Session A50: Physics of Proteins I: Experimental and Computational Studies on the Structure and Conformational Dynamics of Proteins

Computational assessment of mutations of protein cores

Protein mutations are known to change both protein stability and function. Many computational methods have been developed to predict these changes, but often with limited success. We investigate how mutations to residues in protein cores produce changes in packing fraction, side chain conformations, and backbone motion. To investigate the extent of mutation-induced backbone motion, we created two datasets of protein crystal structures. One dataset contains high-resolution crystal structures of mutated proteins and the crystal structure of the associated wildtype proteins. The backbone root-mean-square-deviation (RMSD) of these structures is compared to a duplicate dataset that contains pairs of crystal structures of identical proteins. We also investigate the change in packing fraction of mutated residues. Finally, we show that the hard-sphere plus stereochemical constraint model is able to recapitulate the side chain dihedral angle combinations for buried amino acids in protein cores, protein-protein interfaces, and mutated proteins. We find that the side chain dihedral angle prediction accuracy decreases as the residue solvent accessible surface area (SASA) increases. This reveals the dominance of steric interactions in determining side chain conformations for buried residues and a relative increase in the strength of other interactions for solvent exposed residues. These results further our understanding of how protein mutations affect protein structure and will inform future computational methods of predicting protein stability changes due to mutations.   

Forces on Nascent Polypeptides During Membrane Insertion and Translocation via the Sec Translocon

Co-translational insertion into and translocation across the cell membrane via the Sec translocon are key steps in the biogenesis of membrane and secreted proteins. However, study of these processes is challenging due to the role of long-timescale non-equilibrium dynamics during ribosomal translation. We have developed a coarse-grained (CG) simulation approach capable of reaching experimental (i.e. minute) timescales, while retaining sufficient detail to capture the effect of single amino-acid substitutions. The CG model is applied to uncover the mechanism underlying experimentally observed forces acting on hydrophilic, hydrophobic, and charged nascent polypeptides while they are synthesized by the ribosome and pass through the Sec translocon. Calculated forces show strong agreement with experimental force measurements and visualization of the CG trajectories allows us to ascribe observed forces to distinct physical processes acting on the nascent polypeptide. CG simulations with modified interactions confirm these findings and provide experimentally testable hypotheses. This study demonstrates a combined simulation and experimental approach able to provide high-resolution insight into the co-translational integration and translocation of nascent polypeptides.   

Machine learning of protein folding funnels from experimentally measurable observables.

The stable conformations and dynamical motions of proteins are governed by the underlying single-molecule free energy surface. By integrating ideas from dynamical systems theory with nonlinear machine learning, we establish a technique to recover single-molecule free energy surfaces from univariate time series in a single esperimentally-accessible observable. Using Takens’ Delay Embedding Theorem, we expand the univariate time series into a high dimensional space in which the dynamics are equivalent to those of the molecular motions inreal space. We then apply the diffusion map nonlinear manifold learning algorithm to extract a low-dimensional representation of the free energy surface that is diffeomorphic to the one from the all atom system. We validate our approach in molecular dynamics simulations of a C₂₄H₅₀ n-alkane chain and the Trp-cage mini protein to demonstrate that the low-dimensional free energy surfaces extracted from the atomistic simulation trajectory are geometrically and topologically equivalent to that recovered from a knowledge of only the head-to-tail extent of the molecule. Our approach lays the foundations to extract empirical single-molecule free energy surfaces directly from experimental measurements.   

Protein Folding, Binding and Aggregation in the Cell: Role of Stochastic Resonance

Understanding how proteins fold and form multi-protein assemblies inside the cell is an important fundamental problem that has vital implications for development of therapeutic methods. The cellular environment is critically different from the dilute and static conditions under which proteins are usually studied in vitro or using computational approaches. Protein folding and protein-protein binding inside the cell occur in highly crowded and fluctuating environment, which is critical for stability of larger proteins and assemblies. While, the role of crowding has been extensively investigated previously, the role of fluctuations has been largely neglected. Here, we report on our investigation into the role of local fluctuations that can be attributed to fluctuations of the chemical environment around the protein inside the cell. Using coarse-grained molecular simulations we establish that such fluctuations can have a substantial effect when the characteristic frequency of the applied fluctuations coincides with the “native” rates of the reaction, consistent with the phenomenon of stochastic resonance observed in many other condensed-matter processes. Our computational and theoretical findings as well as the recent experimental conformation of our results will be presented.   

Self-organizing domains in conformations of Ebola virus protein VP40

Conformational variations of an ebola virus protein (VP40) as a function of temperature (T) are studied by coarse-grained Monte Carlo (MC) and Molecular Dynamics simulations. A number of local and global physical quatities are examined including contact map, radius of gyration, structure factor etc. We find that the self-organizing residues leads to onset of domains with globular conformations at low temperatures. Domains are localized around N-tail, intermediate region, and C-terminal at low temperature but disappear at high temperature where the protein is denatured into a random coil conformation. Response of the domain dynamics as a function of temperature shows that the N-terminal domain is most stable and perhaps critical in latching mechanism of the ebola virus.   

Dynamical Transition of Collective Motions in Dry Proteins

Water is widely assumed to be essential for protein dynamics and function. In particular, the well-documented “dynamical” transition at ~200 K, at which the protein changes from a rigid, non-functional form to a flexible, functional state, as detected in hydrogenated protein by incoherent neutron scattering, requires hydration. Here, we report on coherent neutron scattering experiments on perdeuterated proteins and reveal that a transition occurs in dry proteins at the same temperature resulting primarily from the collective heavy-atom motions. The dynamical transition discovered is intrinsic to the energy landscape of dry proteins.

Physical Review Letters 119, 048101 (2017)
  

The Conformation of Factor H Determines its Von Willebrand Factor Reductase Activity

Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kDa), is a complement control protein that regulates the activity of the alternative complement pathway. Recombinant factor H (rFH) has exposed free thiols that can also reduce the disulfide bonds linking smaller soluble von Willebrand factor (VWF) multimers into large VWF multimeric forms. In contrast, commercial, highly purified FH (pFH) is prepared under conditions that result in fewer exposed free thiols and little or no VWF reductase activity. pFH chemically altered by either EDTA or urea regains its VWF reductase activity. We used single molecule force studies with atomic force microscopy (AFM) to compare the characteristics of rFH and pFH in the presence or absence of EDTA and urea. Our data indicate that the VWF reductase activity of full-length FH (rFH and pFH) is associated with different conformations and the amount of free thiols.   

Rapid Terahertz Dichroism Near Field Microscopy for Biomolecular Intramolecular Vibrational Spectroscopy

Intramolecular vibrations of the protein backbone provide access to functional conformational change, however the characterization of these motions has presented a major challenge. Previously we show that while the vibrational density of states and isotropic absorption are relatively insensitive to protein functional state and mutation, anisotropic optical absorbance reveals the reorientation of vibrational displacements. Those measurements used a novel technique where anisotropic terahertz transmission of protein crystals are measured in the near field. The rotation of the protein sample and the corresponding reimaging significantly extend data acquisition time. Here we present our unique technique-ideal polarization-varying anisotropic terahertz microscopy (Ideal PV-ATM) for fast and direct measurement of protein intramolecular vibrations. It avoids the rotation of the sample and shortens the measurement time by a factor of 16. And ideal PV-ATM spectrum of test sample sucrose, which has strong linear dichroism in the THz range, agrees well with the solid-state density functional theory and anisotropic birefringence calculations.   

van der Waals Dispersion Forces and Hydrophobic Solvation

Per definition hydrophobic compounds are averse to aqueous environments. Peculiar examples are non-polar moieties or conformations of biomolecules, which are inevitably exposed to water under physiological conditions. Therefore, an accurate description of the interactions in such an unfavorable setup, in terms of solvation, is of paramount importance to comprehend the biomolecular machinery. One major source of attractive interaction between non-polar entities and water is van der Waals (vdW) dispersion forces. These weak interactions form the basis of biochemical functionality as they give rise to the necessary conformational flexibility. Typically, vdW forces, emerging from instantaneous fluctuations of the electron density, are only phenomenologically included in solvation models. In this contribution, we address the relevance of their intrinsic many-body character for hydrophobic solvation. In our study, we employ a combined approach of the Density-Functional Tight-Binding method and the Many-Body Dispersion model. The findings show that collective charge fluctuations contribute significantly to a distinct conformational dependence of the stability of (bio)molecular solutes in water. Ultimately, this leads to a considerable decrease of energy barriers during protein folding.   

Structural-kinetic relationships determine consistent interpretations of coarse-grained peptide kinetics

Coarse-grained simulation models have provided tremendous insight into the complex behavior of protein systems, but lack a straightforward connection to the true dynamical properties of the system. This lack of consistent dynamics severely limits coarse-grained models from providing accurate interpretations for kinetic experiments. In this work, we perform a detailed investigation into the kinetic properties of secondary structure formation generated by molecular simulation models. Our strategy is to systematically vary force-field parameters of a simple, native-biased coarse-grained model to identify relationships between the emergent structural, kinetic, and thermodynamic properties. Our investigation reveals robust structural-kinetic relationships that can be exploited to guarantee consistent kinetics through the reproduction of particular structural properties. These remarkable relationships are determined by the physics of the model, which shapes the free-energy landscape and restricts the attainable kinetic properties. Our results suggest an approach for constructing kinetically-accurate models that extends the capabilities of current coarse-grained peptide models.   

Hidden dynamics in the unfolding of individual bacteriorhodopsins

Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-µs resolution, we reexamined the unfolding of individual bacteriorhodopsin (bR) molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as 2–3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 µs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins, and, more broadly, enable experimental access to previously obscured protein dynamics.   

Investigation of the Isotope Shift in Protein Collective Vibrations

Recently it has been established that the collective vibrations of proteins can be characterized using anisotropic terahertz spectroscopy. These motions are necessary for biological function. Ideally the observed resonances can be identified with specific motions of the macromolecular subdomains and one could determine if and how mutations effect these motions. Current computational techniques do not accurately predict the measured spectra so isotope shift measurements can provide a means to identify which calculated features are associated with the observed resonances. Protonated and deuterated chicken egg white lysozyme (CEWL) crystals were grown concurrently in buffer solutions that were made using deionized water or heavy water. The absorption spectra of the resulting crystals are determined using anisotropic terahertz microscopy, which removes isotropic background features and identifies structural modes of the protein. Our preliminary results find a 0.3 THz red shift for the deuterated CEWL vibrations. These results are compared to calculations done on normal modes for anisotropic spectra for the two crystal compositions.