Quarterly Reviews of Biophysics最新文献

Throughout all the domains of life, and even among the co-existing viruses, RNA molecules play key roles in regulating the rates, duration, and intensity of the expression of genetic information. RNA acts at many different levels in playing these roles. Trans-acting regulatory RNAs can modulate the lifetime and translational efficiency of transcripts with which they pair to achieve speedy and highly specific recognition using only a few components. Cis-acting recognition elements, covalent modifications, and changes to the termini of RNA molecules encode signals that impact transcript lifetime, translation efficiency, and other functional aspects. RNA can provide an allosteric function to signal state changes through the binding of small ligands or interactions with other macromolecules. In either cis or trans, RNA can act in conjunction with multi-enzyme assemblies that function in RNA turnover, processing and surveillance for faulty transcripts. These enzymatic machineries have likely evolved independently in diverse life forms but nonetheless share analogous functional roles, implicating the biological importance of cooperative assemblies to meet the exact demands of RNA metabolism. Underpinning all the RNA-mediated processes are two key aspects: specificity, which avoids misrecognition, and speedy action, which confers timely responses to signals. How these processes work and how aberrant RNA species are recognised and responded to by the degradative machines are intriguing puzzles. We review the biophysical basis for these processes. Kinetics of assembly and multivalency of interacting components provide windows of opportunity for recognition and action that are required for the key regulatory events. The thermodynamic irreversibility of RNA-mediated regulation is one emergent feature of biological systems that may help to account for the apparent specificity and optimal rates.

All biochemical reactions directly involve structural changes that may occur over a very wide range of timescales from femtoseconds to seconds. Understanding the mechanism of action thus requires determination of both the static structures of the macromolecule involved and short-lived intermediates between reactant and product. This requires either freeze-trapping of intermediates, for example by cryo-electron microscopy, or direct determination of structures in active systems at near-physiological temperature by time-resolved X-ray crystallography. Storage ring X-ray sources effectively cover the time range down to around 100 ps that reveal tertiary and quaternary structural changes in proteins. The briefer pulses emitted by hard X-ray free electron laser sources extend that range to femtoseconds, which covers critical chemical reactions such as electron transfer, isomerization, breaking of covalent bonds, and ultrafast structural changes in light-sensitive protein chromophores and their protein environment. These reactions are exemplified by the time-resolved X-ray studies by two groups of the FAD-based DNA repair enzyme, DNA photolyase, over the time range from 1 ps to 100 μs.

Allostery describes the ability of biological macromolecules to transmit signals spatially through the molecule from an allosteric site – a site that is distinct from orthosteric binding sites of primary, endogenous ligands – to the functional or active site. This review starts with a historical overview and a description of the classical example of allostery – hemoglobin – and other well-known examples (aspartate transcarbamoylase, Lac repressor, kinases, G-protein-coupled receptors, adenosine triphosphate synthase, and chaperonin). We then discuss fringe examples of allostery, including intrinsically disordered proteins and inter-enzyme allostery, and the influence of dynamics, entropy, and conformational ensembles and landscapes on allosteric mechanisms, to capture the essence of the field. Thereafter, we give an overview over central methods for investigating molecular mechanisms, covering experimental techniques as well as simulations and artificial intelligence (AI)-based methods. We conclude with a review of allostery-based drug discovery, with its challenges and opportunities: with the recent advent of AI-based methods, allosteric compounds are set to revolutionize drug discovery and medical treatments.

Prokaryotic microorganisms, comprising Bacteria and Archaea, exhibit a fascinating diversity of cell envelope structures reflecting their adaptations that contribute to their resilience and survival in diverse environments. Among these adaptations, surface layers (S-layers) composed of monomolecular protein or glycoprotein lattices are one of the most observed envelope components. They are the most abundant cellular proteins and represent the simplest biological membranes that have developed during evolution. S-layers provide organisms with a great variety of selective advantages, including acting as an antifouling layer, protective coating, molecular sieve, ion trap, structure involved in cell and molecular adhesion, surface recognition and virulence factor for pathogens. In Archaea that possess S-layers as the exclusive cell wall component, the (glyco)protein lattices function as a cell shape-determining/maintaining scaffold. The wealth of information available on the structure, chemistry, genetics and in vivo and in vitro morphogenesis has revealed a broad application potential for S-layers as patterning elements in a molecular construction kit for bio- and nanotechnology, synthetic biology, biomimetics, biomedicine and diagnostics. In this review, we try to describe the scientifically exciting early days of S-layer research with a special focus on the 'Vienna-S-Layer-Group'. Our presentation is intended to illustrate how our curiosity and joy of discovery motivated us to explore this new structure and to make the scientific community aware of its relevance in the realm of prokaryotes, and moreover, how we developed concepts for exploiting this unique self-assembly structure. We hope that our presentation, with its many personal notes, is also of interest from the perspective of the history of S-layer research.

The membrane potential is a critical aspect of cellular physiology, essential for maintaining homeostasis, facilitating signal transduction, and driving various cellular processes. While the resting membrane potential (RMP) represents a key physiological parameter, membrane potential fluctuations, such as depolarization and hyperpolarization, are equally vital in understanding dynamic cellular behavior. Traditional techniques, such as microelectrodes and patch-clamp methods, offer valuable insights but are invasive and less suited for high-throughput applications. Recent advances in voltage indicators, including fast and slow dyes, and novel imaging modalities such as second harmonic generation (SHG) and photoacoustic imaging, enable noninvasive, high-resolution measurement of both RMP and membrane potential dynamics. This review explores the mechanisms, development, and applications of these tools, emphasizing their transformative potential in neuroscience and cellular electrophysiology research.

The GABA type A receptor (GABA_AR) belongs to the family of pentameric ligand-gated ion channels and plays a key role in inhibition in adult mammalian brains. Dysfunction of this macromolecule may lead to epilepsy, anxiety disorders, autism, depression, and schizophrenia. GABA_AR is also a target for multiple physiologically and clinically relevant modulators, such as benzodiazepines (BDZs), general anesthetics, and neurosteroids. The first GABA_AR structure appeared in 2014, but the past years have brought a particularly abundant surge in structural data for these receptors with various ligands and modulators. Although the open conformation remains elusive, this novel information has pushed the structure-function studies to an unprecedented level. Electrophysiology, mutagenesis, photolabeling, and in silico simulations, guided by novel structural information, shed new light on the molecular mechanisms of receptor functioning. The main goal of this review is to present the current knowledge of GABA_AR functional and structural properties. The review begins with an outline of the functional and structural studies of GABA_AR, accompanied by some methodological considerations, especially biophysical methods, enabling the reader to follow how major breakthroughs in characterizing GABA_AR features have been achieved. The main section provides a comprehensive analysis of the functional significance of specific structural elements in GABA_ARs. We additionally summarize the current knowledge on the binding sites for major GABA_AR modulators, referring to the molecular underpinnings of their action. The final chapter of the review moves beyond examining GABA_AR as an isolated macromolecule and describes the interactions of the receptor with other proteins in a broader context of inhibitory plasticity. In the final section, we propose a general conclusion that agonist binding to the orthosteric binding sites appears to rely on local interactions, whereas conformational transitions of bound macromolecule (gating) and allosteric modulation seem to reflect more global phenomena involving vast portions of the macromolecule.

The 'Viroporin' family comprises a number of mostly small-sized, integral membrane proteins encoded by animal and plant viruses. Despite their sequence and structural diversity, viroporins share a common functional trend: their capacity to assemble transmembrane channels during the replication cycle of the virus. Their selectivity spectrum ranges from low-pH-activated, unidirectional proton transporters, to size-limited permeating pores allowing passive diffusion of metabolites. Through mechanisms not fully understood, expression of viroporins facilitates virion assembly/release from infected cells, and subverts the cell physiology, contributing to cytopathogenicity. Compounds that interact with viroporins and interfere with their membrane-permeabilizing activity in vitro, are known to inhibit virus production. Moreover, viroporin-defective viruses comprise a source of live attenuated vaccines that prevent infection by notorious human and livestock pathogens. This review dives into the origin and evolution of the viroporin concept, summarizes some of the methodologies used to characterize the structure-function relationships of these important virulence factors, and attempts to classify them on biophysical grounds attending to their mechanisms of ion/solute transport across membranes.

The electron cryomicroscopy (cryo-EM) resolution revolution has shifted structural biology into a new era, enabling the routine structure determination of macromolecular complexes at an unprecedented rate. Building on this, electron cryotomography (cryo-ET) offers the potential to visualise the native three-dimensional organisation of biological specimens, from cells to tissues and even entire organisms. Despite this huge potential, the study of tissue-like multicellular specimens via cryo-ET still presents numerous challenges, wherein many steps in the workflow are being developed or in urgent need of improvement. In this review, we outline the latest techniques currently utilised for in situ imaging of multicellular specimens, while clearly enumerating their associated limitations. We consider every step in typical workflows employed by various laboratories, including sample preparation, data collection and image analysis, to highlight recent progress and showcase prominent success stories. By considering the entire structural biology workflow for multicellular specimens, we identify which future exciting developments in hardware and software could enable comprehensive in situ structural biology investigations, bringing forth a new age of discovery in molecular structural and cell biology.

Yeast frataxin (Yfh1) is a small natural protein from yeast that has the unusual property of undergoing cold denaturation at temperatures above the freezing point of water when under conditions of low ionic strength. This peculiarity, together with remarkable resilience, allows the determination, for the whole protein as well as for individual residues, of the stability curve, that is the temperature dependence of the free energy difference between the unfolded and folded forms. The ease of measuring stability curves without the need to add denaturants or introduce ad hoc destabilizing mutations makes this protein an ideal 'tool' for investigating the influence of many environmental factors on protein stability. The present review aims at recapitulating all the open questions that Yfh1 has helped to address, including understanding the differences and commonalities of the cold, heat and pressure unfolded states. This protein thus offers a unique tool for studying aspects of protein stability so far been considered difficult to assess and provides important guidelines that could allow the identification of other similar systems.

Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization microscopy by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical and biochemical processes, including the organization of actin networks, movement of molecular motors, conformations of DNA strands, growth and remodeling of amyloid aggregates, and composition changes within lipid membranes. In this review, we discuss recent innovations in SMOLM and cover three key aspects: (1) biophysical insights enabled by labeling strategies that endow fluorescent probes to bind to targets with orientation specificity; (2) advanced imaging techniques that leverage the physics of light-matter interactions and estimation theory to encode orientation information with high fidelity into microscope images; and (3) computational methods that ensure accurate and precise data analysis and interpretation, even in the presence of severe shot noise. Additionally, we compare labeling approaches, imaging hardware, and publicly available analysis software to aid the community in choosing the best SMOLM implementation for their specific biophysical application. Finally, we highlight future directions for SMOLM, such as the development of probes with improved photostability and specificity, the design of “smart” adaptive hardware, and the use of advanced computational approaches to handle large, complex datasets. This review underscores the significant current and potential impact of SMOLM in deepening our understanding of molecular dynamics, paving the way for future breakthroughs in the fields of biophysics, biochemistry, and materials science.