Annual Review of Biophysics - Volume 27, 1998

Exciting progress has been made in the last decade by those who use physical methods to study the structure of the ribosome and its components. The structures of 10 ribosomal proteins and three isolated ribosomal protein domains are known, and the conformations of a significant number of rRNA sequences have been determined. Electron microscopists have made major advances in the analysis of images of ribosomes, and microscopically derived ribosome models at resolutions approaching 10Å are likely quite soon. Furthermore, ribosome crystallographers are on the verge of phasing the diffraction patterns they have had for several years, and near-atomic resolution models for entire ribosomal subunits could emerge from this source at any time. The literature relevant to these developments is reviewed below.

This review surveys the kinds of protein complex that participate in cell communication and identifies, where possible, general principles by which they form and act. It also advances the notion that biophysical constraints imposed by macromolecular crowding and diffusion have had a controlling influence on the evolution of cell signaling pathways. Complexes associated with the bacterial aspartate receptor, with eucaryotic tyrosine kinase receptors, with T-cell receptors, and with focal contacts are examined together with proteins that serve as adaptors, anchors, and scaffolds for signaling complexes. The importance of diffusion in controlling the numbers and locations of signaling complexes is discussed, as is the special role played by membranes in signaling pathways.

Biophysical events involved in late stages of exocytosis occur at highly localized areas of cells on millisecond and submillisecond time scales. Thus, methodologies with high spatio-temporal resolution are required to achieve measurements at individual secretory cells. Much has been learned about the mechanisms and kinetics of vesicular release through analysis with the carbon fiber microelectrode techniques amperometry and cyclic voltammetry. Coupling of these techniques with other methods such as patch-clamp continues to reveal details of the secretion process. It is now clear that extrusion of the vesicular contents is a more complex process than previously believed. Vesicle-cell fusion, revealed by cell capacitance measurements, is temporally dissociated from secretion measured amperometrically. The stability imparted by interaction and association of vesicle contents at rest results in a rate-limiting extrusion process after full fusion. Furthermore, the presence of partial fusion events and the occurrence of nonquantized release have been revealed with electrochemical tools.

To date, high-resolution structures have been solved for five different architectural proteins complexed to their DNA target sites. These include TATA-box–binding protein, integration host factor (IHF), high mobility group I(Y)[HMG I(Y)], and the HMG-box–containing proteins SRY and LEF-1. Each of these proteins interacts with DNA exclusively through minor groove contacts and alters DNA conformation. This paper reviews the structural features of these complexes and the roles they play in facilitating assembly of higher-order protein–DNA complexes and discusses elements that contribute to sequence-specific recognition and conformational changes.

Eukaryotic protein phosphatases are structurally and functionally diverse enzymes that are represented by three distinct gene families. Two of these, the PPP and PPM families, dephosphorylate phosphoserine and phosphothreonine residues, whereas the protein tyrosine phosphatases (PTPs) dephosphorylate phosphotyrosine amino acids. A subfamily of the PTPs, the dual-specificity phosphatases, dephosphorylate all three phosphoamino acids. Within each family, the catalytic domains are highly conserved, with functional diversity endowed by regulatory domains and subunits. The protein Ser/Thr phosphatases are metalloenzymes and dephosphorylate their substrates in a single reaction step using a metal-activated nucleophilic water molecule. In contrast, the PTPs catalyze dephosphorylation by use of a cysteinyl-phosphate enzyme intermediate. The crystal structures of a number of protein phosphatases have been determined, enabling us to understand their catalytic mechanisms and the basis for substrate recognition and to begin to provide insights into molecular mechanisms of protein phosphatase regulation.

Identification of biomolecules in complex biological mixtures represents a major challenge in biomedical, environmental, and chemical research today. Chemical separations with traditional detection schemes such as absorption, fluorescence, refractive index, conductivity, and electrochemistry have been the standards for definitive identifications of many compounds. In many instances, however, the complexity of the biomixture exceeds the resolution capability of chemical separations. Biosensors based on molecular recognition can dramatically improve the selectivity of and provide biologically relevant information about the components. This review describes how coupling chemical separations with online biosensors solves challenging problems in sample analysis by identifying components that would not normally be detectable by either technique alone. This review also presents examples and principles of combining chemical separations with biosensor detection that uses living systems, whole cells, membrane receptors, enzymes, and immunosensors.

Biochemical and genetic approaches have identified the molecular mechanisms of many genetic reactions, particularly in bacteria. Now a comparably detailed understanding is needed of how groupings of genes and related protein reactions interact to orchestrate cellular functions over the cell cycle, to implement preprogrammed cellular development, or to dynamically change a cell's processes and structures in response to environmental signals. Simulations using realistic, molecular-level models of genetic mechanisms and of signal transduction networks are needed to analyze dynamic behavior of multigene systems, to predict behavior of mutant circuits, and to identify the design principles applicable to design of genetic regulatory circuits. When the underlying design rules for regulatory circuits are understood, it will be far easier to recognize common circuit motifs, to identify functions of individual proteins in regulation, and to redesign circuits for altered functions.

DNA nanotechnology entails the construction of specific geometrical and topological targets from DNA. The goals include the use of DNA molecules to scaffold the assembly of other molecules, particularly in periodic arrays, with the objects of both crystal facilitation and memory-device construction. Many of these products are based on branched DNA motifs. DNA molecules with the connectivities of a cube and a truncated octahedron have been prepared. A solid-support methodology has been developed to construct DNA targets. DNA trefoil and figure-8 knots have been made, predicated on the relationship between a topological crossing and a half-turn of B-DNA or Z-DNA. The same basis has been used to construct Borromean rings from DNA. An RNA knot has been used to demonstrate an RNA topoisomerase activity. The desire to construct periodic matter held together by DNA sticky ends has resulted in a search for stiff components; DNA double crossover molecules appear to be the best candidates. It appears that novel DNA motifs may be of use in the new field of DNA-based computing.

Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design. This effort was very successful. Four drugs are already approved, and others are undergoing clinical trials. The techniques utilized in this remarkable example of structure-assisted drug design included crystallography, NMR, computational studies, and advanced chemical synthesis. The development of these drugs is discussed in detail. Other approaches to designing HIV-1 PR inhibitors, based on the concepts of symmetry and on the replacement of a water molecule that had been found tetrahedrally coordinated between the enzyme and the inhibitors, are also discussed. The emergence of drug-induced mutations of HIV-1 PR leads to rapid loss of potency of the existing drugs and to the need to continue the development process. The structural basis of drug resistance and the ways of overcoming this phenomenon are mentioned.

The substrates for the essential biological processes of transcription, replication, recombination, DNA repair, and cell division are not naked DNA; rather, they are protein-DNA complexes known as chromatin, in one or another stage of a hierarchical series of compactions. These are exciting times for students of chromatin. New studies provide incontrovertible evidence linking chromatin structure to function. Exceptional progress has been made in studies of the structure of chromatin subunits. Surprising new dynamic properties have been discovered. And, much progress has been made in dissecting the functional roles of specific chromatin proteins and domains. This review focuses on in vitro studies of chromatin structure, dynamics, and function.

Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water. Electron transfer is coupled to proton translocation across the membrane, resulting in a proton and charge gradient that is then employed by the F0F1-ATPase to synthesize ATP.

Over the last years, substantial progress has been made in our understanding of the structure and function of this enzyme. Spectroscopic techniques such as EPR, absorbance and resonance Raman spectroscopy, in combination with site-directed mutagenesis work, have been successfully applied to elucidate the nature of the cofactors and their ligands, to identify key residues involved in proton transfer, and to gain insight into the catalytic cycle and the structures of its intermediates. Recently, the crystal structures of a bacterial and a mitochondrial cytochrome c oxidase have been determined. In this review, we provide an overview of the crystal structures, summarize recent spectroscopic work, and combine structural and spectroscopic data in discussing mechanistic aspects of the enzyme. For the latter, we focus on the structure of the oxygen intermediates, proton-transfer pathways, and the much-debated issue of how electron transfer in the enzyme might be coupled to proton translocation.

During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications. Most recently, the combination of triple resonance experiments and ²H, ¹³C, ¹⁵N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution ²H NMR methods to probe the dynamics of protein sidechains.

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein folds. Proteins containing RNP domains function in important steps of posttranscriptional regulation of gene expression by directing the assembly of multiprotein complexes on primary transcripts, mature mRNAs, and stable ribonucleoprotein components of the RNA processing machinery. The diverse functions performed by these proteins depend on their dual ability to recognize RNA and to interact with other proteins, often utilizing specialized auxiliary domains. Crystallographic and NMR structures of several RNP domains and a handful of structures of RNA-protein complexes have begun to reveal the molecular basis for RNP-RNA recognition.

In this review we discuss various recent topics that characterize functional magnetic resonance imaging (fMRI). These topics include a brief description of MRI image acquisition, how to cope with noise or signal fluctuation, the basis of fMRI signal changes, and the relation of MRI signal to neuronal events. Several observations of fMRI that show good correlation to the neurofunction are referred to. Temporal characteristics of fMRI signals and examples of how the feature of real time measurement is utilized are then described. The question of spatial resolution of fMRI, which must be dictated by the vascular structure serving the functional system, is discussed based on various fMRI observations. Finally, the advantage of fMRI mapping is shown in a few examples. Reviewing the vast number of recent fMRI application that have now been reported is beyond the scope of this article.

The hammerhead ribozyme is a small catalytic RNA that cleaves a target phosphodiester bond in a reaction dependent on divalent metal ions. Crystal structures of the hammerhead reveal the tertiary fold of an enzymatic “ground state” of the molecule; however, they do not clarify the catalytic mechanism of the ribozyme, presumably because a significant conformational rearrangement is required to reach an enzymatic transition state. The structural domains seen in the hammerhead can be related to sequence or structural motifs in transfer and ribosomal RNAs, suggesting that they represent tertiary building blocks that will be found in large, complex RNAs.

Pleckstrin homology (PH) motifs are approximately 100 amino-acid residues long and have been identified in nearly 100 different eukaryotic proteins, many of which participate in cell signaling and cytoskeletal regulation. Despite minimal sequence homology, the three-dimensional structures are remarkably conserved. This review gives an overview of the PH domain architecture and examines the best-studied examples in an attempt to understand their function.