Search

Proteins play a key role in living organisms. The study of proteins and their dynamics provides information about their functionality, catalysis and potential alterations towards pathological diseases. Several techniques are used for studying protein dynamics, e.g., magnetic resonance, fluorescence imaging techniques, mid-infrared spectroscopy and biochemical assays. Spectroscopic analysis, based on the use of terahertz (THz) radiation with frequencies between 0.1 and 15 THz (3–500 cm−1), was underestimated by the biochemical community. In recent years, however, the potential of THz spectroscopy in the analysis of both simple structures, such as polypeptide molecules, and complex structures, such as protein complexes, has been demonstrated. The THz absorption spectrum provides some information on proteins: for small molecules the THz spectrum is dominated by individual modes related to the presence of hydrogen bonds. For peptides, the spectral information concerns their secondary structure, while for complex proteins such as globular proteins and viral glycoproteins, spectra also provide information on collective modes. In this short review, we discuss the results obtained by THz spectroscopy in the protein dynamics investigations. In particular, we will illustrate advantages and applications of THz spectroscopy, pointing out the complementary information it may provide.

Differences in ligand-induced protein dynamics extracted as a single feature from a deep learning-based analysis of MD simulations correlate with ligand binding affinity.

Direct observation of protein structural changes during ion transport in ion pumps provides valuable insights into the mechanism of ion transport. In this study, we examined structural changes in the light-driven sodium ion (Na+) pump rhodopsin KR2 on the sub-millisecond time scale, corresponding with the uptake and release of Na+. We compared the ion-pumping activities and transient absorption spectra of WT and the W215F mutant, in which the Trp215 residue located near the retinal chromophore on the cytoplasmic side was replaced with a Phe residue. Our findings indicated that atomic contacts between the bulky side chain of Trp215 and the C20 methyl group of the retinal chromophore promote relaxation of the retinal chromophore from the 13-cis to the all-trans form. Since Trp215 is conserved in other ion-pumping rhodopsins, the present results suggest that this residue commonly acts as a mechanical transducer. In addition, we measured time-resolved ultraviolet resonance Raman (UVRR) spectra to show that the environment around Trp215 becomes less hydrophobic at 1 ms after photoirradiation and recovers to the unphotolyzed state with a time constant of around 10 ms. These time scales correspond to Na+ uptake and release, suggesting evolution of a transient ion channel at the cytoplasmic side for Na+ uptake, consistent with the alternating-access model of ion pumps. The time-resolved UVRR technique has potential for application to other ion-pumping rhodopsins and could provide further insights into the mechanism of ion transport.

Research on learning and memory formation at the level of neural networks, as well as at the molecular level, is challenging due to the immense complexity of the brain. The zebrafish as a genetically tractable model organism can overcome many of the current challenges of studying molecular mechanisms of learning and memory formation. Zebrafish have a translucent, smaller and more accessible brain than that of mammals, allowing imaging of the entire brain during behavioral manipulations. Recent years have seen an extensive increase in published brain research describing the use of zebrafish for the study of learning and memory. Nevertheless, due to the complexity of the brain comprising many neural cell types that are difficult to isolate, it has been difficult to elucidate neural networks and molecular mechanisms involved in memory formation in an unbiased manner, even in zebrafish larvae. Therefore, data regarding the identity, location, and intensity of nascent proteins during memory formation is still sparse and our understanding of the molecular networks remains limited, indicating a need for new techniques. Here, we review recent progress in establishing learning paradigms for zebrafish and the development of methods to elucidate neural and molecular networks of learning. We describe various types of learning and highlight directions for future studies, focusing on molecular mechanisms of long-term memory formation and promising state-of-the-art techniques such as cell-type-specific metabolic labeling.

Surface morphology, in addition to hydrophobic and electrostatic effects, can alter how proteins interact with solid surfaces. Understanding the heterogeneous dynamics of protein adsorption on surfaces with varying roughness is experimentally challenging. In this work, we use single-molecule fluorescence microscopy to study the adsorption of α-lactalbumin protein on the glass substrate covered with a self-assembled monolayer (SAM) with varying surface concentrations. Two distinct interaction mechanisms are observed: localized adsorption/desorption and continuous-time random walk (CTRW). We investigate the origin of these two populations by simultaneous single-molecule imaging of substrates with both bare glass and SAM-covered regions. SAM-covered areas of substrates are found to promote CTRW, whereas glass surfaces promote localized motion. Contact angle measurements and atomic force microscopy imaging show that increasing SAM concentration results in both increasing hydrophobicity and surface roughness. These properties lead to two opposing effects: increasing hydrophobicity promotes longer protein flights, but increasing surface roughness suppresses protein dynamics resulting in shorter residence times. Our studies suggest that controlling hydrophobicity and roughness, in addition to electrostatics, as independent parameters could provide a means to tune desirable or undesirable protein interactions with surfaces. [ABSTRACT FROM AUTHOR]

Biological life depends on motion, and this manifests itself in proteins that display motion over a formidable range of time scales spanning from femtoseconds vibrations of atoms at enzymatic transition states, all the way to slow domain motions occurring on micro to milliseconds. An outstanding challenge in contemporary biophysics and structural biology is a quantitative understanding of the linkages among protein structure, dynamics, and function. These linkages are becoming increasingly explorable due to conceptual and methodological advances. In this Perspective article, we will point toward future directions of the field of protein dynamics with an emphasis on enzymes. Research questions in the field are becoming increasingly complex such as the mechanistic understanding of high-order interaction networks in allosteric signal propagation through a protein matrix, or the connection between local and collective motions. In analogy to the solution to the “protein folding problem,” we argue that the way forward to understanding these and other important questions lies in the successful integration of experiment and computation, while utilizing the present rapid expansion of sequence and structure space. Looking forward, the future is bright, and we are in a period where we are on the doorstep to, at least in part, comprehend the importance of dynamics for biological function.

Abstract It is well known that today nearly one in six of the world’s population has to deal with neurodegenerative disorders. While a number of medical devices have been developed for the detection, prevention, and treatments of such disorders, some fundamentals of the progression of associated diseases are in urgent need of further clarification. In this paper, we focus on Alzheimer’s disease, where it is believed that the concentration changes in amyloid-beta and tau proteins play a central role in its onset and development. A multiscale model is proposed to analyze the propagation of these concentrations in the brain connectome. In particular, we consider a modified heterodimer model for the protein–protein interactions. Higher toxic concentrations of amyloid-beta and tau proteins destroy the brain cell. We have studied these propagations for the primary and secondary and their mixed tauopathies. We model the damage of a brain cell by the nonlocal contributions of these toxic loads present in the brain cells. With the help of rigorous analysis, we check the stability behaviour of the stationary points corresponding to the homogeneous system. After integrating the brain connectome data into the developed model, we see that the spreading patterns of the toxic concentrations for the whole brain are the same, but their concentrations are different in different regions. Also, the time to propagate the damage in each region of the brain connectome is different.

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1 R178Q , showed higher catalytic activity than wild-type UCHL1 (UCHL1 WT ). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1 R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a k cat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (k inact /K i ) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The protein composition in goat milk undergoes changes throughout the different lactation periods, displaying distinct characteristics that are influenced by the dynamic nature of protein composition and concentration during the transition from colostrum secretion to mature milk. To evaluate the dynamics of whey proteins of Saanen goats during the colostral phase and the first month of lactation, 110 milk samples from 11 healthy mammary halves of seven Saanen goats were selected through a clinical evaluation. Whey was obtained by rennet coagulation of the mammary secretion. The biuret method determined total protein concentration, and their fractions were identified by 12% dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum concentrations of all protein fractions were observed in the first 12 h of lactation, reducing throughout the study. Modification of the protein predominance was also observed. The transition from colostrum secretion to milk occurred 5 or 7 d postpartum.

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases., Competing Interests: Declaration of interests A.J.G., J.-H.C., S.X., J.G., and E.D.P. are employees and shareholders of Biogen., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The current study aims to evaluate Asinex library compounds against Campylobacter jejuni DsbA1 protein, a thiol disulfide oxidoreductase enzyme that plays a major role in the oxidative folding of bacterial virulence proteins, making it a promising anti-viral drug target. By employing several techniques of computer-aided drug design, BDC25697459, BDD33601083, and BDC30129064 were identified with binding energy scores of -8.8 kcal/mol, -8.8 kcal/mol, and -8.3 kcal/mol, respectively. However, the control molecule, tetraethylene glycol, exhibited a binding energy score of -7.0 kcal/mol. The control, BDD33601083, and BDC30129064 were unveiled to bind the same co-crystallized binding site (pocket 1), while BDC25697459 interacted with a new binding pocket (pocket 2) adjacent to the control binding region. The molecular dynamics simulation showed that complexes exhibit stable dynamics without significant global or residue-level fluctuations. The average RMSD values were in the range of 2.07 Å-2.45 Å. Similarly, mean RMSF was recorded between 1.30 and 1.42 Å. The C. jejuni DsbA1 was also observed as compact in the presence of the compounds, showing a mean RoG value in the range of 16.42 Å-16.55 Å. In terms of MM/PBSA binding energy, the BDC30129064 complex was ranked top with -44.88 ± 4.14 kcal/mol, whereas the positive control molecule exhibited -22.22 ± 3.33 kcal/mol. From a pharmacokinetic perspective, the compounds are suitable candidates for clinical trial investigation. Preliminary computational analysis of these virtual hits indicates that these compounds have a low potential for ADME and toxicity-associated liabilities. In summary, the compounds displayed a high affinity for the C. jejuni DsbA1 protein, indicating potential efficacy that requires further investigation.Communicated by Ramaswamy H. Sarma.

Protein unfolding and aggregation are often correlated with numerous diseases such as Alzheimer's, Parkinson's, Huntington's, and other debilitating neurological disorders. Such adverse events consist of a plethora of competing mechanisms, particularly interactions that control the stability and cooperativity of the process. However, it remains challenging to probe the molecular mechanism of protein dynamics such as aggregation, and monitor them in real-time under physiological conditions. Recently, Raman spectroscopy and its plasmon-enhanced counterparts, such as surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS), have emerged as sensitive analytical tools that have the potential to perform molecular studies of functional groups and are showing significant promise in probing events related to protein aggregation. We summarize the fundamental working principles of Raman, SERS, and TERS as nondestructive, easy-to-perform, and fast tools for probing protein dynamics and aggregation. Finally, we highlight the utility of these techniques for the analysis of vibrational spectra of aggregation of proteins from various sources such as tissues, pathogens, food, biopharmaceuticals, and lastly, biological fouling to retrieve precise chemical information, which can be potentially translated to practical applications and point-of-care (PoC) devices. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > Diagnostic Nanodevices Nanotechnology Approaches to Biology > Nanoscale Systems in Biology., (© 2023 Wiley Periodicals LLC.)

Nuclear magnetic resonance (NMR) is a major method used to study protein structure at atomic resolution. Besides presenting the high-resolution structure, NMR facilitates studies on protein dynamics near physiological conditions that are intimately related to the biological mechanism of the proteins. Unnatural amino acids (UAA) labeling could significantly reduce the complexity of protein NMR spectra. In this review, we briefly summarize the widely used UAA labeling strategies for proteins, including chemical peptide synthesis, residue-specific peptide modification, 19F labeled aromatic amino acids incorporation and genetic code expansion based UAA incorporation. The recent applicants of UAA in characterizing protein structure and dynamics, the limitations and prospects of UAA are also highlighted.

Summary: Single-molecule tracking (SMT) is a powerful approach to quantify the biophysical parameters of protein dynamics in live cells. Here, we describe a protocol for SMT in live cells of the budding yeast Saccharomyces cerevisiae. We detail how to genetically engineer yeast strains for SMT, how to set up image acquisition parameters, and how different software programs can be used to quantify a variety of biophysical parameters such as diffusion coefficient, residence time, bound fraction, jump angles, and target-search parameters.For complete details on the use and execution of this protocol, please refer to Mehta et al. 1 and Ball et al..2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

In biological systems, protein function depends on spatial and temporal changes known as protein dynamics, which can be probed by amide hydrogen/deuterium (H/D) exchange. Here, we present a protocol for determining protein dynamics by Fourier-transform infrared (FT-IR) spectroscopy. We describe steps for protein sample preparation and FT-IR spectra collection. We then detail procedures for spectra analysis. Applications include the effects of protein mutation or protein and metal ion or ligand interactions on the protein H/D exchange rate. For complete details on the use and execution of this protocol, please refer to Yu et al. (2013). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder with a complex pathogenesis. One promising approach to treating AD is simultaneously targeting multiple aspects of the disease using a multi-target drug (MTD). In this study, multi-target drug (MTD) potential of the nutraceutical molecule Queuine was explored using molecular docking and molecular dynamics (MD) simulations with five different protein targets engaged in AD: AChE, beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1), N-methyl-D-aspartate receptor (NMDAR), monoamine oxidase A (MAO-A), and Synapsin III. Queuine revealed significant binding affinities, the docking scores being -10.1, -5.97, -5.63, -8.40, and -10.56 kcal/mol for AChE, BACE-1, NMDAR, MAO-A, and Synapsin III, respectively. MD simulations showed that Queuine formed stable complexes and preserved its stability throughout the simulation, the backbone fluctuations remaining within 2.5 Å specifically in the case of the BACE-1. Elastic network model simulations and principal component analysis were carried out to illustrate the dynamics of the protein systems. Significant hinge-bending and twisting-type motions that may be relevant to function were observed around the dimerization interfaces or binding sites. Structural clustering based on PCA analysis and cross-correlation maps demonstrated that Queuine binding altered the protein dynamics more drastically in the case of highly mobile proteins NMDAR and MAO-A. We propose that the neuroprotective effect of Queuine may stem from its prominent inhibitory action on enzymes BACE-1 and AChE. Our results suggest that Queuine may serve as a promising MTD candidate for the treatment of AD.Communicated by Ramaswamy H. Sarma.

Antiviral innate immunity works as the first line of host defense against viral infection. Pattern recognition receptors (PRRs) and adaptor proteins involved in the innate immune signaling pathways play critical roles in controlling viral infections via the induction of type I interferon and its downstream interferon-stimulated genes. Dynamic changes of adaptor proteins contribute to precise regulation of the activation and shut-off of signaling transduction, though numerous complex processes are involved in achieving dynamic changes to various proteins of the host and viruses. In this review, we will summarize recent progress on the trafficking patterns and conformational transitions of the adaptors that are involved in the antiviral innate immune signaling pathway during viral DNA sensing. Moreover, we aim to dissect the relationships between protein dynamics and DNA-sensing antiviral innate immune responses, which will reveal the underlying mechanisms controlling protein activity and maintaining cell homeostasis. By comprehensively revealing protein dynamics in cytosolic DNA-sensing antiviral innate immune signaling pathways, we will be able to identify potential new targets for the therapies of certain autoimmune diseases.

A significant body of experimental structures of SARS-CoV-2 spike trimers for the BA.1 and BA.2 variants revealed a considerable plasticity of the spike protein and the emergence of druggable binding pockets. Understanding the interplay of conformational dynamics changes induced by the Omicron variants and the identification of cryptic dynamic binding pockets in the S protein is of paramount importance as exploring broad-spectrum antiviral agents to combat the emerging variants is imperative. In the current study, we explore conformational landscapes and characterize the universe of binding pockets in multiple open and closed functional spike states of the BA.1 and BA.2 Omicron variants. By using a combination of atomistic simulations, a dynamics network analysis, and an allostery-guided network screening of binding pockets in the conformational ensembles of the BA.1 and BA.2 spike conformations, we identified all experimentally known allosteric sites and discovered significant variant-specific differences in the distribution of binding sites in the BA.1 and BA.2 trimers. This study provided a structural characterization of the predicted cryptic pockets and captured the experimentally known allosteric sites, revealing the critical role of conformational plasticity in modulating the distribution and cross-talk between functional binding sites. We found that mutational and dynamic changes in the BA.1 variant can induce the remodeling and stabilization of a known druggable pocket in the N-terminal domain, while this pocket is drastically altered and may no longer be available for ligand binding in the BA.2 variant. Our results predicted the experimentally known allosteric site in the receptor-binding domain that remains stable and ranks as the most favorable site in the conformational ensembles of the BA.2 variant but could become fragmented and less probable in BA.1 conformations. We also uncovered several cryptic pockets formed at the inter-domain and inter-protomer interface, including functional regions of the S2 subunit and stem helix region, which are consistent with the known role of pocket residues in modulating conformational transitions and antibody recognition. The results of this study are particularly significant for understanding the dynamic and network features of the universe of available binding pockets in spike proteins, as well as the effects of the Omicron-variant-specific modulation of preferential druggable pockets. The exploration of predicted druggable sites can present a new and previously underappreciated opportunity for therapeutic interventions for Omicron variants through the conformation-selective and variant-specific targeting of functional sites involved in allosteric changes.