Showing papers in "Annual Review of Biophysics and Biomolecular Structure in 1995"

Molecular and structural basis of target recognition by calmodulin.

Abstract: Calmodulin (CaM) acts as an intracellular calcium sensor that translates the Ca2+ signal into a variety of cellular processes. Ca(2+)-CaM recognition of a short polypeptide segment in target proteins induces conformational changes in both CaM and the target, enabling the target protein to become functionally active. The solution and crystal structures of Ca(2+)-CaM bound to peptides derived from three CaM-dependent enzymes reveal structural features that are common in target recognition by Ca(2+)-CaM. Phosphorylation of the target proteins at sites in or near the CaM-binding region modulates binding of CaM, thereby providing an additional mechanism of functional regulation. The structural aspects of target recognition by Ca(2+)-CaM are discussed using mainly the three-dimensional structural information obtained with nuclear magnetic resonance spectroscopy and X-ray diffraction methods.

Exceptionally Stable Nucleic Acid Hairpins

Abstract: Hairpins represent the dominant secondary structure element in RNA. Certain sequences are found with exceptional frequency in many RNAs and are characterized by exceptionally high thermodynamic stability. Stable RNA hairpins define nucleation sites for folding, determine tertiary interactions in RNA enzymes, protect mRNAs from degradation, and are recognized by RNA-binding proteins. The structures of several stable DNA and RNA hairpins have revealed networks of stabilizing interactions within the hairpin loop: non-Watson-Crick base pairs and base-phosphate and base-sugar contacts. The unusual stability of these structural elements can be used to stabilize RNA and DNA structures and to protect antisense oligonucleotides and mRNAs against exonucleolytic degradation.

Structure and function of DNA methyltransferases.

Abstract: In prokaryotes, the major role of DNA methylation is to protect host DNA against degradation by restriction enzymes. In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including differentiation, gene regulation, and embryonic development. Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes. Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a striking similarity in protein folding and indicates that many SAM-dependent methyltransferases have a common catalytic-domain structure. This feature permits the prediction of tertiary structure for other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases.

The Cystine-Knot Growth-Factor Superfamily

Abstract: Four recent crystal structures of growth factors--nerve growth factor, transforming growth factor-beta, platelet-derived growth factor, and human chorionic gonadotropin--from four separate superfamilies revealed that these proteins are structurally related and share a common overall topology. These proteins have very little sequence homology, but they all have an unusual arrangement of six cysteines linked to form a "cystine-knot" conformation. The active forms of these proteins are dimers, either homo- or heterodimers. Despite the overall topological similarity between the monomers, the interfaces used to form the dimer are in each case quite different. Because the surfaces used for dimer formation are mostly hydrophobic, the uniqueness of each dimer accounts for the lack of sequence homology and raises questions about the effectiveness of reverse sequence fitting in this kind of structure as a predictor of structural homology.

Compact Intermediate States in Protein Folding

Abstract: Recently there has been growing recognition of the existence and importance of compact intermediate states of proteins. Such species have been observed under both transient (refolding kinetics) and equilibrium conditions. It is clear that for many proteins most denaturing conditions do not lead to a fully unfolded protein (random coil), but rather to species with substantial secondary structure and substantial compactness, relative to the fully unfolded state. In addition, there is now good experimental data to demonstrate the existence of two classes of compact denatured states of proteins: compact intermediates, in the thermodynamic sense (i.e., a minimum in the free energy profile for the reaction), and compact substates of the unfolded state (Palleros et al., 1993). It is important to note that it is often experimentally difficult to distinguish between these two types of compact denatured states, especially by spectral methods. Recent reviews of compact denatured states, and particularly the molten globule, include those of Dill and Shortle (1991), Ptitsyn (1987, 1992), Kuwajima (1989), Christensen and Pain (1991), and Baldwin and Roder (1991). Theoretical models for the existence of two classes of denatured states have been presented by Dill and co-workers (Alonso et al., 1991), Ptitsyn (1987, 1992), and Finkelstein and Shakhnovich (1989).

Site-Directed Mutagenesis with an Expanded Genetic Code

Abstract: A biosynthetic method has been developed that makes possible the site-specific incorporation of a large number of amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precise changes within a protein can be made to address detailed structure-function questions. A series of fluorinated tyrosine analogues and linear, branched, and cyclic hydrophobic amino acids have been used to determine the impact of hydrogen bonding and hydrophobic packing, respectively, on protein stability. Glutamate analogues and conformationally restricted amino acids have been used to probe the mechanisms of staphylococcal nuclease and ras. In addition, this technique has been used to construct photocaged proteins and proteins containing photoaffinity labels, spin labels, and isotopic labels at specific positions in the protein sequence suitable for biophysical studies.

Structure-function of the channel-forming colicins.

Abstract: The channel-forming colicins are plasmid-encoded bacteriocins that kill E. coli and related cells and whose mode of action is of interest in related problems of protein import and toxicology. Colicins parasitize metabolite receptors in the outer membrane and translocate across the periplasm with the aid of the Tol or Ton protein systems. X-ray structure data for the channel domain and colicin are available. Residues have been identified that affect the channel ion selectivity and particular helices implicated in channel structure and in conformational changes required for binding or insertion of the channel into the membrane. Unique aspects of the colicin channel system are the involvement of protein import in the gating process, the existence of multiple open and closed states, and the existence and action of an immunity protein that involves specific intramembrane helix-helix interactions with transmembrane helices of the colicin channel-forming domains.

Complexes of the Minor Groove of DNA

Abstract: An increasing number of high-resolution structures suggest that both the minor and major grooves of DNA can function as receptors for proteins and small molecules. In this review, we try to illustrate the diversity of small molecule ligands that are capable of specifically recognizing the minor groove of DNA. Complex formation results in varying degrees of conformational changes in both DNA and ligands. The discussion focuses on intermolecular interactions that contribute to binding affinity and specificity. There probably is no simple general recognition code that explains the binding specificity of minor-groove ligands. To understand DNA recognition by small molecules, characterization of the binding mode at near-atomic resolution must be combined with thermodynamic data on the energetics of ligand binding to short oligonucleotides.

Fluorescent Protein Biosensors: Measurement of Molecular Dynamics in Living Cells

Abstract: A new generation of reagents that report on specific molecular events in living cells, called fluorescent protein biosensors, has evolved from in vitro fluorescence spectroscopy and fluorescent analogue cytochemistry. Creative designs of fluorescent protein biosensors to measure the molecular dynamics of macromolecules, metabolites, and ions in single cells emerge from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. Future advances in fluorescent probe design, computer-driven optical instrumentation, and software will allow us to engineer endogenous cellular components that localize and function as reporters of their activities, thus moving molecular measurement beyond the single cell to living tissues and the whole organism.

Gating-spring models of mechanoelectrical transduction by hair cells of the internal ear

Abstract: A sensory receptor of the internal ear, or hair cell, responds to sound or acceleration when this mechanical stimulus deflects the cell's mechanosensitive organelle, or hair bundle. The gating-spring model posits that mechanoelectrical transduction occurs as mechanical force is transmitted through an elastic element, or gating spring, to the molecular gate of each transduction channel; increased tension in the gating spring then promotes the channel's transition from a closed to an open state. Electrophysiological and micromechanical data from a variety of hair cells, both in vivo and in vitro, confirm that the stimulus dependence of channel open probability and bundle stiffness are quantitatively consistent with the model. The results accord still better, however, with an extended formulation including channel transitions among one open and two closed states. In addition to providing a derivation of this three-state model, this review delineates several experimentally testable predictions of gating-spring models.

Flexible Docking and Design

Abstract: Docking and design are the major computational steps toward under­ standing and affecting receptor-ligand interactions. The flexibility of many ligands makes these calculations difficult and requires the devel­ opment and use of special methods. The need for such tools is illus­ trated by two examples: the design of protease inhibitors and the analy­ sis and design of peptide antigens binding to specific MHC receptors. We review the computational concepts that have been extended from rigid-body to flexible docking, as well as the following important strate­ gies for flexible docking and design: (a) Monte Carlo/molecular dynam-

Nucleic acid hybridization: triplex stability and energetics.

Abstract: In this chapter, we review the current state of the thermodynamic database for triple helical oligonucleotide hybridization reactions and present a critical assessment of the methods used to obtain the relevant data. The thermodynamic stability of triple-helix oligonucleotide constructs is discussed in terms of its dependence on temperature, chain length, pH, salt, base sequence, base and backbone modifications, and ligand binding. In particular, we examine the coupling of hybridization equilibria to proton, cation, and drug-binding equilibria. Throughout the chapter, we emphasize that a detailed understanding of the endogenous and exogenous variables that control triplex stability is required for the rational design of oligonucleotides for specific therapeutic, diagnostic, and/or biotechnological applications, as well as for elucidating the potential cellular roles of these higher-order nucleic acid complexes.

DNA Analogues with Nonphosphodiester Backbones

Abstract: This review discusses the recent developments of DNA analogues with nonphosphodiester backbones in terms of DNA structure and antisense and antigene potential. A larger number of derivatives are now available in which the phosphodiester linkage has been replaced but the deoxyribose retained. However, only a few of these (e.g. the ones having a thioformacetal or a carboxamide linkage) appear to be good structural DNA mimics. Two successful attempts to replace the entire deoxyribose phosphate backbone have been reported, the morpholino derivatives and the peptide nucleic acids (PNA), which contain an N-(2-aminoethyl)glycine-based pseudopeptide backbone. Most information is available on the PNA, which is a very promising DNA mimic. In conclusion, the deoxyribose phosphate backbone is not essential for a potent structural DNA mimic and not even required for a helical duplex structure.

Thermodynamics of partly folded intermediates in proteins

Abstract: Until recently, the energetics of protein-folding intermediates eluded direct measurement by high-sensitivity microcalorimetric techniques. But during the past year, the direct measurement of thermodynamic parameters for folding intermediates of a-lactalbumin, apomyoglobin, cytochrome c, and staphylococcal nuclease has provided new insights on the nature of the forces involved in the stabilization of nascent pro

Capillary electrophoresis of proteins and nucleic acids

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Structure and Mechanism of DNA Topoisomerases

Abstract: DNA topoisomerases are ubiquitous enzymes that control the level of supercoiling of DNA in cells. There are several classes, each with distinct properties, which are briefly discussed in this review. High-resolution X-ray crystallographic structures have been obtained for fragments of two classes of these enzymes, which when combined with biochemical data, reveal a great deal about the gymnastics that the enzymes undergo during catalysis and provide fascinating snapshots of their mechanisms. These mechanisms are discussed in detail. Finally, the first structure of a topoisomerase in a complex with an antibiotic was recently solved. This structure is briefly discussed with regard to the biochemical activity of the compound.

Design of Molecular Function: Channels of Communication

Abstract: The ultimate goal in protein design is to elucidate the fundamental principles that determine structure. With increased understanding of the molecular basis underlying the sequence-structure relationship may come the ability to control it and, thereby, to generate proteins with desired specifications. Channel proteins, which mediate cell signaling, are ideally suitable for protein design. Plausible molecular blueprints for the pore-forming structure are bundles of amphipathic alpha-helices or beta-barrels that cluster together to generate a hydrophilic channel. This review focuses on the progress achieved to produce such designs and on the approximation of the synthetic channels to the targeted biological function.

Mass Spectrometry of Nucleic Acids: The Promise of Matrix-Assisted Laser Desorption-Ionization (MALDI) Mass Spectrometry

Abstract: In the past several years, significant progress has been made in the application of matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to the analysis of large biopolymers, including nucleic acids. By isolating analyte molecules in an appropriate matrix and irradiating the sample with a high-intensity, pulsed laser beam, MALDI can generate intact, gas-phase ions of these analytes. Primarily used with time-of-flight mass spectrometers, this relatively new, soft ionization technique has allowed for the routine analysis of oligonucleotides up to 60 or so nucleotides in length. Recent results have also shown that base specific, matrix-dependent fragmentation is an important factor in the MALDI analysis of oligonucleotides. Further extension of the technique to longer oligonucleotides will rely on both the continued search for new matrix materials and an increased understanding of the desorption and ionization process in MALDI.

NMR Spectroscopic Studies of Paramagnetic Proteins: Iron-Sulfur Proteins

Abstract: Newer NMR methods, particularly in conjunction with stable isotope labeling, offer exciting approaches to structure-function studies of paramagnetic proteins. This review examines progress in NMR spectroscopy of iron-sulfur proteins. The application of multidimensional multinuclear NMR spectroscopy to iron-sulfur proteins and the optimization of NMR pulse sequences for rapidly relaxing spins have allowed investigators to determine sequence-specific assignments for numerous NMR signals in rubredoxins, ferredoxins, and high-potential iron proteins, including those from the cysteine residues that ligate iron ions or iron-sulfur clusters. These advances enable one to interpret the wealth of information derived from NMR parameters, such as the temperature and pH dependence of chemical shifts and the relaxation properties of the resonances that report on interactions between nuclei and between nuclei and unpaired electron density. This information is being used to test theoretical descriptions of electron distribution within these molecules and to model the structures and dynamic properties of the proteins in solution. Mutagenesis of these proteins, in conjunction with NMR studies, is beginning to reveal which residues are important for cluster formation and stability and which residues play a role in electron transfer to and from redox partner proteins.

Actin-Binding Protein Complexes at Atomic Resolution

Abstract: This review describes three structures of actin complexed with different monomer-binding proteins, namely with DNase I, gelsolin segment 1, and profilin. In these proteins, the binding sites are discontinuous in the sequence, and those residues that form intermolecular hydrogen bonds are not well conserved in homologous proteins. The strongly conserved residues that define the family of proteins in gelsolin and profilin reflect the underlying structural fold of each. The binding surfaces for segment 1 and profilin are different, although they peripherally overlap on actin. No extreme features in the binding surfaces of these complexes distinguish them from other globular proteins.

Site-specific dynamics in DNA: theory.

Abstract: This chapter reviews recent progress in understanding duplex DNA dynamics. The weakly bending rod model of Schurr and coworkers is described and compared to a model-free formulation of DNA dynamics. Numerical trajectory methods for obtaining dynamic information are also discussed. The general principles of magnetic resonance relaxation are the reviewed, and the methods by which molecular motions are incorporated into the calculation of relaxation rates or the simulation of experimental NMR and EPR data are described. The impact that the time scale of the dynamics exerts on various computational methods is considered, and in particular, the implementation of (a) the stochastic Liouville equation, (b) the Redfield relaxation matrix, and (c) tensorial preaveraging is described. Expressions for direct and cross-relaxation processes are developed by expanding the density matrix in terms of an irreducible tensor basis set.

Membrane-structure studies using X-ray standing waves.

Abstract: The principle and unique features of X-ray standing waves as a means for investigating membrane structure are described in this review. Thus far, X-ray standing waves have been used in structural studies of Langmuir-Blodgett and self-assembled monomolecular lipid films. Most recently, the method has been used in studies of supported membranes hosting the peripheral membrane protein, cytochrome c. Structural rearrangements occurring in membranes and at surfaces driven by temperature and composition changes have been monitored as well. Finally, ion distribution in the diffuse-double layer next to a charged membrane has been determined using this approach. The review addresses the manner in which these and related measurements were made. What is not covered in the review is a critical appraisal of the limitations of the X-ray standing wave method. Such limitations (which are pronounced and introduce significant mensuration ambiguities at short distances on silver mirrors as used in the membrane protein topology study) have just recently come to our attention and will be reported on separately (S Kirchner, Z Yin, J Wang & M Caffrey, in preparation).

Applications of parallel computing to biological problems.

Abstract: Parallel computers should provide the greatest processing power and memory for scientific simulations in the coming decades. This review discusses general strategies and specific algorithms for the use of various parallel architectures in simulations of biological and artificial polymers. General strategies include space partitioning (domain decomposition cell methods) and distributed independent simulations. Specific algorithms include cellular automata for efficient abstract polymer simulation. One algorithm, the two-space algorithm, is particularly efficient both for parallel and serial computation. Three applications, 2D melts, gel electrophoresis, and polymer collapse, are described. Simulations of high-density melts in 2D show that contrary to expectations, polymers do not completely segregate at the highest densities; instead, polymer interpenetration is significant. Preliminary simulations of gel electrophoresis show its behavior in the diffusive regimen and demonstrate the use of Cellular Automaton Machines (CAMs). Polymer collapse is studied in the regime of large departures from good solvent conditions. In this regime, kinetics plays a significant role. Collapse is dominated (nucleated) by migration of the chain ends.

My life in and Beyond the Laboratory

Abstract: Ephraim Katchalski-Katzir grew up in Israel at a time when the State was coming into being, and like many of those of his generation, participated in its creation and defense. He received his scientific education at the Hebrew University of Jerusalem and thereafter at the Polytechnic Institute of Brooklyn, Columbia University, and Harvard University. During and after the establishment of the State, he acted as scientific consultant to the defense leadership, and continued throughout his career to advise the Government of Israel on research and development. His public activities culminated in his election in 1973 as President of the State of Israel. As one of the founders of the Weizmann Institute of Science, he headed the Department of Biophysics for many years. His research included the study of poly-alpha-amino acids as protein models, immobilized enzymes, and polymers as chemical reagents. He also carried out theoretical and experimental work on the determination of distance distributions and conformational fluctuations in proteins by nonradiative energy-transfer techniques.