Helicase Functional Dynamics from Low-Resolution Experimental Data and Simulation

The biological function of large macromolecular assemblies depends on their structure and their dynamics over a broad range of timescales; for this reason its investigation poses significant challenges to conventional experimental techniques. A promising experimental technique is hydrogen-deuterium exchange detected by mass spectrometry (HDX-MS). I begin by presenting a new computational method for quantitative interpretation of deuterium exchange kinetics. The method is tested on a hexameric viral helicase φ12 P4 that pumps RNA into a virus capsid at the expense of ATP hydrolysis. Molecular dynamics simulations predict accurately the exchange kinetics of most peptide fragments and provide a residue-level interpretation of the low-resolution experimental results. This approach is also a powerful tool to probe mechanisms that cannot be observed by X-ray crystallography, or that occur over timescales longer than those that can be realistically simulated, such as the opening of the hexameric ring. Once validated, the method is applied on a homologous system, the packaging motor φ8 P4, for which RNA loading and translocation mechanisms remain elusive. Quantitative interpretation of HDX-MS data, as well as Förster resonance energy transfer (FRET) and computational observations, suggest that the C-terminal domain of the motor plays a crucial role. A new translocation model of φ8 P4 is proposed, for which the affinity between the motor and RNA is modulated by the C-termini. In the final result chapter, the amount of the structural information carried by HDX-MS data is quantitatively analysed. The impact of the averaging of the exchange over peptide fragments on the information content is investigated. The complementarity of data obtained from HDX-MS and data obtained from other techniques (such as NMR, FRET or SAXS) is also examined

Optimal Reaction Coordinates

The dynamic behavior of complex systems with many degrees of freedom is often analyzed by projection onto one or a few reaction coordinates. The dynamics is then described in a simple and intuitive way as diffusion on the associated free energy pro le. In order to use such a picture for a quantitative description of the dynamics one needs to select the coordinate in an optimal way so as to minimize non-Markovian effects due to the projection. For equilibrium dynamics between two boundary states (e.g., a reaction) the optimal coordinate is known as the committor or the pfold coordinate in protein folding studies. While the dynamics projected on the committor is not Markovian, many important quantities of the original multidimensional dynamics on an arbitrarily complex landscape can be computed exactly. Here we summarize the derivation of this result, discuss different approaches to determine and validate the committor coordinate and present three illustrative applications: protein folding, the game of chess, and patient recovery dynamics after kidney transplant

Estimating Constraints for Protection Factors from HDX-MS Data

Hydrogen/deuterium exchange monitored by mass spectrometry is a promising technique for rapidly fingerprinting structural and dynamical properties of proteins. The time-dependent change in the mass of any fragment of the polypeptide chain depends uniquely on the rate of exchange of its amide hydrogens, but determining the latter from the former is generally not possible. Here, we show that, if time-resolved measurements are available for a number of overlapping peptides that cover the whole sequence, rate constants for each amide hydrogen exchange (or equivalently, their protection factors) may be extracted and the uniqueness of the solutions obtained depending on the degree of peptide overlap. However, in most cases, the solution is not unique, and multiple alternatives must be considered. We provide a statistical method that clusters the solutions to further reduce their number. Such analysis always provides meaningful constraints on protection factors and can be used in situations in which obtaining more refined experimental data is impractical. It also provides a systematic way to improve data collection strategies to obtain unambiguous information at single-residue level (e.g., for assessing protein structure predictions at atomistic level)

Modulation of a protein free-energy landscape by circular permutation

Circular permutations usually retain the native structure and function of a protein while inevitably perturbing its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories, we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, although subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, in which one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed. © 2013 American Chemical Society

Modulation of a Protein Free Energy Landscape by Circular Permutation

[Image: see text] Circular permutations usually retain the native structure and function of a protein while inevitably perturbing its folding dynamics. By using simulations with a structure-based model and a rigorous methodology to determine free-energy surfaces from trajectories, we evaluate the effect of a circular permutation on the free-energy landscape of the protein T4 lysozyme. We observe changes which, although subtle, largely affect the cooperativity between the two subdomains. Such a change in cooperativity has been previously experimentally observed and recently also characterized using single molecule optical tweezers and the Crooks relation. The free-energy landscapes show that both the wild type and circular permutant have an on-pathway intermediate, previously experimentally characterized, in which one of the subdomains is completely formed. The landscapes, however, differ in the position of the rate-limiting step for folding, which occurs before the intermediate in the wild type and after in the circular permutant. This shift of transition state explains the observed change in the cooperativity. The underlying free-energy landscape thus provides a microscopic description of the folding dynamics and the connection between circular permutation and the loss of cooperativity experimentally observed