Structural Refinement from Restrained-Ensemble Simulations Based on EPR/DEER Data: Application to T4 Lysozyme

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Significance C-type inactivation gating in K + channels plays an important role in controlling the firing patterns of excitable cells and is fundamental in determining the length and frequency of the cardiac action potential. At a molecular level, toxins, blockers, and metal ions bind to the outer vestibule and modulate the functional behavior of K + channels. Using KcsA, we show that the shuttling between the inactivated and conductive states of K + channels is accompanied by changes in local outer vestibule dynamics, in the absence of large conformational changes. The altered structural and hydration dynamics of the outer vestibule appear to be likely and important modulators of selectivity filter gating transitions in K + channels.

Section: Resultsmentioning

confidence: 99%

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

Raghuraman

Islam

Mukherjee

et al. 2014

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“…To quantitatively evaluate the relationship between experimental distance distributions and the various crystal structures, we calculated the distance distributions expected based on these structures using molecular dynamics simulations [molecular dynamics of dummy spin labels (MDDS)] (Methods) (33). For this purpose, we introduced dummy spin labels at the sites of interest and carried out MD simulations while fixing the protein backbone (33).…”

Section: Resultsmentioning

confidence: 99%

“…All molecular dynamic simulations of the spin-labeled Mhp1 were carried out with the CHARMM (41) program package, using the all-atom CHARMM27 protein force field (42) with the CMAP corrections and the dummy nitroxide (ON) spin-label force field parameters (33). Three crystal structures of Mhp1 [2JLN (15), 2JLO (15), and 2X79 (8)] were used to construct the geometries of the Mhp1 systems for simulation.…”

Section: Methodsmentioning

confidence: 99%

Conformational cycle and ion-coupling mechanism of the Na ⁺ /hydantoin transporter Mhp1

Kazmier

Sharma

Islam

et al. 2014

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147

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuTfold has been captured in outward-facing, occluded, and inwardfacing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na + -coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion-and substrate-dependent conformational equilibria. In contrast to the Na + /leucine transporter LeuT, our results suggest that Na + binding at the conserved second Na + binding site does not change the energetics of the inward-and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.EPR | DEER | Na + -coupled symport | conformational dynamics | transport mechanism

“…We augment that distance information with secondary structure predictions from PSIPRED. We simulated two different systems (αA-Crystallin and Lysozyme) starting from completely extended conformations using available EPR data (29,30).…”

Section: Resultsmentioning

confidence: 99%

“…From the early days, there has been interest in parlaying knowledge of kinetic routes of protein folding to an understanding of principles of protein structures (36,37). Since then, it has become clear that a unifying principle is that folding energy landscapes are funnel-shaped (30,38,39). Here, MELD establishes funnel-shaped potentials, driven by uncertainbut nevertheless powerful-information from experiments or bioinformatics.…”

Section: Discussionmentioning

confidence: 99%

Determining protein structures by combining semireliable data with atomistic physical models by Bayesian inference

MacCallum

Pérez

Dill

2015

135

233

More than 100,000 protein structures are now known at atomic detail. However, far more are not yet known, particularly among large or complex proteins. Often, experimental information is only semireliable because it is uncertain, limited, or confusing in important ways. Some experiments give sparse information, some give ambiguous or nonspecific information, and others give uncertain information-where some is right, some is wrong, but we don't know which. We describe a method called Modeling Employing Limited Data (MELD) that can harness such problematic information in a physics-based, Bayesian framework for improved structure determination. We apply MELD to eight proteins of known structure for which such problematic structural data are available, including a sparse NMR dataset, two ambiguous EPR datasets, and four uncertain datasets taken from sequence evolution data. MELD gives excellent structures, indicating its promise for experimental biomolecule structure determination where only semireliable data are available.protein structure | molecular modeling | integrative structural biology | Bayesian inference I ncreasingly, structures are determined using integrative structural biology approaches, where direct experimental data are combined with computer-based models (1). Important successes in integrative structural biology have come from pioneering methods such as Modeler (2, 3), methods based on Rosetta (4-7), and others (8). Atomistic molecular dynamics (MD) simulations can be a powerful tool in integrative structural biology, because they capture physical principles and thermodynamic forcesinformation that is otherwise orthogonal to purely structural observations. However, there remain many situations in which it is not yet possible to properly integrate external knowledge with atomistic MD to infer biomolecular structures. Often, the external knowledge is challenging in one or more of the following ways. (i) Sparse data provide too little information to fully constrain the structure. (ii) Ambiguous data are not very specific, allowing alternative structural interpretations. (iii) Uncertain data cannot be interpreted at face value, because they contain false-positive signals that can be misdirective. Determining new challenging protein structures requires ways to handle semireliable data.Here, we describe a physics-based, Bayesian computational method called MELD (Modeling Employing Limited Data). It is a procedure for making rigorous inferences from limited or uncertain data. We build upon previous Bayesian approaches (9-14), which share the key feature of combining prior belief with the available data to produce statistically consistent samples from a posterior distribution, rather than searching for a single well-scoring model. The key properties of MELD are the rigorous treatment of statistical mechanics, a novel likelihood function that can handle uncertain data, and a graphics processing unit (GPU)-accelerated sampling strategy that makes the calculations tractable.MELD uses free energy as the princi...