Annual Review of Biophysics - Volume 52, 2023

My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of advances in HX measurement, interpretation, and cutting edge biophysical applications. After some thoughts about how life brought me there, I take the opportunity to reflect on our early studies of allosteric structure and energy change in hemoglobin, the still-current protein folding problem, and our most recent forward-looking studies on protein machines.

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. White's fundamental findings played a key role in the development of membrane biophysics, helping establish it as fertile ground for research. A story gradually unfolds that reveals the deeply collaborative and painstakingly executed work necessary for a successful career in science.

The mechanism and the evolution of DNA replication and transcription, the key elements of the central dogma of biology, are fundamentally well explained by the physicochemical complementarity between strands of nucleic acids. However, the determinants that have shaped the third part of the dogma—the process of biological translation and the universal genetic code—remain unclear. We review and seek parallels between different proposals that view the evolution of translation through the prism of weak, noncovalent interactions between biological macromolecules. In particular, we focus on a recent proposal that there exists a hitherto unrecognized complementarity at the heart of biology, that between messenger RNA coding regions and the proteins that they encode, especially if the two are unstructured. Reflecting the idea that the genetic code evolved from intrinsic binding propensities between nucleotides and amino acids, this proposal promises to forge a link between the distant past and the present of biological systems.

Carefully orchestrated opening and closing of ion channels control the diffusion of ions across cell membranes, generating the electrical signals required for fast transmission of information throughout the nervous system. Inactivation is a parsimonious means for channels to restrict ion conduction without the need to remove the activating stimulus. Voltage-gated channel inactivation plays crucial physiological roles, such as controlling action potential duration and firing frequency in neurons. The ball-and-chain moniker applies to a type of inactivation proposed first for sodium channels and later shown to be a universal mechanism. Still, structural evidence for this mechanism remained elusive until recently. We review the ball-and-chain inactivation research starting from its introduction as a crucial component of sodium conductance during electrical signaling in the classical Hodgkin and Huxley studies, through the discovery of its simple intuitive mechanism in potassium channels during the molecular cloning era, to the eventual elucidation of a potassium channel structure in a ball-and-chain inactivated state.

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. The continued emergence of new options to determine free energies has also bred confusion amid the community of users, who may find it difficult to choose the best-suited algorithm to address the problem at hand. In an attempt to clarify the current landscape, this review recounts how the field has been shaped and how the broad gamut of methods available today is rooted in a few foundational principles laid down many years ago.Three examples of molecular processes central to biophysics illustrate where free-energy calculations stand and what are the conceptual and practical obstacles that we must overcome to increase their predictive power.

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. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA–mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

Advances in a scientific discipline are often measured by small, incremental steps. In this review, we report on two intertwined disciplines in the protein structure prediction field, modeling of single chains and modeling of complexes, that have over decades emulated this pattern, as monitored by the community-wide blind prediction experiments CASP and CAPRI. However, over the past few years, dramatic advances were observed for the accurate prediction of single protein chains, driven by a surge of deep learning methodologies entering the prediction field. We review the mainscientific developments that enabled these recent breakthroughs and feature the important role of blind prediction experiments in building up and nurturing the structure prediction field. We discuss how the new wave of artificial intelligence–based methods is impacting the fields of computational and experimental structural biology and highlight areas in which deep learning methods are likely to lead to future developments, provided that major challenges are overcome.

Nucleotide-binding and leucine-rich repeat (NLR) proteins are critical intracellular immune receptors in both animals and plants. Perception of pathogen-derived or stress-associated signals induces NLR oligomerization to form multiprotein complexes called inflammasomes in animals or resistosomes in plants to mediate host immune response. Significant progress has been made during the past few years in our understanding of NLR biology, particularly the structural perspective of these two types of NLR-containing complexes. In this article, we review the latest advances in our structural knowledge of how NLR inflammasomes and resistosomes are activated and assembled and how the structural information provides insight into their distinct mechanisms of action. Commonalities and differences between NLR inflammasomes and resistosomes are also discussed.

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. Therefore, the discovery of mitochondrial ion channels influencing ion permeability through the membrane has defined a new dimension of the function of ion channels in different cell types, mainly linked to the important tasks that mitochondrial ion channels perform in cell life and death. This review summarizes studies on animal mitochondrial ion channels with special focus on their biophysical properties, molecular identity, and regulation. Additionally, the potential of mitochondrial ion channels as therapeutic targets for several diseases is briefly discussed.

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. To date, most successful have been experiments in which conformational change is triggered in light-dependent proteins. In this review, I emphasize emerging techniques that probe the dynamic basis of function in proteins lacking natively light-dependent transitions and speculate about extensions and further possibilities. In addition, I review how the weaker and more distributed signals in these data push the limits of the capabilities of analytical methods. Taken together, these new methods are beginning to establish a powerful paradigm for the study of the physics of protein function.

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions.

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.

Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input–output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity. In this article, we review the literature on the design of this effect, paying particular attention to the design strategies involved, the extent to which each can be rationally applied to (and optimized for) new receptors, and what each teaches us about the origins and optimization of this important phenomenon.

The recent proliferation of cryo-electron tomography (cryo-ET) techniques has led to the cryo-ET resolution revolution. Meanwhile, significant efforts have been made to improve the identification of targets in the cellular context and the throughput of cryo-focused ion beam (FIB) milling. Together, these developments led to a surge of in situ discoveries on how enveloped viruses are assembled and how viruses interact with cells in infected hosts. In this article, we review the recent advances in cryo-ET, high-resolution insights into virus assembly, and the findings from inside infected eukaryotic and prokaryotic cells.

Large biomolecular systems are at the heart of many essential cellular processes. The dynamics and energetics of an increasing number of these systems are being studied by computer simulations. Pushing the limits of length- and timescales that can be accessed by current hard- and software has expanded the ability to describe biomolecules at different levels of detail. We focus in this review on the ribosome, which exemplifies the close interplay between experiment and various simulation approaches, as a particularly challenging and prototypic nanomachine that is pivotal to cellular biology due to its central role in translation. We sketch widely used simulation methods and demonstrate how the combination of simulations and experiments advances our understanding of the function of the translation apparatus based on fundamental physics.

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. We further discuss aspects of cryo-EM methodology that are the greatest pitfalls challenging successful structure determination to date. Lastly, we highlight and propose potential future developments that would improve the method even further in the near future.

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.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.

Many proteins contain large structurally disordered regions or are entirely disordered under physiological conditions. The functions of these intrinsically disordered proteins (IDPs) often involve interactions with other biomolecules. An important emerging effort has thus been to identify the molecular mechanisms of IDP interactions and how they differ from the textbook notions of biomolecular binding for folded proteins. In this review, we summarize how the versatile tool kit of single-molecule fluorescence spectroscopy can aid the investigation of these conformationally heterogeneous and highly dynamic molecular systems. We discuss the experimental observables that can be employed and how they enable IDP complexes to be probed on timescales from nanoseconds to hours. Key insights include the diverse structural and dynamic properties of bound IDPs and the kinetic mechanisms facilitated by disorder, such as fly-casting; disorder-mediated encounter complexes; and competitive substitution via ternary complexes, which enables rapid dissociation even for high-affinity complexes. We also discuss emerging links to aggregation, liquid–liquid phase separation, and cellular processes, as well as current technical advances to further expand the scope of single-molecule spectroscopy.