omit mass properties at the nano scale. The disproportionate size of the small mass of Myosin V relative to the large viscous friction forces requires a small integration step size that leads to a long simulation run time; however, the proposed model can be numerically integrated in a reasonable amount of time. This paper discusses modeling flexibility in the protein as an extension of the original rigid body model. Empirical studies have shown that Myosin V's neck domain can be considered as three pairs of tandem elements called IQ motifs which can bending at junctures between them. Therefore, each neck is modeled by three rigid bodies connected by ball-and-socket joints together, rather than single rigid body has been used in the previous works. Euler parameters are used to model the orientation of bodies in order to eliminate singularities in the description of orientation. In order to accomplish this, the equations of motion are reduced to minimal form using changing holonomic and non-holonomic constraints applied to the model which represent the normalization of the Euler parameters as well as contact and impact non-penetration conditions. The differences between the dynamic behavior of the new mechanical model, with flexible neck domains, and the original rigid body model are compared using simulation results.   

Barrel fluctuation and oxygen diffusion pathways in the monomeric fluorescent proteins

Fluorescent proteins are valuable tools as biochemical markers for studying cellular processes. Improving the photostability of the FPs is highly desirable in biochemical, biomedical and cell biology. Oxygen is necessary for the proper maturation of the chromophore in fluorescent proteins (FPs), but photobleaching of FPs is also oxygen sensitive. The photobleaching of the monomeric variant of RFPs has been attributed to the lack of proper shielding against oxygen or other small molecules, ions or halides. We use molecular dynamics simulation to investigate the protein barrel fluctuations in mCherry, one of the most useful monomeric mFruit variant of RFPs. We also employ oxygen diffusion simulations to search for possible pathways of oxygen to the chromophore. The ultimate goal is to use the results of these calculations to propose amino acid substitutions that will block the oxygen pathways and prevent photobleaching in the engineered proteins.   

Protein Dynamics Studied by Quasi-elastic Neutron Scattering

The biological function and activities of proteins are intimately related to their structures and dynamics. Nowadays, neutron scattering is one of the most powerful tools to study the protein dynamics. In this study, we use quasielastic neutron scattering (QENS) at the Spallation Neutron Source, ORNL, to study relaxational dynamics of two structurally different proteins --- hen egg white lysozyme and an inorganic pyrophosphatase from a hyperthermophile, in the time range of 10ps to 1ns. We experimentally prove that the slow dynamics of globular proteins can be described by the mode-coupling theory (MCT) that was originally developed for glass-forming molecular liquids. The MCT predicts the appearance of a logarithmic decay for a glass-forming liquid. Such dynamic behavior is also observed by recent molecular dynamics (MD) simulations on protein molecules. In addition, we compare the temperature dependence of the dynamics of the two proteins with completely different activity profiles. Our results greatly help understanding the relation between protein dynamics and their biological functions.   

Intrinsic Mean Square Displacement in Proteins

The dynamics of biological molecules is investigated in neutron scattering experiments, in molecular dynamics simulations, and using analytical theory. Specifically, the mean square displacement (MSD), $\langle r^2\rangle _{exp}$, of hydrogen in proteins is determined from measurements of the incoherent elastic neutron scattering intensity (ENSI). The MSD, $\langle r^2\rangle _{exp}$, is usually obtained from the dependence of the ENSI on the scattering wave vector $Q$. The MSD increases with increasing temperature reaching large values at room temperature. Large MSD is often associated with and used as an indicator of protein function. The observed MSD, however, depends on the energy resolution of the neutron spectrometer employed. We present a method, a first attempt, to extract the intrinsic MSD of hydrogen in protein from measurements, one that is independent of the instrument resolution. The method consists of a model of the ENSI that contains (1) the intrinsic MSD, (2) the instrument resolution width and (3) a parameter describing the motional processes that contribute to the MSD. Several examples of intrinsic MSDs $\langle r^2\rangle$ in proteins obtained from fitting to data in the existing literature will be presented.   

Scaling Behavior in Twisted, Helical and Undulating Lysozyme Amyloid Fibrils

We combine atomic force microscopy single-molecule statistical analysis with polymer physics concepts to study the molecular conformations of lysozyme amyloid fibrils. We use different denaturation conditions to yield amyloid fibrils of different types. At 90\r{ }C and pH2, highly laminated twisted and helical ribbons are found, in which as many as 17 protofilaments pack laterally for a total width approaching 180 nm. In the case of 60\r{ }C and pH2, we find thin, wavy fibrils, in which the scaling behavior varies at multiple length scales. We use bond and pair correlation functions, end-to-end distribution and worm-like chain model to identify 3 characteristic length scales. At short length scales there is a first bending transition of the fibrils, corresponding to a bending length Lb. At slightly larger length scales ($>$2Lb), the fibrils become pseudoperiodic and start to undulate. Finally, at length scales larger than the persistence length Lp, the fibrils become flexible and are well described by a 2D self-avoiding random walk. We interpret these results in terms of the periodic fluctuations of the cross-section orientation of the fibrils (twisting) and the impact these have on the area moment of inertia and the corresponding propensity of the fibrils to bend.   

Probing Volume Changes and the Intracellular Water in Single Erythrocytes

In the living cell, water is one of the most abundant substances, and cells have developed very efficient machinery for transporting water in and out. Erythrocytes can undergo large, but reversible, volume changes under hydrostatic pressure and a possible mechanism may involve transport of water. We employ confocal micro-Raman spectroscopy over the frequency range from 150 to 4000 cm$^{-1}$ to probe both the intracellular hemoglobin and water in individual red blood cells under physiological conditions. We investigate changes in the OH stretch bands near 3400 cm$^{-1}$ due to the cellular water. Results of experiments that employ variations in external parameters such as pressure will be presented.   

Effects of pressure on the protein barrel and the chromophore interactions in mCherry

Fluorescent proteins (FP) can be attached to proteins of interest, which makes it possible to study the movement, localization and many other physiological processes of the tagged proteins. A number of fluorescent proteins have been genetically engineered to enhance their intensity, photo-stability, pH-stability etc. The structural fluctuations of FPs determine the ease of access of small molecules like oxygen and may be an important consideration for their fluorescence spectrum and stability. A protein's response to pressure perturbations provides useful insights for understanding their folding and dynamics. Experiments have shown that application of pressure affects both the fluorescence peak as well as the quantum yield of the protein. We report on molecular dynamics computational investigations of the effect of pressure on the fluctuations of the beta barrel and the structure of the chromophore of a well characterized Red Fluorescent Protein, mCherry. We discuss our results on how pressure affects the ability of water and other ions to penetrate the barrel to reach the chromophore, as well as the effect on the time dependent hydrogen bonding network in the chromophore's cavity region.   

Thermal response of proteins (histone H2AX, H3.1) by a coarse-grained Monte Carlo simulation with a knowledge-based phenomenological potential

Using a coarse-grained bond fluctuating model, we investigate structure and dynamics of two histones, H2AX (143 residues) and H3.1 (136 residues) as a function of temperature ($T)$. A knowledged based contact matrix is used as an input for a phenomenological residue-residue interaction in a generalized Lennard-Jones potential. Metropolis algorithm is used to execute stochastic movement of each residue. A number of local and global physical quantities are analyzed. Despite unique energy and mobility profiles of its residues in a specific sequence, the histone H3.1 appears to undergo a structural transformation from a random coil to a globular conformation on reducing the temperature. The radius of gyration of the histone H2AX, in contrast, exhibits a non-monotonic dependence on temperature with a maximum at a characteristic temperature ($T_{c})$ where crossover occurs from a positive (stretching below $T_{c})$ to negative (contraction above$ T_{c})$ thermal response on increasing $T$. Multi-scale structures of the proteins are examined by a detailed analysis of their structure functions.