Understanding Of Protein Dynamics Research Articles

155 The role of solvent mobility in protein dynamics

Proteins are soft materials in which the internal motions are essential for their functions. Therefore, an understanding of protein dynamics is fundamental importance to biology. The mobility of th...

3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography.

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Protein Translocation through an Electrically Tunable Membrane

Understanding protein dynamics through an artificial nanopore has implications in many areas such as sensing and filtering. Collecting statistical information while tracking the movement of a full atomic protein model is computationally expensive since number of atoms ranges in the thousands. The need to represent protein with a computationally cost effective model is imperative, along with understanding its dynamics through the nanopore. In this work we studied the dynamics of the protein insulin placed near a nanopore of an electrically tunable semiconductor membrane. Using Brownian dynamics method we calculated the trajectory of the modeled protein in the electrolyte-membrane electrostatic potential. The time spent by the protein before a successful translocation and the translocation times were both analyzed. Our results indicate that the localized electric field within the nanopore affects the movement of the protein. Also, by comparing the results of the full atomic protein model with a coarse grained model and a single bead model, we evaluate which model best approximates the full atomic protein model.

Robust Elastic Network Model: Precise Prediction of Atomic Fluctuations in Protein Crystal Structures

Elastic network model (ENM) based normal mode analysis has become popular as its capability and suitability for the study of protein dynamics. However, the existing ENMs often fail to reproduce the experimentally observed B-factor (i.e., atomic fluctuation) because of oversimplification of their force-fields. In this work, we have proposed a robust ENM (RENM) in which, for reflecting inter-connections accused from surrounding molecules in a unit cell, symmetric constraints based on crystal space group are applied to the representative single molecule as well as its intra-connections are also represented by using lumped masses and specific spring constants depending on the types of amino acids and chemical bonds, respectively. More than 500 protein structures are tested by RENM. Their results show better agreement with experimental B-factor without additional computation burden compared to those of traditional ENM. Moreover, the global spring constant is quantitatively determined as a function of temperature at 100K and 290K, which enables us to compute directly atomic fluctuations and vibrational density of state without any fitting process. Thus, RENM is expected to play an important role in understanding protein dynamics based on its crystal structure information.

Shedding light on protein folding, structural and functional dynamics by single molecule studies.

The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

Sequence-based Gaussian network model for protein dynamics

Motivation: Gaussian network model (GNM) is widely adopted to analyze and understand protein dynamics, function and conformational changes. The existing GNM-based approaches require atomic coordinates of the corresponding protein and cannot be used when only the sequence is known.Results: We report, first of its kind, GNM model that allows modeling using the sequence. Our linear regression-based, parameter-free, sequence-derived GNM (L-pfSeqGNM) uses contact maps predicted from the sequence and models local, in the sequence, contact neighborhoods with the linear regression. Empirical benchmarking shows relatively high correlations between the native and the predicted with L-pfSeqGNM B-factors and between the cross-correlations of residue fluctuations derived from the structure- and the sequence-based GNM models. Our results demonstrate that L-pfSeqGNM is an attractive platform to explore protein dynamics. In contrast to the highly used GNMs that require protein structures that number in thousands, our model can be used to study motions for the millions of the readily available sequences, which finds applications in modeling conformational changes, protein–protein interactions and protein functions.Contact: zerozhua@126.comSupplementary information: Supplementary data are available at Bioinformatics online.

INSIGHTS IN ENZYME FUNCTIONAL DYNAMICS AND ACTIVITY REGULATION BY SINGLE MOLECULE STUDIES

The advent of advanced single molecule measurements heralded the arrival of a wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways not deducible by conventional bulk assays. They offered the direct observation and quantification of the abundance and life time of multiple states and transient intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements, thus providing unprecedented insights into complex biological processes. Here we survey the current state of the art in single-molecule fluorescence microscopy methodology for studying the mechanism of enzymatic activity and the insights on protein functional dynamics. We will initially discuss the strategies employed to date, their limitations and possible ways to overcome them, and finally how single enzyme kinetics can advance our understanding on mechanisms underlying function and regulation of proteins. [Formula: see text]Special Issue Comment: This review focuses on functional dynamics of individual enzymes and is related to the review on ion channels by Lu,44 the reviews on mathematical treatment of Flomenbom45 and Sach et al.,46 and review on FRET by Ruedas-Rama et al.41

PROTEIN DYNAMICS IN PHOSPHORYLATION-MEDIATED ALLOSTERY PROBED BY AMIDE EXCHANGE MASS SPECTROMETRY

A major goal of molecular biology is to correlate molecular structure with function. Since most enzymes and biological catalysts are proteins, the focus for correlating 'form' with 'function' has been entirely on protein macromolecular structure. It is obvious that any understanding of protein function must come through an understanding protein dynamics. Furthermore, all of the regulatory reactions are through changes in dynamics brought about by post-translational modifications, the most important of which is phosphorylation. This review highlights the important role of covalent phosphorylation and noncovalent phosphates in regulating allosteric effects and function through a study of protein dynamics. Mass spectrometry is a relatively new and increasingly important tool for describing protein dynamics. All examples described in this review have been studied by amide hydrogen/deuterium exchange mass spectrometry.

Exploring cavity dynamics in biomolecular systems

BackgroundThe internal cavities of proteins are dynamic structures and their dynamics may be associated with conformational changes which are required for the functioning of the protein. In order to study the dynamics of these internal protein cavities, appropriate tools are required that allow rapid identification of the cavities as well as assessment of their time-dependent structures.ResultsIn this paper, we present such a tool and give results that illustrate the applicability for the analysis of molecular dynamics trajectories. Our algorithm consists of a pre-processing step where the structure of the cavity is computed from the Voronoi diagram of the van der Waals spheres based on coordinate sets from the molecular dynamics trajectory. The pre-processing step is followed by an interactive stage, where the user can compute, select and visualize the dynamic cavities. Importantly, the tool we discuss here allows the user to analyze the time-dependent changes of the components of the cavity structure. An overview of the cavity dynamics is derived by rendering the dynamic cavities in a single image that gives the cavity surface colored according to its time-dependent dynamics.ConclusionThe Voronoi-based approach used here enables the user to perform accurate computations of the geometry of the internal cavities in biomolecules. For the first time, it is possible to compute dynamic molecular paths that have a user-defined minimum constriction size. To illustrate the usefulness of the tool for understanding protein dynamics, we probe the dynamic structure of internal cavities in the bacteriorhodopsin proton pump.

Probing plasma membrane dynamics at the single-molecule level

The plant plasma membrane is highly dynamic and changes multiple aspects of its organization in response to environmental and internal factors. A detailed understanding of membrane dynamics in living plant cells has remained obscure because of the limited spatial resolution of conventional optical microscopy. Recently, several single-molecule imaging approaches have been developed and used to provide valuable insights into the fundamental biochemical and biophysical properties of the plant plasma membrane, including the organization of membrane microdomains and the dynamics of single-molecule diffusion. Here we review single-molecule imaging methods, including total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS), and super-resolution microscopy, and examine their contributions to recent progress in understanding protein dynamics and membrane organization in living plant cells.

Open questions: two challenges in chemical biology - chemical engineering and the science of diet

A respected scientist was once asked what research project he thought he would be working on in five years. He responded that if he knew the answer, he would be working on it now. As the great New York Yankee baseball catcher Yogi Berra said, “It’s difficult to make predictions, especially about the future.” It is thus with considerable trepidation that I offer a short discussion about two research topics at the chemistry/biology interface that I believe may offer opportunities for major advances: protein chemical engineering and chemoprevention. Understanding protein dynamics, relating structure and function, and deciphering the specific contributions of post-translational modifications has attracted intense effort but has yielded slow progress. Part of the challenge has been the limitation of the genetic code and the natural 20 amino acids. While making recombinant proteins has become relatively routine, the inability to introduce biophysical probes, isotopic labels, unnatural amino acids, and post-translational modifications site-specifically into proteins has been a major challenge historically. Recently developed methods for the chemical manipulation of proteins using semisynthetic strategies and unnatural amino acid mutagenesis promise to be increasingly powerful in the analysis of protein structure and function. Exploitation of inteins, sortase, and chemoselective modification reactions have already been valuable in modifying enzymes and histone proteins for the introduction of a wide range of post-translational modifications and fluorescent probes. Unnatural amino acid mutagenesis by the development of evolved aminoacyl tRNA synthases and nonsense suppression approaches have produced a range of chemically altered proteins. As synthetic biology develops organisms with artificial genomes lacking particular nonsense codons, it should be possible to create cells and animals with a wide range of chemical modifications. Such techniques are likely to be of special utility for membrane proteins, which are often very complicated to study. As these protein chemical methods are adapted to cellular and in vivo experiments, they should be transformative in illuminating the contributions such proteins make to cell signaling and gene regulation. Another emerging area for chemistry/biology interface research is in disease prevention using natural products and their analogs. The search for, and application of, chemical substances in dietary plants has garnered increasing attention among clinical and basic science investigators in recent years. Whereas herbal products have had a rich history as a source of pharmaceuticals in Chinese and Western medicine, the focus on prevention rather than treatment is gaining currency with researchers and the general public. As health care costs rise inexorably, massive late stage expenditures in the last year of life to stave off advanced illness are unsustainable. It is generally recognized that identifying a health problem at an early phase offers the best chance for successful and cost-effective therapeutic intervention. In this light, chemoprevention through dietary adjustment is an attractive public health strategy. However, there has been a justified skepticism surrounding research in this area, which has sometimes lacked rigor and molecular detail. To move this field from the phenomenological to the mechanistic, defining key molecular pathways that are affected by chemoprevention compounds is critical. Such has been elegantly demonstrated with the broccoli compound sulforaphane and its interactions with the Keap1/Nrf2 anti-oxidant response pathway. Dozens of clinical trials are underway for myriad conditions to determine if broccoli extracts rich in sulforaphane can protect against cancer, inflammatory diseases, and photo-irradiation-induced skin damage. Going forward, there are likely to be many molecular mechanisms yet to be discovered that provide opportunities for chemoprevention. There is also much to still be learned in nutritional science and metabolomics that could have a major impact on public health.

Nanoscale protein dynamics: A new frontier for neutron spin echo spectroscopy

Recent studies show that neutron spin echo spectroscopy (NSE) can reveal long-range protein domain motions on nanometer lengthscales and on nanosecond to microsecond timescales. This unique capability of NSE provides new opportunities to understand protein dynamics and functions, such as how binding signals are propagated in a protein to distal sites. Here we review our applications of NSE to the study of nanoscale protein domain motions in a set of cell signaling proteins. We summarize the theoretical framework we have developed, which allows one to interpret the NSE data (Biophys. J. 99, 3473 (2010) and Proc. Natl. Acad. Sci. USA 102, 17646 (2005)). Our theoretical framework uses simple concepts from nonequilibrium statistical mechanics, and does not require elaborate molecular dynamics simulations, complex fits to rotational motion, or elastic network models. It is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate our experimental scheme involving deuterium labeling of a protein domain or a subunit in a protein complex. We show that our selective deuteration scheme can highlight and resolve specific domain dynamics from the abundant global translational and rotational motions in a protein. Our approach thus clears significant hurdles to the application of NSE for the study of protein dynamics in solution.

E. Coli Heptosyltransferase I: Exploring Function and Dynamics to Create Better Inhibitors for GT-B Enzymes

Heptosyltransferase (Hep) enzymes are essential for the formation of bacterial biofilms in Gram negative bacteria, making the Hep enzymes an important target for the development of biofilm inhibitors. Our research is directed toward the detailed characterization of both the chemical mechanism of HepI, as well as the protein dynamics. We have already demonstrated the ability of this enzyme to accept a variety of simplified substrate analogues with catalytic efficiencies as good or better than with the native substrate. GTs of the GT-B structural fold, like HepI, are characterized by having two beta/alpha/beta rossman-like domains connected by a linker region, requiring a conformational change from open to closed states in order for catalysis to occur. Currently our lab is exploring these conformational changes through intrinsic tryptophan fluorescence. Using stopped-flow and steady state fluorescence analysis, in conjunction with mutagenisis, we are attempting to elucidate conformational changes that occur during the catalytic cycle. By combining analyses of the chemical mechanism with an understanding of protein dynamics in this system, this work is anticipated to lead to enhancements in drug discovery for this and other GT enzymes.

Site-Resolved Hydration Dynamics of Staphylococcal Nuclease in Reverse Micelles

Measurements of water dynamics and protein-water interactions are essential to understanding protein folding, structure, function, and dynamics. However, protein-water interactions have historically been difficult to study and have mostly been limited to indirect methods that are unable to measure transient and short-lived interactions. We recently developed a novel method for studying protein-water interactions using NMR spectroscopy by encapsulating proteins of interest in reverse micelles. Appropriate amphiphilic surfactant molecules spontaneously form nanoscale bubbles in the presence of a small volume of water and bulk organic solvent, resulting in reverse micelles with aqueous protein in the interior and organic solvent on the exterior. The removal of bulk water and the effects of nanoconfinement slow protein hydration waters allowing for site-resolved measurement of protein-water interactions and dynamics via the nuclear Overhauser effect. Staphylococcal nuclease (SNase) is an extensively studied 16 kD protein with a large number of mutants that have been well classified using standard biophysical techniques. Here we use a pseudo wild-type hyperstable variant (Δ+PHS) and V66E mutant to study surface protein-water dynamics and overall protein hydration. High resolution NOESY-HSQC and ROESY-HSQCs were collected for SNase encapsulated in reverse micelles. Site-specific ratios of NOE and ROE signal intensity at the water chemical shift describe longevity of interacting waters, and can therefore be mapped to the protein structure to determine areas of slow and fast hydration dynamics. Supported by NSF grant MCB 0842814 and NIH postdoctoral fellowship GM087099 to N.V.N.

Time-resolved crystallography for protein structure: the case of heme proteins

Time-resolved Laue diffraction is an elegant approach by which polychromatic X-ray pulses are used in combination with rapid laser triggering of light-sensitive samples so as to make a movie of protein reaction dynamics in real time. The current state-of-the-art with protein Laue diffraction has been time-resolved studies of light-induced conformational changes in myoglobin-CO with 150 ps resolution. Myoglobin has traversed the whole history of pump–probe Laue diffraction, yielding a massive amount of data that have provided considerable insight into the understanding of protein dynamics. A picture of factors governing myoglobin dynamics following ligand dissociation has been drawn from data arising from wild-type and mutant proteins. Other heme proteins have been studied using this approach: Scapharca inequivalvis dimeric hemoglobin, the FixL PAS domain and the photosynthetic reaction center complex of Blastochloris viridis.

Simple Theory for Salt Effects on the Solubility of Amide

Elucidating the interactions of cosolvents and cosolutes, for example, urea and inorganic salts, with proteins plays a very important role in understanding protein structure formation, solubility, and dynamics. In a recent study, we rationalized the experimentally observed salt effects on water/air surface tension and showed the potential importance of cation/anion association/cooperativity. In this paper, we focus on salt effects on the solvation of simple model compounds such as peptides and benzene, hoping to obtain a more general and simple understanding of the Hofmeister series. We show here that preferred cation binding to the carbonyl and anion to the apolar surface of model polypeptides can help explain the experimentally observed salt effects on polypeptide activity coefficient in water. The effects of ions on the solvation of amide group can be both direct and indirect, which together attribute to an effective change of the proton donor/acceptor equilibrium in aqueous solutions. We show that such an argument can be used to understand not only the salt effects on solubility of amides but also why some organic compounds are protein secondary structure denaturants whereas others are protectants.

Proteins on the move: insights gained from fluorescent protein technologies

Proteins are always on the move, and this may occur through diffusion or active transport. The realization that the regulation of signal transduction is highly dynamic in space and time has stimulated intense interest in the movement of proteins. Over the past decade, numerous new technologies using fluorescent proteins have been developed, allowing us to observe the spatiotemporal dynamics of proteins in living cells. These technologies have greatly advanced our understanding of protein dynamics, including protein movement and protein interactions.

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein

Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, υ(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of υ(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ΔG(‡) = 71 ± 5 kJ/mol and an exponential prefactor υ(‡) ∼4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (∼6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (∼10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.

Introducing Bioorthogonal Functionalities into Proteins in Living Cells

Proteins are the workhorses of the cell, playing crucial roles in virtually every biological process. The revolutionary ability to visualize and monitor proteins in living systems, which is largely the result of the development of green fluorescence protein (GFP) and its derivatives, has dramatically expanded our understanding of protein dynamics and function. Still, GFPs are ill suited in many circumstances; one major drawback is their relatively large size, which can significantly perturb the functions of the native proteins to which they are fused. To bridge this gap, scientists working at the chemistry-biology interface have developed methods to install bioorthogonal functional groups into proteins in living cells. The bioorthogonal group is, by definition, a non-native and nonperturbing chemical group. But more importantly, the installed bioorthogonal handle is able to react with a probe bearing a complementary functionality in a highly selective fashion and with the cell operating in its physiological state. Although extensive efforts have been directed toward the development of bioorthogonal chemical reactions, introducing chemical functionalities into proteins in living systems remains an ongoing challenge. In this Account, we survey recent progress in this area, focusing on a genetic code expansion approach. In nature, a cell uses posttranslational modifications to append the necessary functional groups into proteins that are beyond those contained in the canonical 20 amino acids. Taking lessons from nature, scientists have chosen or engineered certain enzymes to modify target proteins with chemical handles. Alternatively, one can use the cell's translational machinery to genetically encode bioorthogonal functionalities, typically in the form of unnatural amino acids (UAAs), into proteins; this can be done in a residue-specific or a site-specific manner. For studying protein dynamics and function in living cells, site-specific modification by means of genetic code expansion is usually favored. A variety of UAAs bearing bioorthogonal groups as well as other functionalities have been genetically encoded into proteins of interest. Although this approach is well established in bacteria, tagging proteins in mammalian cells is challenging. A facile pyrrolysine-based system, which might potentially become the "one-stop shop" for protein modification in both prokaryotic and eukaryotic cells, has recently emerged. This technology can effectively introduce a series of bioorthogonal handles into proteins in mammalian cells for subsequent chemical conjugation with small-molecule probes. Moreover, the method may provide more precise protein labeling than GFP tagging. These advancements build the foundation for studying more complex cellular processes, such as the dynamics of important receptors on living mammalian cell surfaces.

Predictive Power of Molecular Dynamics Receptor Structures in Virtual Screening

Molecular dynamics (MD) simulation is a well-established method for understanding protein dynamics. Conformations from unrestrained MD simulations have yet to be assessed for blind virtual screening (VS) by docking. This study presents a critical analysis of the predictive power of MD snapshots to this regard, evaluating two well-characterized systems of varying flexibility in ligand-bound and unbound configurations. Results from such VS predictions are discussed with respect to experimentally determined structures. In all cases, MD simulations provide snapshots that improve VS predictive power over known crystal structures, possibly due to sampling more relevant receptor conformations. Additionally, MD can move conformations previously not amenable to docking into the predictive range.