Showing papers in "Quarterly Reviews of Biophysics in 1997"

The Hofmeister series: salt and solvent effects on interfacial phenomena

Abstract: Advances in experimental and computational methodologies have led to a recent renewed interest in the Hofmeister series and its molecular origins. New results are surveyed and assessed. Insights into the underlying mechanisms have been gained, although deeper molecular understanding still seems to be elusive. The principal reason appears to be that the Hofmeister series emerges from a combination of a general effect of cosolutes (salts, etc.) on solvent structure, and of specific interactions between the cosolutes and the solute (protein or other biopolymer). Hence every system needs to be studied individually in detail, a state of affairs which is likely to continue for some time. A deeper understanding of the Hofmeister series can be an extraordinarily valuable guide to designing experiments, including not only those probing the series per se, but also those designed to elucidate the adsorption, aggregation and stabilization phenomena which underlie so many biological events. The aim of this review is to provide an up-to-date framework to guide such understanding, consolidating recent advances in the many fields on which the Hofmeister series impinges.

Infrared spectroscopy of proteins and peptides in lipid bilayers

Abstract: Infrared spectroscopy is a useful technique for the determination of conformation and orientation of membrane-associated proteins and lipids. The technique is especially powerful for detecting conformational changes by recording spectral differences before and after perturbations in physiological solution. Polarized infrared measurements on oriented membrane samples have revealed valuable information on the orientation of chemical groupings and substructures within membrane molecules which is difficult to obtain by other methods. The application of infrared spectroscopy to the static and dynamic structure of proteins and peptides in lipid bilayers is reviewed with some emphasis on the importance of sample preparation. Limitations of the technique with regard to the absolute determination of secondary structure and orientation and new strategies for structural assignments are also discussed.

Protein identification in the post-genome era: the rapid rise of proteomics

Abstract: Most advances in biology can usually be traced back to the development of a new technique: the recent explosion in sequence information in the databases arose from the pioneering work on separation methods by Frederick Sanger which paved the way for the development of protein (Sanger, 1945) and DNA/RNA (Maxam & Gilbert, 1977; Sanger, 1981) sequencing and culminated in the receipt of two Nobel prizes by Sanger. The initial phase of sequence database expansion was slow due to the tedious and slow nature of protein sequencing. Peptide sequencing was carried out manually and the complete analysis of a protein was tiresome, requiring the isolation of sufficient peptides from several digests of the target protein using proteases of different specialities to collect an overlapping set of fragments which cover the whole sequence. Protein sequencing gained momentum when the phenylisothiocyanate sequencing chemistry developed by Edman in 1949 was automated (Edman & Begg, 1967) and a commercial instrument requiring lower amounts (nanomoles) of sample was put on the market. Further technical advances such as novel valves to deal with small volumes of aggressive chemicals, the introduction of high pressure liquid chromatography (HPLC), and novel supports for sample immobilization, were all combined in the first gas phase sequencers, greatly increasing the sensitivity and allowing automated data collection (Hewick et al. 1981) and analysis. The new instruments with a sensitivity in the low picomole range appeared as rapid advances in DNA technology such as the development of restriction mapping (Danna et al. 1973), cloning (Cohen et al. 1973) and the dideoxynucleotide sequencing chemistry were threatening to make protein chemistry a relic of the past (Malcolm, 1978).

From membrane to molecule to the third amino acid from the left with a membrane transport protein.

Abstract: The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)

Dynamic receptor superstructures at the plasma membrane

Abstract: 1. INTRODUCTION 681.1 Receptor patterns in the plasma membrane 681.2 Different types of receptor patterns 712. METHODS TO INVESTIGATE NON-RANDOM RECEPTOR CLUSTERING 732.1 Fluorescence resonance energy transfer 732.2 Flow cytometric energy transfer measurement 782.3 Fluorescence anisotropy and energy transfer 792.4 Photobleaching energy transfer on single cells 812.5 Two-dimensional mapping of receptor superstructures 822.6 Detecting single receptor molecules 852.7 Chemical identification of receptor clusters 862.8 Electron microscopy 872.9 Scanning force microscopy 883. CONFORMATIONAL STATES OF RECEPTORS 903.1 Multi-subunit receptor structures 903.2 Physical parameters influencing conformational states 913.3 Chemical interactions and receptor conformations 924. ON THE ORIGIN OF NATURALLY OCCURRING RECEPTOR CLUSTERS 934.1 Synthesis of receptors and their localization in the plasma membrane 934.2 Lipid domain structure of the plasma membrane 944.3 The validity of the SingerNicolson model 945. CONCLUSIONS 966. ACKNOWLEDGEMENTS 967. REFERENCES 97

Periodic patterns in biochemical reactions

Abstract: Whenever fundamental features of living systems and their molecular basis are reviewed, the problem of timing, of time setting or free open-end running times is only marginally on the desk of research agendas, although the finite ageing as one of the features resulting from time markers is known since long. With the discovery of cellular and most important of cellfree oscillatory processes new concepts and experimental techniques were designed to approach these questions more directly leading not only to a better understanding of timing but strongly contributed to concepts for spatial pattern generation. As given in the list of contents major items in the field of intracellular and intercellular periodic reactions are reviewed in Sections 2–7 in terms of specific properties of various systems and in Section 8 in summing important features common to all oscillatory stems in chemistry and biology. Section 9 draws attention to the problem of patterning in the mesoscopic domains of living systems, which is so basic in terms of the volume dimensions specific for the cellular and subcellular reaction compartments in biology. The last chapter sets some marks on urgent problems currently approached by the combined methods of molecular genetics, biochemistry and computer technologies.1. INTRODUCTION AND HISTORY 1222. PERIODIC GLYCOLYSIS: TEMPORAL AND SPATIAL OSCILLATIONS 1232.1 Cell-free glycolysis 1232.2 Intact cells 1282.2.1 Yeast 1282.2.2 Myocytes 1302.2.3 β-Cells of the islets of pancreas 1313. CALCIUM OSCILLATIONS 1313.1 Temporal oscillations 1313.2 Calcium waves 1363.3 Physiology 1384. MICROTUBULE OSCILLATIONS 1405. THE MITOTIC OSCILLATOR 1416. DICTYOSTELIUM DISCOIDEUM 1447. CARDIOMUSCULAR NETWORK 1508. GENERAL PROPERTIES OF DYNAMIC SPATIAL PATTERNS 1529. MICROSCOPIC PATTERNS 15610. PERSPECTIVES 16211. ACKNOWLEDGEMENTS 16312. REFERENCES 163

Cyclic nucleotide-gated channels: structural basis of ligand efficacy and allosteric modulation.

Abstract: Most working proteins, including metabolic enzymes, transcription regulators, and membrane receptors, transporters, and ion channels, share the property of allosteric coupling. The term 'allosteric' means that these proteins mediate indirect interactions between sites that are physically separated on the protein. In the example of ligand-gated ion channels, the binding of a suitable ligand elicits local conformational changes at the binding site, which are coupled to further conformational changes in regions distant from the binding site. The physical motions finally arrive at the site of biological activity: the ion-permeating pore. The conformational changes that lead from the ligand binding to the actual opening of the pore comprise 'gating'. In 1956, del Castillo and Katz suggested that the competition between different ligands at nicotinic acetylcholine receptors (nAChRs) could be explained by formation of an intermediate, ligand-bound, yet inactive state of the receptor, which separates the active state of the receptor from the initial binding of the ligand (del Castillo & Katz, 1957). This 'binding-then-gating', two-step model went beyond the then-prevailing drug-receptor model that assumes a single bimolecular binding reaction, and paralleled Stephenson's conceptual dichotomy of 'affinity' and 'efficacy' (Stephenson, 1956). In 1965 Monod, Wyman and Changeux presented a simple allosteric model (the MWC model) (Monod et al. 1965) that explained the cooperative binding of oxygen to haemoglobin; it was adopted as an important paradigm for ligand-gated channels soon after its initial formulation (Changeux et al. 1967; Karlin, 1967; Colquhoun, 1973).