Annual Review of Biophysics - Volume 37, 2008

My research began with theory and methods for ultracentrifugal studies of proteins, first at the University of Wisconsin, Madison, with Bob Alberty and Jack Williams, then at Oxford University with A.G. (“Sandy”) Ogston, and finally back at Wisconsin with Williams and Lou Gosting. In 1959 I joined Arthur Kornberg's Biochemistry Department at Stanford University. Our first work was physical studies of DNA replication and then DNA physical chemistry, and DNA studies ended with the energetics of DNA twisting. In 1971 we began to search for protein folding intermediates by fast-reaction methods. We found the slow-folding and fast-folding forms of unfolded ribonuclease A, which led to the understanding that proline isomerization is sometimes part of the folding process. Using hydrogen exchange as a probe, we found the rapid formation of secondary structure during folding and used this to provide an NMR pulse labeling method for determining structures of folding intermediates. Our studies of peptide helices provided basic helix-coil parameters, also evidence for hierarchic folding, and further indicated that peptide hydrogen bonds are important in the energetics of folding.

Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine is the translocon, which works in concert with ribosomes to manage the orderly insertion of α-helical membrane proteins directly into the endoplasmic reticulum membrane of eukaryotes or into the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid membrane, where physicochemical interactions determine the final three-dimensional structure. Much progress has been made during the past several years toward understanding the physical chemistry of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider the connection between the physical principles of membrane protein stability and translocon selection of transmembrane helices.

F1F0 ATP synthases convert energy stored in an electrochemical gradient of H⁺ or Na⁺ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore under diverse environmental conditions. The ATP synthase of each particular cell is confronted with specific challenges, imposed by the specific environment, and thus by necessity must adapt to these conditions for optimal operation. Examples of these adaptations include diverse mechanisms for regulating the ATP hydrolysis activity of the enzyme, the utilization of different coupling ions with distinct ion binding characteristics, different ion-to-ATP ratios reflected by variations in the size of the rotor c ring, the mode of ion delivery to the binding sites, and the different contributions of the electrical and chemical gradients to the driving force.

Elements of the cytoskeleton interact intimately and communicate bidirectionally with cellular membranes. Such interactions are critical for a host of cellular processes. Here we focus on the many types of interactions that exist between the cytoskeleton and the plasma membrane to illustrate why these cellular components can never truly be studied in isolation in vivo. We discuss how membrane-cytoskeleton interactions are mediated and modulated, and how many proteins involved in these interactions are disrupted in human disease. We then highlight key molecular and physical variables that must be considered in order to mechanistically dissect events associated with changes in plasma membrane morphology. These considerations are integrated into the context of cell migration, filopodia formation, and clathrin-mediated endocytosis to show how a holistic view of the plasma membrane-cytoskeleton interface can allow for the appropriate interpretation of experimental findings and provide novel mechanistic insight into these important cellular events.

This review highlights insights gained from computational studies on protein-metal recognition. We systematically dissect the various factors governing metal binding affinity and selectivity in proteins starting from (a) the intrinsic properties of the metal and neighboring metal cations (if present), to (b) the primary coordination sphere, (c) the second coordination shell, (d) the protein matrix, (e) the bulk solvent, and (f) competing non-protein ligands from the surrounding biological environment. The results herein reveal the fundamental principles and the molecular bases underlying protein-metal recognition, which serve as a guide to engineer novel metalloproteins with programmed properties.

Riboswitches are RNAs capable of binding cellular metabolites using a diverse array of secondary and tertiary structures to modulate gene expression. The recent determination of the three-dimensional structures of parts of six different riboswitches illuminates common features that allow riboswitches to be grouped into one of two types. Type I riboswitches, as exemplified by the purine riboswitch, are characterized by a single, localized binding pocket supported by a largely pre-established global fold. This arrangement limits ligand-induced conformational changes in the RNA to a small region. In contrast, Type II riboswitches, such as the thiamine pyrophosphate riboswitch, contain binding pockets split into at least two spatially distinct sites. As a result, binding induces both local changes to the binding pocket and global architecture. Similar organizational themes are found in other noncoding RNAs, making it possible to begin to build a hierarchical classification of RNA structure based on the spatial organization of their active sites and associated secondary structural elements.

Modern instrumentation for calorimetry permits direct determination of enthalpy values for binding reactions and conformational transitions in biomolecules. Complete thermodynamic profiles consisting of free energy, enthalpy, and entropy may be obtained for reactions of interest in a relatively straightforward manner. Such profiles are of enormous value in drug design because they provide information about the balance of driving forces that cannot be obtained from structural or computational methods alone. This perspective shows several examples of the insight provided by thermodynamic data in drug design.

While nature evolved polypeptides over billions of years, protein design by evolutionary mimicry is progressing at a far more rapid pace. The mutation, selection, and amplification steps of the evolutionary cycle may be imitated in the laboratory using existing proteins, or molecules created de novo from random sequence space, as starting templates. However, the astronomically large number of possible polypeptide sequences remains an obstacle to identifying and isolating functionally interesting variants. Intelligently designed libraries and improved search techniques are consequently important for future advances. In this regard, combining experimental and computational methods holds particular promise for the creation of tailored protein receptors and catalysts for tasks unimagined by nature.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a minority phospholipid of the inner leaflet of plasma membranes. Many plasma membrane ion channels and ion transporters require PIP2 to function and can be turned off by signaling pathways that deplete PIP2. This review discusses the dependence of ion channels on phosphoinositides and considers possible mechanisms by which PIP2 and analogues regulate ion channel activity.

RNA folding is a remarkably complex problem that involves ion-mediated electrostatic interaction, conformational entropy, base pairing and stacking, and noncanonical interactions. During the past decade, results from a variety of experimental and theoretical studies pointed to (a) the potential ion correlation effect in Mg²⁺-RNA interactions, (b) the rugged energy landscapes and multistate RNA folding kinetics even for small RNA systems such as hairpins and pseudoknots, (c) the intraloop interactions and sequence-dependent loop free energy, and (d) the strong nonadditivity of chain entropy in RNA pseudoknot and other tertiary folds. Several related issues, which have not been thoroughly resolved, require combined approaches with thermodynamic and kinetic experiments, statistical mechanical modeling, and all-atom computer simulations.

Intrinsically disordered proteins (IDPs) lack stable tertiary and/or secondary structures under physiological conditions in vitro. They are highly abundant in nature and their functional repertoire complements the functions of ordered proteins. IDPs are involved in regulation, signaling, and control, where binding to multiple partners and high-specificity/low-affinity interactions play a crucial role. Functions of IDPs are tuned via alternative splicing and posttranslational modifications. Intrinsic disorder is a unique structural feature that enables IDPs to participate in both one-to-many and many-to-one signaling. Numerous IDPs are associated with human diseases, including cancer, cardiovascular disease, amyloidoses, neurodegenerative diseases, and diabetes. Overall, intriguing interconnections among intrinsic disorder, cell signaling, and human diseases suggest that protein conformational diseases may result not only from protein misfolding, but also from misidentification, missignaling, and unnatural or nonnative folding. IDPs, such as α-synuclein, tau protein, p53, and BRCA1, are attractive targets for drugs modulating protein-protein interactions. From these and other examples, novel strategies for drug discovery based on IDPs have been developed. To summarize work in this area, we are introducing the D² (disorder in disorders) concept.

We review the effects of molecular crowding on solute diffusion in solution and in cellular aqueous compartments and membranes. Anomalous diffusion, in which mean squared displacement does not increase linearly with time, is predicted in simulations of solute diffusion in media crowded with fixed or mobile obstacles, or when solute diffusion is restricted or accelerated by a variety of geometric or active transport processes. Experimental measurements of solute diffusion in solutions and cellular aqueous compartments, however, generally show Brownian diffusion. In cell membranes, there are examples of both Brownian and anomalous diffusion, with the latter likely produced by lipid-protein and protein-protein interactions. We conclude that the notion of universally anomalous diffusion in cells as a consequence of molecular crowding is not correct and that slowing of diffusion in cells is less marked than has been generally assumed.

Advances in microfabrication and nanofabrication are opening new opportunities to investigate complicated questions of cell biology in ways not before possible. In particular, the spatial regulation of cellular processes can be examined by engineering the chemical and physical environment to which the cell responds. Lithographic methods and selective chemical modification schemes can provide biocompatible surfaces that control cellular interactions on the micron and submicron scales on which cells are organized. Combined with fluorescence microscopy and other approaches of cell biology, a widely expanded toolbox is becoming available. This review illustrates the potential of these integrated engineering tools, with an emphasis on patterned surfaces, for investigating fundamental mechanisms of receptor-mediated signaling in cells. We highlight progress made with immune cells and in particular with the IgE receptor system, which has been valuable for developing technology to gain new information about spatial regulation in signaling events.

The “protein folding problem” consists of three closely related puzzles: (a) What is the folding code? (b) What is the folding mechanism? (c) Can we predict the native structure of a protein from its amino acid sequence? Once regarded as a grand challenge, protein folding has seen great progress in recent years. Now, foldable proteins and nonbiological polymers are being designed routinely and moving toward successful applications. The structures of small proteins are now often well predicted by computer methods. And, there is now a testable explanation for how a protein can fold so quickly: A protein solves its large global optimization problem as a series of smaller local optimization problems, growing and assembling the native structure from peptide fragments, local structures first.

Helicases and remodeling enzymes are ATP-dependent motor proteins that play a critical role in every aspect of RNA and DNA metabolism. Most RNA-remodeling enzymes are members of helicase superfamily 2 (SF2), which includes many DNA helicase enzymes that display similar structural and mechanistic features. Although SF2 enzymes are typically called helicases, many of them display other types of functions, including single-strand translocation, strand annealing, and protein displacement. There are two mechanisms by which RNA helicase enzymes unwind RNA: The nonprocessive DEAD group catalyzes local unwinding of short duplexes adjacent to their binding sites. Members of the processive DExH group often translocate along single-stranded RNA and displace paired strands (or proteins) in their path. In the latter case, unwinding is likely to occur by an active mechanism that involves Brownian motor function and stepwise translocation along RNA. Through structural and single-molecule investigations, researchers are developing coherent models to explain the functions and dynamic motions of helicase enzymes.

The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

High-resolution X-ray crystal structures determined in the past six years dramatically influence our view of ligand-induced activation of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Ligand binding to the extracellular region of EGFR promotes a major domain reorganization, plus local conformational changes, that are required to generate an entirely receptor-mediated dimer. In this activated complex the intracellular kinase domains associate to form an asymmetric dimer that supports the allosteric activation of one kinase. These models are discussed with emphasis on recent studies that add details or bolster the generality of this view of activation of this family of receptors. The EGFR family is implicated in several disease states, perhaps most notably in cancers. Activating tumor mutations have been identified in the intracellular and extracellular regions of EGFR. The impact of these tumor mutations on the understanding of EGFR activation and of its inhibition is discussed.

Expected and observed effects of volume exclusion on the free energy of rigid and flexible macromolecules in crowded and confined systems, and consequent effects of crowding and confinement on macromolecular reaction rates and equilibria are summarized. Findings from relevant theoretical/simulation and experimental literature published from 2004 onward are reviewed. Additional complexity arising from the heterogeneity of local environments in biological media, and the presence of nonspecific interactions between macromolecules over and above steric repulsion, are discussed. Theoretical and experimental approaches to the characterization of crowding- and confinement-induced effects in systems approaching the complexity of living organisms are suggested.

Receptor-ligand bonds strengthened by tensile mechanical force are referred to as catch bonds. This review examines experimental data and biophysical theory to analyze why mechanical force prolongs the lifetime of these bonds rather than shortens the lifetime by pulling the ligand out of the binding pocket. Although many mathematical models can explain catch bonds, experiments using structural variants have been more helpful in determining how catch bonds work. The underlying mechanism has been worked out so far only for the bacterial adhesive protein FimH. This protein forms catch bonds because it is allosterically activated when mechanical force pulls an inhibitory domain away from the ligand-binding domain. Other catch bond–forming proteins, including blood cell adhesion proteins called selectins and the motor protein myosin, show evidence of allosteric regulation between two domains, but it remains unclear if this is related to their catch bond behavior.