Annual Review of Biophysics

Recent advances in cryo-electron microscopy have marked only the beginning of the potential of this technique. To bring structure into cell biology, the modality of cryo-electron tomography has fast developed into a bona fide in situ structural biology technique where structures are determined in their native environment, the cell. Nearly every step of the cryo-focused ion beam-assisted electron tomography (cryo-FIB-ET) workflow has been improved upon in the past decade, since the first windows were carved into cells, unveiling macromolecular networks in near-native conditions...

Single particle cryo-electron microscopy (cryo-EM) has matured into a robust method for the determination of biological macromolecule structures in the past decade, complementing X-ray crystallography and nuclear magnetic resonance. Constant methodological improvements in both cryo-EM hardware and image processing software continue to contribute to an exponential growth in the number of structures solved annually. In this review, we provide a historical view of the many steps that were required to make cryo-EM a successful method for the determination of high-resolution protein complex structures...

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i...

No abstract text is available yet for this article.

Mitochondria are involved in multiple cellular tasks, such as ATP synthesis, metabolism, metabolite and ion transport, regulation of apoptosis, inflammation, signaling, and inheritance of mitochondrial DNA. The majority of the correct functioning of mitochondria is based on the large electrochemical proton gradient, whose component, the inner mitochondrial membrane potential, is strictly controlled by ion transport through mitochondrial membranes. Consequently, mitochondrial function is critically dependent on ion homeostasis, the disturbance of which leads to abnormal cell functions...

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data...

Proteins guide the flows of information, energy, and matter that make life possible by accelerating transport and chemical reactions, by allosterically modulating these reactions, and by forming dynamic supramolecular assemblies. In these roles, conformational change underlies functional transitions. Time-resolved X-ray diffraction methods characterize these transitions either by directly triggering sequences of functionally important motions or, more broadly, by capturing the motions of which proteins are capable...

Rhodopsin is the photoreceptor in human rod cells responsible for dim-light vision. The visual receptors are part of the large superfamily of G protein-coupled receptors (GPCRs) that mediate signal transduction in response to diverse diffusible ligands. The high level of sequence conservation within the transmembrane helices of the visual receptors and the family A GPCRs has long been considered evidence for a common pathway for signal transduction. I review recent studies that reveal a comprehensive mechanism for how light absorption by the retinylidene chromophore drives rhodopsin activation and highlight those features of the mechanism that are conserved across the ligand-activated GPCRs...

Efforts to combine theory and experiment to advance our knowledge of molecular processes relevant to biophysics have been considerably enhanced by the contribution of statistical-mechanics simulations. Key to the understanding of such molecular processes is the underlying free-energy change. Being able to accurately predict this change from first principles represents an appealing prospect. Over the past decades, the synergy between steadily growing computational resources and unrelenting methodological developments has brought free-energy calculations into the arsenal of tools commonly utilized to tackle important questions that experiment alone has left unresolved...

Temperature impacts biological systems across all length and timescales. Cells and the enzymes that comprise them respond to temperature fluctuations on short timescales, and temperature can affect protein folding, the molecular composition of cells, and volume expansion. Entire ecosystems exhibit temperature-dependent behaviors, and global warming threatens to disrupt thermal homeostasis in microbes that are important for human and planetary health. Intriguingly, the growth rate of most species follows the Arrhenius law of equilibrium thermodynamics, with an activation energy similar to that of individual enzymes but with maximal growth rates and over temperature ranges that are species specific...

Cells are crowded, but proteins are almost always studied in dilute aqueous buffer. We review the experimental evidence that crowding affects the equilibrium thermodynamics of protein stability and protein association and discuss the theories employed to explain these observations. In doing so, we highlight differences between synthetic polymers and biologically relevant crowders. Theories based on hard-core interactions predict only crowding-induced entropic stabilization. However, experiment-based efforts conducted under physiologically relevant conditions show that crowding can destabilize proteins and their complexes...

Major recent advances and previous data have led to a plausible model of how key proteins mediate neurotransmitter release. In this model, the soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin form tight complexes that bring the membranes together and are crucial for membrane fusion. NSF and SNAPs disassemble SNARE complexes and ensure that fusion occurs through an exquisitely regulated pathway that starts with Munc18-1 bound to a closed conformation of syntaxin-1...

Some oxidoreductase enzymes use redox-active tyrosine, tryptophan, cysteine, and/or glycine residues as one-electron, high-potential redox (radical) cofactors. Amino-acid radical cofactors typically perform one of four tasks-they work in concert with a metallocofactor to carry out a multielectron redox process, serve as storage sites for oxidizing equivalents, activate the substrate molecules, or move oxidizing equivalents over long distances. It is challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single-amino-acid residue...

In stark contrast to foldable proteins with a unique folded state, intrinsically disordered proteins and regions (IDPs) persist in perpetually disordered ensembles. Yet an IDP ensemble has conformational features-even when averaged-that are specific to its sequence. In fact, subtle changes in an IDP sequence can modulate its conformational features and its function. Recent advances in theoretical physics reveal a set of elegant mathematical expressions that describe the intricate relationships among IDP sequences, their ensemble conformations, and the regulation of their biological functions...

Super-resolution microscopy techniques, and specifically single-molecule localization microscopy (SMLM), are approaching nanometer resolution inside cells and thus have great potential to complement structural biology techniques such as electron microscopy for structural cell biology. In this review, we introduce the different flavors of super-resolution microscopy, with a special emphasis on SMLM and MINFLUX (minimal photon flux). We summarize recent technical developments that pushed these localization-based techniques to structural scales and review the experimental conditions that are key to obtaining data of the highest quality...

Embryonic development hinges on effective coordination of molecular events across space and time. Waves have recently emerged as constituting an ubiquitous mechanism that ensures rapid spreading of regulatory signals across embryos, as well as reliable control of their patterning, namely, for the emergence of body plan structures. In this article, we review a selection of recent quantitative work on signaling waves and present an overview of the theory of waves. Our aim is to provide a succinct yet comprehensive guiding reference for the theoretical frameworks by which signaling waves can arise in embryos...

The advent of biotechnology has enabled metabolic engineers to assemble heterologous pathways in cells to produce a variety of products of industrial relevance, often in a sustainable way. However, many pathways face challenges of low product yield. These pathways often suffer from issues that are difficult to optimize, such as low pathway flux and off-target pathway consumption of intermediates. These issues are exacerbated by the need to balance pathway flux with the health of the cell, particularly when a toxic intermediate builds up...

The author first describes his childhood in the South and the ways in which it fostered the values he has espoused throughout his life, his development of a keen fascination with science, and the influences that supported his progress toward higher education. His experiences in ROTC as a student, followed by two years in the US Army during the Vietnam War, honed his leadership skills. The bulk of the autobiography is a chronological journey through his scientific career, beginning with arrival at the University of California, Irvine in 1972, with an emphasis on the postdoctoral students and colleagues who have contributed substantially to each phase of his lab's progress...

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods have become indispensable tools for the study of biomolecules. In this article, we briefly review the basic methodological details of QM/MM approaches and discuss their applications to various energy transduction problems in biomolecular machines, such as long-range proton transports, fast electron transfers, and mechanochemical coupling. We highlight the particular importance for these applications of balancing computational efficiency and accuracy...

An ideal biosensor material at room temperature, with an extremely large surface area per unit mass combined with the possibility of harnessing quantum mechanical attributes, would be comprised of graphene and other two-dimensional (2D) materials. The sensing of a variety of sizes and types of biomolecules involves modulation of the electrical charge density of (current through) the 2D material and manifests through specific components of the capacitance (resistance). While sensitive detection at the single-molecule level, i...