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BBA - Proteins and Proteomics (v.1814, #8)

Editorial Board (pp. i).

Protein dynamics: Experimental and computational approaches by Stefano Bettati; F. Javier Luque; Cristiano Viappiani (pp. 913-915).

Coupling of protein and environment fluctuations by R.D. Young; P.W. Fenimore (pp. 916-921).

We review the concepts of protein dynamics developed over the last 35years and extend applications of the unified model of protein dynamics to heat flow and spatial fluctuations in hydrated myoglobin (Mb) powders. Differential scanning calorimetry (DSC) and incoherent neutron scattering (INS) data on hydration Mb powders are explained by the temperature-dependence of the hydration-shell β_h process measured by dielectric relaxation spectroscopy (DRS). The unified model explains the temperature dependence of DSC and INS data as a kinetic effect due to a fixed experimental time window and a broad distribution of hydration-shell β_h fluctuation rates. We review the slaving of large scale protein motions to the bulk solvent α process, and the metastability of Mb molecules in glass forming bulk solvent at low temperatures. This article is part of a Special Issue entitled: "Protein Dynamics: Experimental and Computational Approaches".

Keywords: Protein dynamics; Protein hydration; Temperature-dependence; Calorimetry; Neutron scattering

Exploring and exploiting allostery: Models, evolution, and drug targeting by Alessio Peracchi; Andrea Mozzarelli (pp. 922-933).

The concept of allostery was elaborated almost 50years ago by Monod and coworkers to provide a framework for interpreting experimental studies on the regulation of protein function. In essence, binding of a ligand at an allosteric site affects the function at a distant site exploiting protein flexibility and reshaping protein energy landscape. Both monomeric and oligomeric proteins can be allosteric. In the past decades, the behavior of allosteric systems has been analyzed in many investigations while general theoretical models and variations thereof have been steadily proposed to interpret the experimental data. Allostery has been established as a fundamental mechanism of regulation in all organisms, governing a variety of processes that range from metabolic control to receptor function and from ligand transport to cell motility. A number of studies have shed light on how evolutionary pressures have favored and molded the development of allosteric features in specific macromolecular systems. The widespread occurrence of allostery has been recently exploited for the development and design of allosteric drugs that bind to either physiological or non-physiological allosteric sites leading to gain of function or loss of function. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► The concept of allostery provides a general framework for understanding the regulation of protein function. ► Both in monomeric and oligomeric proteins, ligand binding at an allosteric site affects the function at a distant site exploiting the conformational flexibility and reshaping the energy landscape. ► Evolutionary pressures have favored and molded the development of allostery. ► The widespread occurrence of protein allostery is being exploited for the development and design of allosteric drugs.

Keywords: Allostery; Conformational changes; Protein; Regulation

Protein dynamics and pressure: What can high pressure tell us about protein structural flexibility? by Patrizia Cioni; Edi Gabellieri (pp. 934-941).

After a brief overview of NMR and X-ray crystallography studies on protein flexibility under pressure, we summarize the effects of hydrostatic pressure on the native fold of azurin from Pseudomonas aeruginosa as inferred from the variation of the intrinsic phosphorescence lifetime and the acrylamide bimolecular quenching rate constants of the buried Trp residue. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of the hydration of the polypeptide. The study of the effect of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (Δ V⁰) of azurin and on the internal dynamics of the protein fold under pressure demonstrate that the creation of an internal cavity will enhance the plasticity and lower the stability of the globular structure. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: High pressure; Tryptophan phosphorescence; Protein dynamics; Protein structure; Protein cavities; Azurin

An introduction to NMR-based approaches for measuring protein dynamics by Ian R. Kleckner; Mark P. Foster (pp. 942-968).

Proteins are inherently flexible at ambient temperature. At equilibrium, they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biological functions including catalysis, binding, regulation and cellular structure. Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique for measuring these essential features of proteins. Here we provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common experimental and analytical framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape analysis, (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.Display Omitted► Internal dynamics of proteins are essential for proper function. ► Protein dynamics can be rigorously studied using NMR spectroscopy. ► Eight distinct NMR based approaches are discussed with recent examples. ► The process of analyzing complex experimental data is generalized into a common model-based framework.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; s; second; ms; millisecond (10; −; ³; s); μs; microsecond (10; −; ⁶; s); ns; nanosecond (10; −; ⁹; s); ps; picosecond (10; −; ¹²; s); k; _ex; chemical exchange rate constant; τ; _ex; chemical exchange timescale (=; 1; k; _ex; ); R; _ex; transverse relaxation rate constant due to chemical exchange; RDC; residual dipolar coupling; RT NMR; real time NMR; NSR; nuclear spin relaxation; RF RD; rotating frame relaxation dispersion; CPMG RD; Carr–Purcell Meiboom–Gill relaxation dispersion; EXSY; EXchange SpectroscopY; PRE; paramagnetic relaxation enhancement; S; ²; square of the generalized modelfree order parameterProtein dynamics; Protein flexibility; NMR

Protein stability, flexibility and function by Kaare Teilum; Johan G. Olsen; Birthe B. Kragelund (pp. 969-976).

Proteins rely on flexibility to respond to environmental changes, ligand binding and chemical modifications. Potentially, a perturbation that changes the flexibility of a protein may interfere with its function. Millions of mutations have been performed on thousands of proteins in quests for a delineation of the molecular details of their function. Several of these mutations interfered with the binding of a specific ligand with a concomitant effect on the stability of the protein scaffold. It has been ambiguous and not straightforward to recognize if any relationships exist between the stability of a protein and the affinity for its ligand. In this review, we present examples of proteins where changes in stability results in changes in affinity and of proteins where stability and affinity are uncorrelated. We discuss the possibility for a relationship between stability and binding. From the data presented is it clear that there are specific sites (flexibility hotspots) in proteins that are important for both binding and stability. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► The effects of mutations on protein stability and ligand binding have been compared. ► Protein stability and ligand affinity are correlated in special cases. ► Flexibility hotspots are sites in proteins important for both stability and function.

Keywords: Protein stability; Protein affinity; Flexibility; Ligand binding

Modelling proteins: Conformational sampling and reconstruction of folding kinetics by Konstantin Klenin; Birgit Strodel; David J. Wales; Wolfgang Wenzel (pp. 977-1000).

In the last decades biomolecular simulation has made tremendous inroads to help elucidate biomolecular processes in-silico. Despite enormous advances in molecular dynamics techniques and the available computational power, many problems involve long time scales and large-scale molecular rearrangements that are still difficult to sample adequately. In this review we therefore summarise recent efforts to fundamentally improve this situation by decoupling the sampling of the energy landscape from the description of the kinetics of the process. Recent years have seen the emergence of many advanced sampling techniques, which permit efficient characterisation of the relevant family of molecular conformations by dispensing with the details of the short-term kinetics of the process. Because these methods generate thermodynamic information at best, they must be complemented by techniques to reconstruct the kinetics of the process using the ensemble of relevant conformations. Here we review recent advances for both types of methods and discuss their perspectives to permit efficient and accurate modelling of large-scale conformational changes in biomolecules. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: Protein dynamics; Conformational ensemble; Advanced sampling; Markov models

Dynamics of protein folding: Probing the kinetic network of folding–unfolding transitions with experiment and theory by Ginka S. Buchner; Ronan D. Murphy; Nicolae-Viorel Buchete; Jan Kubelka (pp. 1001-1020).

The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been achieved owing to the rapidly growing interdisciplinary interest and significant progress in both experimental techniques and theoretical methods. Improvements in the experimental time resolution led to determination of the timescales of the important elementary events in folding, such as formation of secondary structure and tertiary contacts. Sensitive single molecule methods made possible probing the distributions of the unfolded and folded states and following the folding reaction of individual protein molecules. Discovery of proteins that fold in microseconds opened the possibility of atomic-level theoretical simulations of folding and their direct comparisons with experimental data, as well as of direct experimental observation of the barrier-less folding transition. The ultra-fast folding also brought new questions, concerning the intrinsic limits of the folding rates and experimental signatures of barrier-less “downhill” folding. These problems will require novel approaches for even more detailed experimental investigations of the folding dynamics as well as for the analysis of the folding kinetic data. For theoretical simulations of folding, a main challenge is how to extract the relevant information from overwhelmingly detailed atomistic trajectories. New theoretical methods have been devised to allow a systematic approach towards a quantitative analysis of the kinetic network of folding–unfolding transitions between various configuration states of a protein, revealing the transition states and the associated folding pathways at multiple levels, from atomistic to coarse-grained representations. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► New highly time-resolved and single-molecule experimental methods have established characteristic timescales of the polypeptide chain dynamics and elementary events in protein folding. ► The maximum protein folding rates (the protein folding “speed limit”) are generally slower than those of elementary folding events in model compounds. ► Ultra-fast and “downhill” folding proteins in principle allow investigations of the polypeptide chain dynamics during folding; however, experimental signatures of “downhill” folding remain controversial. ► Coarse master equations provide maps for protein folding kinetic networks. ► Kinetic network analysis bridge folding theory, MD simulations and experiments.

Keywords: Protein folding; Experimental kinetics; Dynamics; Elementary events in folding; Ultra-fast folding; Downhill folding; Protein folding speed limit; Free-energy surface; Molecular dynamics simulations; Master equations; Coarse graining

Protein folding at single-molecule resolution by Allan Chris M. Ferreon; Ashok A. Deniz (pp. 1021-1029).

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► Powerful single-molecule tools dissect complex protein folding mechanisms. ► They provide views of intricate protein structural distributions and dynamics. ► This review briefly introduces common FRET and force methods. ► Folding landscapes, IDPs, assisted folding and biomechanical unfolding. ► Future work is expected on more complex systems in cells and organisms.

Keywords: Protein folding landscapes; Protein dynamics; Intrinsically disordered protein; Osmolyte; Chaperone; Single molecule

Ligand dynamics in heme proteins observed by Fourier transform infrared-temperature derivative spectroscopy by Karin Nienhaus; G. Ulrich Nienhaus (pp. 1030-1041).

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for the investigation of protein–ligand interactions in heme proteins. Nitric oxide and carbon monoxide are attractive physiologically relevant ligands because their bond stretching vibrations give rise to strong mid-infrared absorption bands that can be measured with exquisite sensitivity and precision using photolysis difference spectroscopy at cryogenic temperatures. These stretching bands are fine-tuned by electrostatic interactions with the environment and, therefore, ligands can be utilized as local probes of structure and dynamics. Bound to the heme iron, the ligand stretching bands are susceptible to changes in the iron–ligand bond and the electric field at the active site. Upon photolysis, the vibrational bands display changes due to ligand relocation to docking sites within the protein, rotational motions of the ligand in these sites and protein conformational changes. Photolysis difference spectra taken over a wide temperature range (3–300K) using specific temperature protocols for sample photodissociation can provide detailed insights into both protein and ligand dynamics. Moreover, temperature-derivative spectroscopy (TDS) has proven to be a particularly powerful technique to study protein–ligand interactions. The FTIR-TDS technique has been extensively applied to studies of carbon monoxide binding to heme proteins, whereas measurements with nitric oxide are still scarce. Here we describe infrared cryo-spectroscopy and present a variety of applications to the study of protein–ligand interactions in heme proteins. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: Abbreviations; FTIR; Fourier transform infrared; TDS; temperature derivative spectroscopy; Mb; myoglobin; Hb; hemoglobin; Ngb; neuroglobin; DHP; dehaloperoxidase; NP; nitrophorin; hIDO; human indoleamine 2,3-dioxygenaseFourier transform infrared spectroscopy; Temperature derivative spectroscopy; Heme protein; Ligand binding; Ligand migration; Photolysis difference spectroscopy

Ligand migration and hexacoordination in type 1 non-symbiotic rice hemoglobin by Nitin Kumar Bisht; Stefania Abbruzzetti; Sheetal Uppal; Stefano Bruno; Francesca Spyrakis; Andrea Mozzarelli; Cristiano Viappiani; Suman Kundu (pp. 1042-1053).

Type 1 non-symbiotic rice hemoglobin (rHb1) shows bis-histidyl heme hexacoordination and is capable of binding diatomic ligands reversibly. The biological function is as yet unclear, but the high oxygen affinity makes it unlikely to be involved in oxygen transport. In order to gain insight into possible physiological roles, we have studied CO rebinding kinetics after laser flash photolysis of rHb1 in solution and encapsulated in silica gel. CO rebinding to wt rHb1 in solution occurs through a fast geminate phase with no sign of rebinding from internal docking sites. Encapsulation in silica gel enhances migration to internal cavities. Site-directed mutagenesis of FB10, a residue known to have a key role in the regulation of hexacoordination and ligand affinity, resulted in substantial effects on the rebinding kinetics, partly inhibiting ligand exit to the solvent, enhancing geminate rebinding and enabling ligand migration within the internal cavities. The mutation of HE7, one of the histidyl residues involved in the hexacoordination, prevents hexacoordination, as expected, but also exposes ligand migration through a complex system of cavities. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► A hydrophobic tunnel connects the distal cavity of rice Hb1 to the solvent. ► Photodissociated ligands can be transiently trapped inside the tunnel. ► Residues at E7 and B10 affect the shape of the tunnel. ► Residues at E7 and B10 influence protein reactivity.

Keywords: Non-symbiotic plant hemoglobin; Laser flash photolysis; Silica gel; Ligand migration; Hexacoordination

Protein dynamics and ligand migration interplay as studied by computer simulation by Arroyo-Manez Pau Arroyo-Mañez; Damián E. Bikiel; Leonardo Boechi; Luciana Capece; Santiago Di Lella; Darío A. Estrin; Marti Marcelo A. Martí; Diego M. Moreno; Alejandro D. Nadra; Ariel A. Petruk (pp. 1054-1064).

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: Ligand migration; Protein dynamics; Molecular dynamics

Time resolved thermodynamics associated with ligand photorelease in heme peroxidases and globins: Open access channels versus gated ligand release by Carissa M. Vetromile; Jaroslava Miksovska; Randy W. Larsen (pp. 1065-1076).

Heme proteins represent a diverse class of biomolecules responsible for an extremely diverse array of physiological functions including electron transport, monooxygenation, ligand transport and storage, cellular signaling, respiration, etc. An intriguing aspect of these proteins is that such functional diversity is accomplished using a single type of heme macrocycle based upon iron protoporphyrin IX. The functional diversity originates from a delicate balance of inter-molecular interactions within the protein matrix together with well choreographed dynamics that modulate the heme electronic structure as well as ligand entry/exit pathways from the bulk solvent to the active site. Of particular interest are the dynamics and energetics associated with the entry/exit of ligands as this process plays a significant role in regulating the rates of heme protein activity. Time-resolved photoacoustic calorimetry (PAC) has emerged as a powerful tool through which to probe the underlying energetics associated with small molecule dissociation and release to the bulk solvent in heme proteins on time scales from tens of nanoseconds to several microseconds. In this review, the results of PAC studies on various classes of heme proteins are summarized highlighting how different protein structures affect the thermodynamics of ligand migration. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.► The photothermal summary demonstrate unique dynamics for heme ligand migration. ► Peroxidases with large channels are dynamically consistent with fast ligand exit. ► Globin dynamics reveal more complex conformational regulation for ligand release. ► Globin dynamics are unique to each type of protein within the class. ► Photothermal methods provide new insights into heme ligand dynamics.

Keywords: Photoacoustic calorimetry; Heme; Iron porphyrin; Ligand; Kinetics; Enthalpy

Protein dynamics and enzyme catalysis: Insights from simulations by John D. McGeagh; Kara E. Ranaghan; Adrian J. Mulholland (pp. 1077-1092).

The role of protein dynamics in enzyme catalysis is one of the most active and controversial areas in enzymology today. Some researchers claim that protein dynamics are at the heart of enzyme catalytic efficiency, while others state that dynamics make no significant contribution to catalysis. What is the biochemist – or student – to make of the ferocious arguments in this area? Protein dynamics are complex and fascinating, as molecular dynamics simulations and experiments have shown. The essential question is: do these complex motions have functional significance? In particular, how do they affect or relate to chemical reactions within enzymes, and how are chemical and conformational changes coupled together? Biomolecular simulations can analyse enzyme reactions and dynamics in atomic detail, beyond that achievable in experiments: accurate atomistic modelling has an essential part to play in clarifying these issues. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: Quantum mechanics; Molecular mechanics; Protein dynamics; Molecular dynamics; Quantum tunnelling; Conformational change; Functional dynamics; Enzyme catalysis; Computational enzymology

Studies of photo-induced protein reactions by spectrally silent reaction dynamics detection methods: Applications to the photoreaction of the LOV2 domain of phototropin from Arabidopsis thaliana by Masahide Terazima (pp. 1093-1105).

Biological function involves a series of chemical reactions of biological molecules, and during these reactions, there are numerous spectrally silent dynamic events that cannot be monitored by absorption or emission spectroscopic techniques. Such spectrally silent dynamics include changes in conformation, intermolecular interactions (hydrogen bonding, hydrophobic interactions), inter-protein interactions (oligomer formation, dissociation reactions) and conformational fluctuations. These events might be associated with biological function. To understand the molecular mechanisms of reactions, time-resolved detection of such dynamics is essential. Recently, it has been shown that time-resolved detection of the refractive index is a powerful tool for measuring dynamic events. This technique is complementary to optical absorption detection methods and the signal contains many unique properties, which are difficult to obtain by other methods. The advantages and methods for signal analyses are described in detail in this review. A typical example of an application of time-resolved refractive index change detection is given in the second part: The photoreaction of the LOV2 domain of a blue light photoreceptor from Arabidopsis Thaliana (phototropin). This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Keywords: Protein reaction; Diffusion; Dynamics; Photosensor

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BBA - Proteins and Proteomics (v.1814, #8)