Abstract
Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally.
Highlights
Proteins are dynamical entities whose ability to change shape often plays essential roles in their function
We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations
We resorted to a set of flexible and efficient enhanced sampling methods, collectively known as ‘metadynamics’ (Laio and Parrinello, 2002), that have previously been used in a wide range of applications
Summary
Proteins are dynamical entities whose ability to change shape often plays essential roles in their function. As these minor conformations may, be critical to protein functions, including protein folding, ligand binding, enzyme catalysis, and signal transduction (Mulder et al, 2001; Tang et al, 2007; Baldwin and Kay, 2009) it is important to be able to characterize them in detail While it may in certain cases be possible to capture sparsely populated conformations in crystals under perturbed experimental conditions, or to examine their structures by analysis of electron density maps (Fraser et al, 2009), NMR spectroscopy provides unique opportunities to study the dynamical equilibrium between major and minor conformations (Baldwin and Kay, 2009; Sekhar and Kay, 2013) via e.g. chemical-exchange saturation transfer (Vallurupalli et al, 2012), Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (Hansen et al, 2008), or indirectly via paramagnetic relaxation enhancement (Tang et al, 2007) or residual dipolar coupling (Lukin et al, 2003) experiments. In favourable cases such experiments can provide thermodynamic and kinetic information (i.e. the population of G and E states and the rate of exchange between them), and structural information in the form of
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