Annual Review of Biophysics最新文献

The spatiotemporal organization of intracellular compartments is fundamental to cellular function and to the understanding of the processes underpinning health and disease. Fluorescence microscopy offers a powerful means to observe organelle morphology and dynamics with high specificity. However, no single technique can capture the wide range of relevant spatiotemporal scales due to inherent trade-offs in resolution, speed, field of view, signal-to-noise ratio, and sample viability. In this review, we describe recent developments across high-resolution fluorescence microscopy techniques and associated computational methods, critically evaluating how these advances address key limitations. Through biological examples of organelle dynamics at different scales, we illustrate the impact of these technologies on our understanding of cellular organization and function. Finally, we discuss the current challenges and outline future directions for imaging-based research, highlighting the potential for further innovations to deepen insights into dynamic subcellular processes.

Single-molecule techniques have transformed biological research by enabling direct observation and manipulation of individual molecules. These methods overcome ensemble averaging inherent in bulk measurements and facilitate studies under physiological stresses and out-of-equilibrium conditions. They have provided valuable insights into diverse biological processes, from stepping mechanisms of molecular motors to mechanical properties of biomolecules to the dynamic strength of intermolecular bonds. Advances in multiplexed and high-throughput single-molecule force spectroscopy methods are improving throughput, capabilities, and accessibility. In this review, we detail the evolution of multiplexed force spectroscopy technologies, highlighting key advances in instrumentation, molecular engineering, and analytical techniques. We discuss diverse applications spanning molecular biophysics, biomolecular sensing, proteomics, and cellular mechanobiology. Finally, we explore ongoing challenges and future opportunities and highlight how the impact of multiplexed single-molecule force spectroscopy can continue to grow through further developments in novel instrumentation, chemical tools, and innovative applications.

Bacteria are unicellular organisms that typically lack membrane-bound organelles. Nevertheless, they are not merely "bags of enzymes" and instead use alternate mechanisms to organize their components in space and time. Biomolecular condensates are a newly described class of membraneless compartment that organizes cellular functions in bacteria. In this review, we cover key biophysical features of bacterial cells and discuss how their finite size and crowded interior may affect condensate nucleation and stability. Next, we describe three examples of endogenous condensates, highlighting the molecular components driving their formation and the functional roles they may play in cells. Finally, we provide an overview of current and prospective tools to study and manipulate both endogenous and synthetic condensates alike. Overall, bacterial condensates present a fascinating system to explore open questions that span the disciplines of biophysics, molecular and cell biology, and bioengineering.

The mitochondrial permeability transition (PT) is a Ca2+-dependent permeability increase of the inner mitochondrial membrane mediated by opening of a high-conductance channel, the PT pore. Its molecular nature has been the subject of intense research and the source of controversies, but a considerable consensus has been reached that the PT originates from specific conformations of the F_OF₁-ATP synthase and of the adenine nucleotide translocator. The ATP synthase forms high-conductance channels in mammals and yeast but not in the anoxia- and salt-tolerant brine shrimp Artemia franciscana, which is refractory to the PT; it forms low-conductance and Ca2+-selective channels in Drosophila melanogaster, which undergoes a process of Ca2+-induced Ca2+ release but not a PT. The structural definition of ATP synthases from several species may allow for some inferences to be made about the mechanism of channel formation, or lack thereof, and provides a testable framework for future research.

Histones are small basic proteins that form the proteinaceous core of the nucleosome, the repeating building block of chromatin in all eukaryotes. Long thought to be exclusive to eukaryotes, histones are now increasingly appreciated for their roles in organizing genomes across all domains of life, namely in archaea, bacteria, and even viruses. We survey recent advances in our understanding of the imaginative uses of histones in disparate biological entities, ranging from nucleosome-like metastable particles in giant viruses to slinky-like hypernucleosomes in archaea to bacterial histones that bind DNA in decidedly unorthodox ways. Across these different contexts, we examine how DNA compaction and conformation emanate from evolutionarily conserved aspects of histone structure, including how the oligomeric states of histones dictate their capacity to contort DNA in different conformations. It appears that relatively small tweaks to the amino acid sequences of histones can result in structural and functional variations in DNA binding. As such, nucleosomes in eukaryotes sample only a narrow range of possible structures.

While the role of water in soluble protein structure and function is well-established, the analogous role of lipids as a solvent for membrane proteins is less understood. Bacterial membranes exhibit extraordinary lipid diversity, with Escherichia coli synthesizing over 1,800 distinct glycerophospholipids. This lipid diversity gives rise to bulk membrane properties and specific lipid-lipid and lipid-protein interactions that directly affect α-IMP folding, assembly, and function. In this review, we use the same thermodynamic framework for understanding the solvation of soluble proteins to examine bacterial α-helical integral membrane protein (α-IMP) interactions with chemically diverse lipid environments. We propose that preferential solvent interactions were essential evolutionary drivers that enabled lipids to evolve as protein cofactors and substrates, with lipid chemical diversity creating unique evolutionary pressures distinct from those of aqueous systems.

This review focuses on the use of high-pressure nuclear magnetic resonance (HP NMR) to map local protein stability and conformational landscapes, with an emphasis on the population and characteristics of protein excited states. Section 1 discusses the volumetric properties of proteins in the pressure-temperature plane, highlighting the underlying mechanisms of pressure effects, the magnitude of the volume changes upon unfolding, their temperature dependence, and the nature of the unfolded state at high pressure. In Section 2, NMR-detected, pressure-induced equilibrium unfolding of proteins is discussed. Section 3 covers how HP NMR can reveal the complexity of protein conformational landscapes, the population of excited states, and the local stability distribution across the structure. Studies exploring the sequence determinants of these landscapes are presented. Of particular interest are the sequence determinants that define the excited states implicated in functional dynamics, one of the most important unresolved issues in protein science.

Globular proteins are expected to assume folds with fixed secondary structures, α-helices and β-sheets. Fold-switching proteins challenge this expectation by remodeling their secondary and/or tertiary structures in response to cellular stimuli. Though these shape-shifting proteins were once thought to be haphazard evolutionary by-products with little intrinsic biological relevance, recent work has shown that evolution has selected for their dual-folding behavior, which plays critical roles in biological processes across all kingdoms of life. The widening scope of fold switching draws attention to the ways it challenges conventional wisdom, raising fundamental unanswered questions about protein structure, biophysics, and evolution. Here we discuss the progress being made to answer these questions and suggest future directions for the field.

Evolutionary processes have transformed simple cellular life into a great diversity of forms, ranging from the ubiquitous eukaryotic cell design to the more specific cellular forms of spirochetes, cyanobacteria, ciliates, heliozoans, amoeba, and many others. The cellular traits that constitute these forms require an evolutionary explanation. Ultimately, the persistence of a cellular trait depends on its net contribution to fitness, a quantitative measure. Independent of any positive effects, a cellular trait exhibits a baseline energetic cost that needs to be accounted for when quantitatively examining its net fitness effect. Here, we explore how the energetic burden introduced by a cellular trait quantitatively affects cellular fitness, describe methods for determining cell energy budgets, summarize the costs of cellular traits across the tree of life, and examine how the fitness impacts of these energetic costs compare to other evolutionary forces and trait benefits.

A complete understanding of protein function and dynamics requires the characterization of the multiple thermodynamic states, including the denatured state ensemble (DSE). Whereas residual structure in the DSE (as well as in partially folded states) is pertinent in many biological contexts, here we are interested in how such structure affects protein thermodynamics. We examine issues related to chain collapse in light of new developments, focusing on potential complications arising from differences in the DSE's properties under various conditions. Despite some variability in the degree of collapse and structure in the DSE, stability measurements are remarkably consistent between two standard methods, calorimetry and chemical denaturation, as well as with hydrogen-deuterium exchange. This robustness is due in part to the DSEs obtained with different perturbations being thermodynamically equivalent and hence able to serve as a common reference state. An examination of the properties of the DSE points to it as being a highly expanded ensemble with minimal amounts of stable hydrogen bonded structure. These two features are likely to be critical in the broad and successful application of thermodynamics to protein folding. Our review concludes with a discussion of the impact of these findings on folding mechanisms and pathways.