Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Coadsorbate‐Induced Reversal of Solid–Liquid Interface Dynamics

Coadsorbed anions are well‐known to influence surface reactivity and dynamics at solid–liquid interfaces. Here we demonstrate that the chemical nature of these spectator species can entirely determine the microscopic dynamic behavior. Quantitative in situ video‐STM data on the surface diffusion of adsorbed sulfur atoms on Cu(100) electrodes in aqueous solution covered by bromide and chloride spectators, respectively, reveal in both cases a strong exponential potential dependence, but with opposite sign. This reversal is highly surprising in view of the isostructural adsorbate arrangement in the two systems. Detailed DFT studies suggest an anion‐induced difference in the sulfur diffusion mechanism, specifically an exchange diffusion on the Br‐covered surface. Experimental evidence for the latter is provided by the observation of Cu vacancy formation in the Br system, which can be rationalized by a side reaction of the sulfur exchange diffusion.     

Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

Universality of the Sodium Ion Binding Mechanism in Class A G‐Protein‐Coupled Receptors

The allosteric modulation of G‐protein‐coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na⁺ ions bound to the inactive conformations at the conserved Asp^2.50. To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na⁺ binding to the allosteric site are limited. We performed hundreds‐of‐microsecond long simulations of 18 GPCRs and elucidated their Na⁺ binding mechanism. In class A GPCRs, the Na⁺ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na⁺ binding to the allosteric site, which reveal a conserved mechanism of Na⁺ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na⁺ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.     

Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

The recent discovery of inhibitory compounds binding to distinct pockets on GDP‐bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras?GDP on microsecond to millisecond timescales using relaxation‐based NMR experiments, and identified substantial slow dynamics with τ_ex of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor‐bound and free states. These findings, which have been demonstrated in both wild‐type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras–inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.     

Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of ¹H/²H exchange kinetics at the level of the amide proton in the mid‐infrared spectral range (1700–1500 cm^?1). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H‐bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300–30 cm^?1), a spectral range that includes a continuum mode of the hydrogen bonding signature.     

The Mechanism of Ligand‐Induced Activation or Inhibition of μ‐ and κ‐Opioid Receptors

G‐protein‐coupled receptors (GPCRs) are important targets for treating severe diseases. However why certain molecules act as activators whereas others, with similar structures, block GPCR activation, is poorly understood since the same molecule can activate one receptor subtype while blocking another closely related receptor. To shed light on these central questions, we used all‐atom, long‐time‐scale molecular dynamics simulations on the κ‐opioid and μ‐opioid receptors (κOR and μOR). We found that water molecules penetrating into the receptor interior mediate the activating versus blocking effects of a particular ligand–receptor interaction. Both the size and the flexibility of the bound ligand regulated water influx into the receptor. The solvent‐accessible inner surface area was found to be a parameter that can help predict the function of the bound ligand.     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.     

A Sharp Thermal Transition of Fast Aromatic‐Ring Dynamics in Ubiquitin

Aromatic amino acid side chains have a rich role within proteins and are often central to their structure and function. Suitable isotopic‐labelling strategies enable studies of sub‐nanosecond aromatic‐ring dynamics using solution NMR relaxation methods. Surprisingly, it was found that the three aromatic side chains in human ubiquitin show a sharp thermal dynamical transition at approximately 312 K. Hydrostatic pressure has little effect on the low‐temperature behavior, but somewhat decreases the amplitude of motion in the high‐temperature regime. Therefore, below the transition temperature, ring motion is largely librational. Above this temperature, a complete ring‐rotation process that is fully consistent with a continuous diffusion not requiring the transient creation of a large activated free volume occurs. Molecular dynamics simulations qualitatively corroborate this view and reinforce the notion that the dynamical character of the protein interior has much more liquid‐alkane‐like properties than previously appreciated.     

Connectivity and binding‐site recognition: Applications relevant to drug design

Here, we describe a family of methods based on residue–residue connectivity for characterizing binding sites and apply variants of the method to various types of protein–ligand complexes including proteases, allosteric‐binding sites, correctly and incorrectly docked poses, and inhibitors of protein–protein interactions. Residues within ligand‐binding sites have about 25% more contact neighbors than surface residues in general; high‐connectivity residues are found in contact with the ligand in 84% of all complexes studied. In addition, a k‐means algorithm was developed that may be useful for identifying potential binding sites with no obvious geometric or connectivity features. The analysis was primarily carried out on 61 protein–ligand structures from the MEROPS protease database, 250 protein–ligand structures from the PDBSelect (25%), and 30 protein–protein complexes. Analysis of four proteases with crystal structures for multiple bound ligands has shown that residues with high connectivity tend to have less variable side‐chain conformation. The relevance to drug design is discussed in terms of identifying allosteric‐binding sites, distinguishing between alternative docked poses and designing protein interface inhibitors. Taken together, this data indicate that residue–residue connectivity is highly relevant to medicinal chemistry. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Decoding the components of dynamics in three‐domain proteins

In this study, we examine the feasibility and limitations of describing the motional behavior of three‐domain proteins in which the domains are linearly connected. In addition to attempting the determination of the internal and overall reorientational correlation times, we investigate the existence of correlations in the motions between the three domains. Since in linearly arranged three‐domain proteins, there are typically no experimental data that can directly report on motional correlation between the first and the third domain, we address this question by dynamics simulations. Two limiting cases occur: (1) for weak repulsive potentials and (2) when strong repulsive potentials are applied between sequential domains. The motions of the terminal domains become correlated in the case of strong interdomain repulsive potentials when these potentials do not allow the angle between the sequential domains to be smaller than about 60°. Using the model‐free (MF) and extended MF formalisms of Lipari and Szabo, we find that the motional behavior can be separated into two components; the first component represents the concerted overall motion of the three domains, and the second describes the independent component of the motion of each individual domain. We find that this division of the motional behavior of the protein is maintained only when their timescales are distinct and can be made when the angles between sequential domains remain between 60° and 160°. In this work, we identify and quantify interdomain motional correlations. © 2013 Wiley Periodicals, Inc.