Annual Review of Biophysics - Volume 26, 1997

The scope and utility of phage display is reviewed with emphasis on medical applications and structure-based ligand and drug design, from literature mostly after 1994. General principles by which phage-displayed peptides achieve affinity and selectivity for targets are described, along with selected structural or mechanistic studies of the binding of peptides or proteins discovered or engineered by phage display. Such engineered proteins whose wild-type or mutant crystal or 2D-NMR structures yield insight about the basis for enhanced affinity or altered specificity include antibodies, zinc fingers, human growth hormone, protein A, and atrial natriuretic peptide. Structures of complexes of de novo phage-discovered peptide ligands with targets such as the Src SH3 domain, streptavidin, and erythropoietin receptor reveal the structural basis for receptor-peptide recognition in these systems.

Researchers have made good progress in unraveling the molecular mechanisms of excitation-contraction (EC) coupling in striated muscle. Despite this progress, paradoxes abound. In skeletal muscle, the existence of a mechanical coupling between membrane charge movement and activation of sarcoplasmic reticulum (SR) release channels is essentially established, but the contribution of Ca²⁺-induced Ca²⁺ release (CICR) to the transient and steady-state components of Ca²⁺ release remains controversial. In cardiac muscle, the role of CICR as the primary mechanism of EC coupling is well established, but the stability and tight coupling between membrane Ca²⁺ current and release are paradoxical. Answers may lie in microdomain issues, and in the examination of discrete elementary release events, although quantitative treatments are needed. This review explores the theoretical and experimental methods used and the observations made in the study of microdomain Ca²⁺.

Chromatin structure is now believed to be dynamic and intimately related with cellular processes such as transcription. Over the past few years, high-resolution structures for the histones have become available. These structures and their implications for nucleosome organization are reviewed here.

The evidence showing that the self-assembly of complex RNAs occurs in discrete transitions, each relating to the folding of sub-systems of increasing size and complexity starting from a state with most of the secondary structure, is reviewed. The reciprocal influence of the concentration of magnesium ions and nucleotide mutations on tertiary structure is analyzed. Several observations demonstrate that detrimental mutations can be rescued by high magnesium concentrations, while stabilizing mutations lead to a lesser dependence on magnesium ion concentration. Recent data point to the central controlling and monitoring roles of RNA-binding proteins that can bind to the different folding stages, either before full establishment of the secondary structure or at the molten globule state before the cooperative transition to the final three-dimensional structure.

One of the fundamental properties of the RNA helix is its intrinsic resistance to bend- or twist-deformations. Results of a variety of physical measurements point to a persistence length of 700–800 Å for double-stranded RNA in the presence of magnesium cations, approximately 1.5–2.0-fold larger than the corresponding value for DNA. Although helix flexibility represents an important, quantifiable measure of the forces of interaction within the helix, it must also be considered in describing conformational variation of nonhelix elements (e.g. internal loops, branches), since the latter always reflect the properties of the flanking helices; that is, such elements are never completely rigid. For one important element of tertiary structure, namely, the core of yeast tRNA^Phe, the above consideration has led to the conclusion that the core is not substantially more flexible than an equivalent length of pure helix.

Phospholamban is a 52-amino-acid protein that assembles into a pentamer in sarcoplasmic reticulum membranes. The protein has a role in the regulation of the resident calcium ATPase through an inhibitory association that can be reversed by phosphorylation. The phosphorylation of phospholamban is initiated by β-adrenergic stimulation, identifying phospholamban as an important component in the stimulation of cardiac activity by β-agonists. It is this role of phospholamban that has motivated studies in recent decades. There is evidence that phospholamban may also function as a Ca²⁺-selective ion channel. The structural properties of phospholamban have been studied by mutagenesis, modeling, and spectroscopy, resulting in a new view of the organization of this key molecule in membranes.

Innovative algorithms have been developed during the past decade for simulating Newtonian physics for macromolecules. A major goal is alleviation of the severe requirement that the integration timestep be small enough to resolve the fastest components of the motion and thus guarantee numerical stability. This timestep problem is challenging if strictly faster methods with the same all-atom resolution at small timesteps are sought. Mathematical techniques that have worked well in other multiple-timescale contexts—where the fast motions are rapidly decaying or largely decoupled from others—have not been as successful for biomolecules, where vibrational coupling is strong.

This review examines general issues that limit the timestep and describes available methods (constrained, reduced-variable, implicit, symplecttic, multiple-timestep, and normal-mode-based schemes). A section compares results of selected integrators for a model dipeptide, assessing physical and numerical performance. Included is our dual timestep method LN, which relies on an approximate linearization of the equations of motion every Δt interval (5 fs or less), the solution of which is obtained by explicit integration at the inner timestep Δτ (e.g., 0.5 fs). LN is computationally competitive, providing 4–5 speedup factors, and results are in good agreement, in comparison to 0.5 fs trajectories.

These collective algorithmic efforts help fill the gap between the time range that can be simulated and the timespans of major biological interest (milliseconds and longer). Still, only a hierarchy of models and methods, along with experimentational improvements, will ultimately give theoretical modeling the status of partner with experiment.

Two sensory rhodopsins (SRI and SRII) mediate color-sensitive phototaxis responses in halobacteria. These seven-helix receptor proteins, structurally and functionally similar to animal visual pigments, couple retinal photoisomerization to receptor activation and are complexed with membrane-embedded transducer proteins (HtrI and HtrII) that modulate a cytoplasmic phosphorylation cascade controlling the flagellar motor. The Htr proteins resemble the chemotaxis transducers from Escherichia coli. The SR-Htr signaling complexes allow studies of the biophysical chemistry of signal generation and relay, from the photobiophysics of initial excitation of the receptors to the final output at the level of the flagellar motor switch, revealing fundamental principles of sensory transduction and more broadly the nature of dynamic interactions between membrane proteins. We review here recent advances that have led to new insights into the molecular mechanism of signaling by these membrane complexes.

A characteristic feature of cellular signal transduction pathways in eukaryotes is the separation of catalysis from target recognition. Several modular domains that recognize short peptide sequences and target signaling proteins to these sequences have been identified. The structural bases of the specificities of recognition by SH2, SH3, and PTB domains have been elucidated by X-ray crystallography and NMR, and these results are reviewed here. In addition, the mechanism of cooperative interactions between these domains is discussed.

Eukaryotes have three distinct RNA polymerases that catalyze transcription of nuclear genes. RNA polymerase II is responsible for transcribing nuclear genes encoding the messenger RNAs and several small nuclear RNAs. Like RNA polymerases I and III, polymerase II cannot recognize its target promoter directly and initiate transcription without accessory factors. Instead, this large multisubunit enzyme relies on general transcription factors and transcriptional activators and coactivators to regulate transcription from class II promoters. X-ray crystallography and nuclear magnetic resonance spectroscopy have been used to study complexes of general transcription factors and transcriptional activators with their specific DNA targets. This work has provided important structural insights into transcription initiation by polymerase II and the more general problem of DNA sequence recognition.

Over the past two decades, nanosecond absorption and vibrational spectroscopies have developed into powerful tools for monitoring the secondary, tertiary, and quaternary structural relaxations of biological macromolecules under near-physiological conditions of solvent and temperature. Observed through such methods, the dynamic response of a biomolecule to photoinitiated excursions from equilibrium can reveal valuable information about the structure-function relationship, information beyond that obtained from the static structures provided by X-ray crystallography, nuclear magnetic resonance spectroscopy, and other steady-state methods. Most recently, the development of ultra-sensitive polarization techniques for absorption spectroscopy has greatly enhanced the amount of time-resolved structural information that can be obtained from the broadened electronic spectra of biomolecules. This review examines nanosecond absorption, vibrational, and polarized absorption methods, and their applications to protein function and folding, emphasizing the complementary nature of information obtained from electronic and vibrational spectra measured on the nanosecond time scale.

Zinc-finger domains are small metal-binding modules that are found in a wide range of gene regulatory proteins. Peptides corresponding to these domains have provided valuable model systems for examining a number of biophysical parameters entirely unrelated to their nucleic acid binding properties. These include the chemical basis for metal-ion affinity and selectivity, thermodynamic properties related to hydrophobic packing and beta-sheet propensities, and constraints on the generation of ligand-binding and potential catalytic sites. These studies have laid the foundation for applications such as the generation of optically detected zinc probes and the design of metal-binding peptides and proteins with desired spectroscopic and chemical properties.

Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.

Phage display makes large-peptide diversity libraries readily attainable for identifying novel peptide ligands for receptors and other protein or non-protein targets. This technology kindles enthusiasm for the idea that large and protein-protein interaction surfaces (epitopes) can be distilled down to small pharmacophores. These may be accessible to organic scaffolding, yielding new orally active drugs that might otherwise have taken greater time and effort to be discovered through chemical-library screening. This review, though not comprehensive with respect to the explosive volume of phage display work over the last few years, focuses on recent developments in phage-displayed peptide technology.

Oil-water partitioning, solubilities, and vapor pressure experiments on small-molecule compounds are often used as models to obtain energies for biomolecular modeling. For example, measured partition coefficients, K, are often inserted into the formula −RTlnK to obtain quantities thought to represent microscopic contact interaction free energies. We review evidence here that this procedure does not always give microscopically meaningful free energies. Some partitioning processes, particularly involving polymeric solvents such as octanol or hexadecane, are governed not only by translational entropies and contact interactions, but also by free energies resulting from changes in the conformations of the polymer chains upon solute insertion. The Flory-Huggins theory is more suitable for these situations than is the classical approach. We discuss the physical bases for both approaches.

Ten years have passed since the initial reports that antibodies could be programmed to have enzymatic activity by immunization with a transition-site analog. Much of the research over the last decade has focused on defining the scope and generality of antibody catalysis; however, during the past two years the first few crystal structures of catalytic antibody transition-state analogs have been reported. This review analyzes four such structures of catalytic antibodies that catalyze markedly different reactions, including ester hydrolysis, sulfide oxidation, and a pericyclic rearrangement. Structure-function relations for these catalysts are discussed and compared to the structure and function of natural enzymes, as well as the chemistry that occurs in solution.

This review focuses on the recent advances in EPR spectroscopy as they are applied both to photoinduced electron transfer in the photosynthetic apparatus and to biomimetic systems. The review deals with time-resolved direct-detection cw and pulsed EPR and ENDOR methods, both at conventional bands [X-(9.5 GHz), K-(24 GHz), and Q-(35 GHz)] and at high frequency bands (W-band, 95 GHz, and even highter frequency bands). EPR studies on photosynthetic and model systems in their doublet, triplet and radical pair states are surveyed, including their static and dynamic properties. Applications of time-resolved EPR in studying photoinduced electron and energy transfer in isotropic and anisotropic environments, and the concepts of electron spin polarization and magnetic field effects in photochemical reactions are also reviewed.

Surface plasmon resonance biosensors have become increasingly popular for the qualitative and quantitative characterization of the specific binding of a mobile reactant to a binding partner immobilized on the sensor surface. This article reviews the use of this new technique to measure the binding affinities and the kinetic constants of reversible interactions between biological macromolecules. Immobilization techniques, the most commonly employed experimental strategies, and various analytical approaches are summarized. In recent years, several sources of potential artifacts have been identified: immobilization of the binding partner, steric hindrance of binding to adjacent binding sites at the sensor surface, and finite rate of mass transport of the mobile reactant to the sensor surface. Described here is the influence of these artifacts on the measured binding kinetics and equilibria, together with suggested control experiments.