ALBERT

Notes: Abstract Owing to the rapid development of in vivo applications for non-viral gene delivery vectors, it is necessary to have a better understanding of how the structure-activity relationships of these lipid-DNA complexes are affected by their environment. Indeed, research in gene therapy first focused on in vitro cell culture studies to determine the mechanisms involved in the delivery of DNA into the cell. New biophysical techniques such as electron microscopy and X-ray diffraction have been developed to discern the structure of the lipid-DNA complex. However, further studies have revealed discrepancies between optimal lipid-DNA formulations for in vitro transfection and for in vivo administration of these vectors. Furthermore, some immune stimulatory effects have been associated with in vivo lipid-DNA administration. This review summarizes the current state of knowledge on in vitro and in vivo lipid-DNA complex transfections. New prospects of vectors for in vivo gene transfer are also discussed.

Notes: Abstract Hundreds of acetyltransferases exist. All use a common acetyl donor-acetyl coenzyme A-and each exhibits remarkable specificity for acetyl acceptors, which include small molecules and proteins. Analysis of the primary sequences of these enzymes indicates that they can be sorted into several superfamilies. This review covers the three-dimensional structures of members of one of these superfamilies, now referred to in the literature as the GCN5-related N-acetyltransferases (GNAT), reflecting the importance of one functional category, the histone acetyltransferases. Despite the diversity of substrate specificities, members of the GNAT superfamily demonstrate remarkable similarity in protein topology and mode of acetyl coenzyme A binding, likely reflecting a conserved catalytic mechanism.

Notes: Abstract Protein kinase C homology-1 and -2, FYVE, and pleckstrin homology domains are ubiquitous in eukaryotic signal transduction and membrane-trafficking proteins. These domains regulate subcellular localization and protein function by binding to lipid ligands embedded in cell membranes. Structural and biochemical analysis of these domains has shown that their molecular mechanisms of membrane binding depend on a combination of specific and nonspecific interactions with membrane lipids. In vivo studies of green fluorescent protein fusions have highlighted the key roles of these domains in regulating protein localization to plasma and internal membranes in cells.

Notes: Abstract Although the force fields and interaction energies that control protein behavior can be inferred indirectly from equilibrium and kinetic measurements, recent developments have made it possible to quantify directly (a) the ranges, magnitudes, and time dependence of the interaction energies and forces between biological materials; (b) the mechanical properties of isolated proteins; and (c) the strength of single receptor-ligand bonds. This review describes recent results obtained by using the atomic force microscope, optical tweezers, the surface force apparatus, and micropipette aspiration to quantify short-range protein-ligand interactions and the long-range, nonspecific forces that together control protein behavior. The examples presented illustrate the power of force measurements to quantify directly the force fields and energies that control protein behavior.

Notes: Abstract Cys2His2 zinc fingers are one of the most common DNA-binding motifs found in eukaryotic transcription factors. These proteins typically contain several fingers that make tandem contacts along the DNA. Each finger has a conserved betabetaalpha structure, and amino acids on the surface of the alpha-helix contact bases in the major groove. This simple, modular structure of zinc finger proteins, and the wide variety of DNA sequences they can recognize, make them an attractive framework for attempts to design novel DNA-binding proteins. Several studies have selected fingers with new specificities, and there clearly are recurring patterns in the observed side chain-base interactions. However, the structural details of recognition are intricate enough that there are no general rules (a "recognition code") that would allow the design of an optimal protein for any desired target site. Construction of multifinger proteins is also complicated by interactions between neighboring fingers and the effect of the intervening linker. This review analyzes DNA recognition by Cys2His2 zinc fingers and summarizes progress in generating proteins with novel specificities from fingers selected by phage display.

Notes: Abstract This review describes how kinetic experiments using techniques with dramatically improved time resolution have contributed to understanding mechanisms in protein folding. Optical triggering with nanosecond laser pulses has made it possible to study the fastest-folding proteins as well as fundamental processes in folding for the first time. These include formation of alpha-helices, beta-sheets, and contacts between residues distant in sequence, as well as overall collapse of the polypeptide chain. Improvements in the time resolution of mixing experiments and the use of dynamic nuclear magnetic resonance methods have also allowed kinetic studies of proteins that fold too fast (〉 103 s-1) to be observed by conventional methods. Simple statistical mechanical models have been extremely useful in interpreting the experimental results. One of the surprises is that models originally developed for explaining the fast kinetics of secondary structure formation in isolated peptides are also successful in calculating folding rates of single domain proteins from their native three-dimensional structure.

Notes: Abstract ClC-type chloride channels are ubiquitous throughout the biological world. Expressed in nearly every cell type, these proteins have a host of biological functions. With nine distinct homologues known in eukaryotes, the ClCs represent the only molecularly defined family of chloride channels. ClC channels exhibit features of molecular architecture and gating mechanisms unprecedented in other types of ion channels. They form two-pore homodimers, and their voltage-dependence arises not from charged residues in the protein, but rather via coupling of gating to the movement of chloride ions within the pore. Because the functional characteristics of only a few ClC channels have been studied in detail, we are still learning which properties are general to the whole family. New approaches, including structural analyses, will be crucial to an understanding of ClC architecture and function.

Notes: Abstract In the past decade, a general design for sequence-specific minor groove ligands has evolved, based on the natural products distamycin and netropsin. By utilizing a basic set of design rules for connecting pyrrole, imidazole, and hydroxypyrrole modules, new ligands can be prepared to target almost any sequence of interest with both high affinity and specificity. In this review we present the design rules with a brief history of how they evolved. The structural basis for sequence-specific recognition is explained, together with developments that allow linking of recognition modules that enable targeting of long DNA sequences. Examples of the affinity and specificity that can be achieved with a number of variations on the basic design are given. Recently these molecules have been used to compete with proteins both in vitro and in vivo, and a brief description of the experimental results are given.

Notes: Abstract NMR spin relaxation spectroscopy is a powerful approach for characterizing intramolecular and overall rotational motions in proteins. This review describes experimental methods for measuring laboratory frame spin relaxation rate constants by high-resolution solution-state NMR spectroscopy, together with theoretical approaches for interpreting spin relaxation data in order to quantify protein conformational dynamics on picosecond-nanosecond time scales. Recent applications of these techniques to proteins are surveyed, and investigations of the contribution of conformational chain entropy to protein function are highlighted. Insights into the dynamical properties of proteins obtained from NMR spin relaxation spectroscopy are compared with results derived from other experimental and theoretical techniques.

Notes: Abstract The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Structures of the SNARE complex, synaptotagmin III, nSec1, domains of the NSF chaperone and its adaptor SNAP, and Rab3 and some of its effectors provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models.

Notes: Abstract Considerable recent progress has been made in the field of ab initio protein structure prediction, as witnessed by the third Critical Assessment of Structure Prediction (CASP3). In spite of this progress, much work remains, for the field has yet to produce consistently reliable ab initio structure prediction protocols. In this work, we review the features of current ab initio protocols in an attempt to highlight the foundations of recent progress in the field and suggest promising directions for future work.

Notes: Abstract Species and tissue-specific isozymes of phosphorylase display differences in regulatory properties consistent with their distinct roles in particular organisms and tissues. In this review, we compare crystallographic structures of regulated and unregulated phosphorylases, including maltodextrin phosphorylase (MalP) from Escherichia coli, glycogen phosphorylase from yeast, and mammalian isozymes from muscle and liver tissues. Mutagenesis and functional studies supplement the structural work and provide insights into the structural basis for allosteric control mechanisms. MalP, a simple, unregulated enzyme, is contrasted with the more complicated yeast and mammalian phosphorylases that have evolved regulatory sites onto the basic catalytic architecture. The human liver and muscle isozymes show differences structurally in their means of invoking allosteric activation. Phosphorylation, though common to both the yeast and mammalian enzymes, occurs at different sites and activates the enzymes by surprisingly different mechanisms.

Notes: Abstract Computer modeling has been developed and widely applied in studying molecules of biological interest. The force field is the cornerstone of computer simulations, and many force fields have been developed and successfully applied in these simulations. Two interesting areas are (a) studying enzyme catalytic mechanisms using a combination of quantum mechanics and molecular mechanics, and (b) studying macromolecular dynamics and interactions using molecular dynamics (MD) and free energy (FE) calculation methods. Enzyme catalysis involves forming and breaking of covalent bonds and requires the use of quantum mechanics. Noncovalent interactions appear ubiquitously in biology, but here we confine ourselves to review only noncovalent interactions between protein and protein, protein and ligand, and protein and nucleic acids.

Notes: Abstract Molecular chaperones are required to assist folding of a subset of proteins in Escherichia coli. We describe a conceptual framework for understanding how the GroEL-GroES system assists misfolded proteins to reach their native states. The architecture of GroEL consists of double toroids stacked back-to-back. However, most of the fundamentals of the GroEL action can be described in terms of the single ring. A key idea in our framework is that, with coordinated ATP hydrolysis and GroES binding, GroEL participates actively by repeatedly unfolding the substrate protein (SP), provided that it is trapped in one of the misfolded states. We conjecture that the unfolding of SP becomes possible because a stretching force is transmitted to the SP when the GroEL particle undergoes allosteric transitions. Force-induced unfolding of the SP puts it on a higher free-energy point in the multidimensional energy landscape from which the SP can either reach the native conformation with some probability or be trapped in one of the competing basins of attraction (i.e., the SP undergoes kinetic partitioning). The model shows, in a natural way, that the time scales in the dynamics of the allosteric transitions are intimately coupled to folding rates of the SP. Several scenarios for chaperonin-assisted folding emerge depending on the interplay of the time scales governing the cycle. Further refinement of this framework may be necessary because single molecule experiments indicate that there is a great dispersion in the time scales governing the dynamics of the chaperonin cycle.

Notes: Abstract The mammalian thioredoxins are a family of small (approximately 12 kDa) redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase and in turn reduce oxidized cysteine groups on proteins. The two main thioredoxins are thioredoxin-1, a cytosolic and nuclear form, and thioredoxin-2, a mitochondrial form. Thioredoxin-1 has been studied more. It performs many biological actions including the supply of reducing equivalents to thioredoxin peroxidases and ribonucleotide reductase, the regulation of transcription factor activity, and the regulation of enzyme activity by heterodimer formation. Thioredoxin-1 stimulates cell growth and is an inhibitor of apoptosis. Thioredoxins may play a role in a variety of human diseases including cancer. An increased level of thioredoxin-1 is found in many human tumors, where it is associated with aggressive tumor growth. Drugs are being developed that inhibit thioredoxin and that have antitumor activity.

Notes: Abstract Early NMR structural studies of serum lipoproteins were based on 1H, 13C, 31P, and 2H studies of lipid components. From the early studies information on composition, lipid chain dynamics and order parameters, and monolayer organization resulted. More recently, selective or complete isotopic labeling techniques, combined with multidimensional NMR spectroscopy, have resulted in structural information of apoprotein fragments. Finally, use of heteronuclear three- and four-dimensional experiments have yielded solution structures and protein-lipid interactions of intact apolipoproteins C-I, C-II, and A-I.

Notes: Abstract During the course of their biological function, proteins undergo different types of structural rearrangements ranging from local to large-scale conformational changes. These changes are usually triggered by their interactions with small-molecular-weight ligands or other macromolecules. Because binding interactions occur at specific sites and involve only a small number of residues, a chain of cooperative interactions is necessary for the propagation of binding signals to distal locations within the protein structure. This process requires an uneven structural distribution of protein stability and cooperativity as revealed by NMR-detected hydrogen/deuterium exchange experiments under native conditions. The distribution of stabilizing interactions does not only provide the architectural foundation to the three-dimensional structure of a protein, but it also provides the required framework for functional cooperativity. In this review, the statistical thermodynamic linkage between protein stability, functional cooperativity, and ligand binding is discussed.

Notes: Abstract Proteins are designed to function in environments crowded by cosolutes, but most studies of protein equilibria are conducted in dilute solution. While there is no doubt that crowding changes protein equilibria, interpretations of the changes remain controversial. This review combines experimental observations on the effect of small uncharged cosolutes (mostly sugars) on protein stability with a discussion of the thermodynamics of cosolute-induced nonideality and critical assessments of the most commonly applied interpretations. Despite the controversy surrounding the most appropriate manner for interpreting these effects of thermodynamic nonideality arising from the presence of small cosolutes, experimental advantage may still be taken of the ability of the cosolute effect to promote not only protein stabilization but also protein self-association and complex formation between dissimilar reactants. This phenomenon clearly has potential ramifications in the cell, where the crowded environment could well induce the same effects.

Notes: Abstract Nuclear receptors (NRs) form a superfamily of ligand-inducible transcription factors composed of several domains. Recent structural studies focused on domain E, which harbors the ligand-binding site and the ligand-dependent transcription activation function AF-2. Structures of single representatives in an increasing number of various complexes as well as new structures of further NRs addressed issues such as discrimination of ligands, superagonism, isotype specificity, and partial agonism. Until today, one unique transcriptionally active form of domain E was determined; however, divergent tertiary structures of apo-forms and transcriptionally inactive forms are known. Thus, recent results link the transformation of NRs upon ligand binding to principles of protein folding. Furthermore, the ensemble of NR structures, including those of DNA-binding domains, provides one of the foundations for the understanding of interactions with transcription intermediary factors up to the characterization of the link between NR complexes and the basal transcriptional machinery at the structural level.

Notes: Abstract Microtubules are polymers that are essential for, among other functions, cell transport and cell division in all eukaryotes. The regulation of the microtubule system includes transcription of different tubulin isotypes, folding of alpha/beta-tubulin heterodimers, post-translation modification of tubulin, and nucleotide-based microtubule dynamics, as well as interaction with numerous microtubule-associated proteins that are themselves regulated. The result is the precise temporal and spatial pattern of microtubules that is observed throughout the cell cycle. The recent high-resolution analysis of the structure of tubulin and the microtubule has brought new insight to the study of microtubule function and regulation, as well as the mode of action of antimitotic drugs that disrupt normal microtubule behavior. The combination of structural, genetic, biochemical, and biophysical data should soon give us a fuller understanding of the exquisite details in the regulation of the microtubule cytoskeleton.

Notes: Abstract The past few years have seen exciting advances in understanding the structure and function of catalytic RNA. Crystal structures of several ribozymes have provided detailed insight into the folds of RNA molecules. Models of other biologically important RNAs have been constructed based on structural, phylogenetic, and biochemical data. However, many questions regarding the catalytic mechanisms of ribozymes remain. This review compares the structures and possible catalytic mechanisms of four small self-cleaving RNAs: the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.

Notes: Abstract Active transport requires the alternation of substrate uptake and release with a switch in the access of the substrate binding site to the two sides of the membrane. Both the transfer and switch aspects of the photocycle have been subjects of magnetic resonance studies in bacteriorhodopsin. The results for ion transfer indicate that the Schiff base of the chromophore is hydrogen bonded before, during, and after its deprotonation. This suggests that the initial complex counterion of the Schiff base decomposes in such a way that the Schiff base carries its immediate hydrogen-bonding partner with it as it rotates during the first half of the photocycle. If so, bacteriorhodopsin acts as an inward-directed hydroxide pump rather than as an outward-directed proton pump. The studies of the access switch explore both protein-based and chromophore-based mechanisms. Combined with evidence from functional studies of mutants and other forms of spectroscopy, the results suggest that maintaining access to the extracellular side of the protein after photoisomerization involves twisting of the chromophore and that the decisive switch in access to the cytoplasmic side results from relaxation of the chromophore when the constraints on the Schiff base are released by decomposition of the complex counterion.

Notes: Abstract We review the physical properties of phosphatidylinositol 4,5-bisphosphate (PIP2) that determine both its specific interactions with protein domains of known structure and its nonspecific electrostatic sequestration by unstructured domains. Several investigators have postulated the existence of distinct pools of PIP2 within the cell to account for the myriad functions of this lipid. Recent experimental work indicates certain regions of the plasma membrane-membrane ruffles and nascent phagosomes-do indeed concentrate PIP2. We consider two mechanisms that could account for this phenomenon: local synthesis and electrostatic sequestration. We conclude by considering the hypothesis that proteins such as MARCKS bind a significant fraction of the PIP2 in a cell, helping to sequester it in lateral membrane domains, then release this lipid in response to local signals such as an increased concentration of Ca++/calmodulin or activation of protein kinase C.

Notes: Abstract The first crystal structures of intact T cell receptors (TCRs) bound to class I peptide-MHC (pMHCs) antigens were determined in 1996. Since then, further structures of class I TCR/pMHC complexes have explored the degree of structural variability in the TCR-pMHC system and the structural basis for positive and negative selection. The recent determination of class II and allogeneic class I TCR/pMHC structures, as well as those of accessory molecules (e.g., CD3), has pushed our knowledge of TCR/pMHC interactions into new realms, shedding light on clinical pathologies, such as graft rejection and graft-versus-host disease. Furthermore, the determination of coreceptor structures lays the foundation for a more comprehensive structural description of the supramolecular TCR signaling events and those assemblies that arise in the immunological synapse. While these telling photodocumentaries of the TCR/pMHC interaction are composed mainly from static crystal structures, a full description of the biological snapshots in T cell signaling requires additional analytical methods that record the dynamics of the process. To this end, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and ultracentrifugation (UC) have furnished both affinities and kinetics of the TCR/pMHC association. In the past year, structural, biochemical, and molecular biological data describing TCR/pMHC interactions have sublimely coalesced into a burgeoning well of understanding that promises to deliver further insights into T cell recognition. The coming years will, through a more intimate union of structural and kinetic data, allow many pressing questions to be addressed, such as how TCR/pMHC ligation is affected by coreceptor binding and what is the mechanism of TCR signaling in both early and late stages of T cell engagement with antigen-presenting cells.

Notes: Abstract The structures of an increasing number of channels and other alpha-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family. In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins. In the case of channels, alpha-helices create the sealed barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes. The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle. The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity. Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.

Notes: Abstract Using luminescent lanthanides, instead of conventional fluorophores, as donor molecules in resonance energy transfer measurements offers many technical advantages and opens up a wide range of new applications. Advantages include farther measurable distances (~100 A) with greater accuracy, insensitivity to incomplete labeling, and the ability to use generic relatively large labels, when necessary. Applications highlighted include the study of ion channels in living cells, protein-protein interaction in cells, DNA-protein complexes, and high-throughput screening assays to measure peptide dimerization associated with DNA transcription factors and ligand-receptor interactions.

Notes: Abstract Cryo-electron microscopy (cryo-EM) of biological molecules in single-particle (i.e., unordered, nonaggregated) form is a new approach to the study of molecular assemblies, which are often too large and flexible to be amenable to X-ray crystallography. New insights into biological function on the molecular level are expected from cryo-EM applied to the study of such complexes "trapped" at different stages of their conformational changes and dynamical interactions. Important molecular machines involved in the fundamental processes of transcription, mRNA splicing, and translation are examples for successful applications of the new technique, combined with structural knowledge gained by conventional techniques of structure determination, such as X-ray crystallography and NMR.

Notes: Abstract The recent report of the crystal structure of rhodopsin provides insights concerning structure-activity relationships in visual pigments and related G protein-coupled receptors (GPCRs). The seven transmembrane helices of rhodopsin are interrupted or kinked at multiple sites. An extensive network of interhelical interactions stabilizes the ground state of the receptor. The ligand-binding pocket of rhodopsin is remarkably compact, and several chromophore-protein interactions were not predicted from mutagenesis or spectroscopic studies. The helix movement model of receptor activation, which likely applies to all GPCRs of the rhodopsin family, is supported by several structural elements that suggest how light-induced conformational changes in the ligand-binding pocket are transmitted to the cytoplasmic surface. The cytoplasmic domain of the receptor includes a helical domain extending from the seventh transmembrane segment parallel to the bilayer surface. The cytoplasmic surface appears to be approximately large enough to bind to the transducin heterotrimer in a one-to-one complex. The structural basis for several unique biophysical properties of rhodopsin, including its extremely low dark noise level and high quantum efficiency, can now be addressed using a combination of structural biology and various spectroscopic methods. Future high-resolution structural studies of rhodopsin and other GPCRs will form the basis to elucidate the detailed molecular mechanism of GPCR-mediated signal transduction.

Notes: Abstract Integrins are a structurally elaborate family of heterodimers that mediate divalent cation-dependent cell adhesion in a wide range of biological contexts. The inserted (I) domain binds ligand in the subset of integrins in which it is present. Its structure has been determined in two alternative conformations, termed open and closed. In striking similarity to signaling G proteins, rearrangement of a Mg2+-binding site is linked to large conformational movements in distant backbone regions. Mutations have been used to stabilize either the closed or open structures. These show that the snapshots of the open conformation seen only in the presence of a ligand or a ligand mimetic represent a high-affinity, ligand-binding conformation, whereas those of the closed conformation correspond to a low-affinity conformation. The C-terminal alpha-helix moves 10 A down the side of the domain in the open conformation. Locking in the conformation of the preceding loop is sufficient to increase affinity for ligand 9000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. The transition from the closed to open conformation has been implicated in fast (〈1 s) regulation of integrin affinity in response to activation signals from inside the cell. Recent integrin structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the headpiece, and a critical role for integrin EGF domains in the stalk region. These studies suggest that the headpiece of the integrin faces down toward the membrane in the inactive conformation and extends upward in a "switchblade"-like opening motion upon activation. These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.

Notes: Abstract Optical single transporter recording (OSTR) is an emerging technique for the fluorescence microscopic measurement of transport kinetics in membrane patches. Membranes are attached to transparent microarrays of cylindrical test compartments (TCs) ~0.1-100 mum in diameter and ~10-100 mum in depth. Transport across membrane patches that may contain single transporters or transporter populations is recorded by confocal microscopy. By these means transport of proteins through single nuclear pore complexes has been recorded at rates of 〈1 translocation/s. In addition to the high sensitivity in terms of measurable transport rates OSTR features unprecedented spatial selectivity and parallel processing. This article reviews the conceptual basis of OSTR and its realization. Applications to nuclear transport are summarized. The further development of OSTR is discussed and its extension to a diversity of transporters, including translocases and ATP-binding cassette (ABC) pumps, projected.

Notes: Abstract Since mid-1990, with cloning and identification of several families of natural killer (NK) receptors, research on NK cells began to receive appreciable attention. Determination of structures of NK cell surface receptors and their ligand complexes led to a fast growth in our understanding of the activation and ligand recognition by these receptors as well as their function in innate immunity. Functionally, NK cell surface receptors are divided into two groups, the inhibitory and the activating receptors. Structurally, they belong to either the immunoglobulin (Ig)-like receptor superfamily or the C-type lectin-like receptor (CTLR) superfamily. Their ligands are either members of class I major histocompatibility complexes (MHC) or homologs of class I MHC molecules. The inhibitory form of NK receptors provides the protective immunity through recognizing class I MHC molecules with self-peptides on healthy host cells. The activating, or the noninhibitory, NK receptors mediate the killing of tumor or virally infected cells through their specific ligand recognition. The structures of activating and inhibitory NK cell surface receptors and their complexes with the ligands determined to date, including killer immunoglobulin-like receptors (KIRs) and their complexes with HLA molecules, CD94, Ly49A, and its complex with H-2Dd, and NKG2D receptors and their complexes with class I MHC homologs, are reviewed here.

Notes: Abstract Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

Notes: Abstract The lamba integrase, or tyrosine-based family of site-specific recombinases, plays an important role in a variety of biological processes by inserting, excising, and inverting DNA segments. Flp, encoded by the yeast 2-mum plasmid, is the best-characterized eukaryotic member of this family and is responsible for maintaining the copy number of this plasmid. Over the past several years, structural and biochemical studies have shed light on the details of a common catalytic scheme utilized by these enzymes with interesting variations under different biological contexts. The emergence of new Flp structures and solution data provides insights not only into its unique mechanism of active site assembly and activity regulation but also into the specific contributions of certain protein residues to catalysis.

Notes: Abstract The past decade has witnessed increasingly detailed insights into the structural mechanism of the bacteriorhodopsin photocycle. Concurrently, there has been much progress within our knowledge pertaining to the lipids of the purple membrane, including the discovery of new lipids and the overall effort to localize and identify each lipid within the purple membrane. Therefore, there is a need to classify this information to generalize the findings. We discuss the properties and roles of haloarchaeal lipids and present the structural data as individual case studies. Lipid-protein interactions are discussed in the context of structure-function relationships. A brief discussion of the possibility that bacteriorhodopsin functions as a light-driven inward hydroxide pump rather than an outward proton pump is also presented.

Notes: Abstract G protein-coupled receptors (GPCRs) are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways activating cellular processes. Rhodopsin is a GPCR found in rod cells in retina where it functions as a photopigment. Its molecular structure is known from cryo-electron microscopic and X-ray crystallographic studies, and this has reshaped many structure/function questions important in vision science. In addition, this first GPCR structure has provided a structural template for studies of other GPCRs, including many known drug targets. After presenting an overview of the major structural elements of rhodopsin, recent literature covering the use of the rhodopsin structure in analyzing other GPCRs will be summarized. Use of the rhodopsin structural model to understand the structure and function of other GPCRs provides strong evidence validating the structural model.

Notes: Abstract The coupling of high-performance mass spectrometry instrumentation with highly efficient chromatographic and electrophoretic separations has enabled rapid qualitative and quantitative analysis of thousands of proteins from minute samples of biological materials. Here, we review recent progress in the development and application of mass spectrometry-based techniques for the qualitative and quantitative analysis of global proteome samples derived from whole cells, tissues, or organisms. Techniques such as multidimensional peptide and protein separations coupled with mass spectrometry, accurate mass measurement of peptides from global proteome digests, and mass spectrometric characterization of intact proteins hold great promise for characterization of highly complex protein mixtures. Advances in chemical tagging and isotope labeling techniques have enabled quantitative analysis of proteomes, and highly specific isolation strategies have been developed aimed at selective analysis of posttranslationally modified proteins.

Notes: Residual dipolar couplings (RDCs) have recently emerged as a new tool in nuclear magnetic resonance (NMR) with which to study macromolecular structure and function in a solution environment. RDCs are complementary to the more conventional use of NOEs to provide structural information. While NOEs are local-distance restraints, RDCs provide long-range orientational information. RDCs are now widely utilized in structure calculations. Increasingly, they are being used in novel applications to address complex issues in structural biology such as the accurate determination of the global structure of oligonucleotides and the relative orientation of protein domains. This review briefly describes the theory and methods for obtaining RDCs and then describes the range of biological applications where RDCs have been used.

Notes: Views of how cell membranes are organized are presently changing. The lipid bilayer that constitutes these membranes is no longer understood to be a homogeneous fluid. Instead, lipid assemblies, termed rafts, have been introduced to provide fluid platforms that segregate membrane components and dynamically compartmentalize membranes. These assemblies are thought to be composed mainly of sphingolipids and cholesterol in the outer leaflet, somehow connected to domains of unknown composition in the inner leaflet. Specific classes of proteins are associated with the rafts. This review critically analyzes what is known of phase behavior and liquid-liquid immiscibility in model systems and compares these data with what is known of domain formation in cell membranes.

Notes: Abstract I have had a very fortunate career in astronomy, benefiting greatly from numerous accidents of fate. I grew up in Cincinnati, Ohio, served in the US Army Air Force in World War II, and had all my further education at the University of Chicago, from PhB in the College to PhD in astronomy and astrophysics. There, as a postdoc at Princeton University, and as a young faculty member at Caltech and Mount Wilson and Palomar Observatories, I had excellent teachers and mentors. I have done research primarily on gaseous nebulae and active galactic nuclei, but also made a few early contributions on stellar interiors and the heating in the outer layers of the Sun. The major part of my scientific career was at the University of Wisconsin and Lick Observatory, but I also had three productive years at the Institute for Advanced Study.

Notes: Abstract ROSAT observations indicate that approximately half of all nearby groups of galaxies contain spatially extended X-ray emission. The radial extent of the X-ray emission is typically 50-500 h-1100 kpc or approximately 10-50% of the virial radius of the group. Diffuse X-ray emission is generally restricted to groups that contain at least one early-type galaxy. X-ray spectroscopy suggests the emission mechanism is most likely a combination of thermal bremsstrahlung and line emission. This interpretation requires that the entire volume of groups be filled with a hot, low-density gas known as the intragroup medium. ROSAT and ASCA observations indicate that the temperature of the diffuse gas in groups ranges from approximately 0.3 keV to 2 keV. Higher temperature groups tend to follow the correlations found for rich clusters between X-ray luminosity, temperature, and velocity dispersion. However, groups with temperatures below approximately 1 keV appear to fall off the cluster LX-T relationship (and possibly the LX-sigma and sigma-T cluster relationships, although evidence for these latter departures is at the present time not very strong). Deviations from the cluster LX-T relationship are consistent with preheating of the intragroup medium by an early generation of stars and supernovae. There is now considerable evidence that most X-ray groups are real, physical systems and not chance superpositions or large-scale filaments viewed edge-on. Assuming the intragroup gas is in hydrostatic equilibrium, X-ray observations can be used to estimate the masses of individual systems. ROSAT observations indicate that the typical mass of an X-ray group is ~1013 h-1100 M out to the radius to which X-ray emission is currently detected. The observed baryonic masses of groups are a small fraction of the X-ray determined masses, which implies that groups are dominated by dark matter. On scales of the virial radius, the dominant baryonic component in groups is likely the intragroup medium.

Notes: Abstract The brown dwarfs occupy the gap between the least massive star and the most massive planet. They begin as dimly stellar in appearance and experience fusion (of at least deuterium) in their interiors. But they are never able to stabilize their luminosity or temperature and grow ever fainter and cooler with time. For that reason, they can be viewed as a constituent of baryonic "dark matter." Indeed, we currently have a hard time directly seeing an old brown dwarf beyond 100 pc. After 20 years of searching and false starts, the first confirmed brown dwarfs were announced in 1995. This was due to a combination of increased sensitivity, better search strategies, and new means of distinguishing substellar from stellar objects. Since then, a great deal of progress has been made on the observational front. We are now in a position to say a substantial amount about actual brown dwarfs. We have a rough idea of how many of them occur as solitary objects and how many are found in binary systems. We have obtained the first glimpse of atmospheres intermediate in temperature between stars and planets, in which dust formation is a crucial process. This has led to the proposal of the first new spectral classes in several decades and the need for new diagnostics for classification and setting the temperature scale. The first hints on the substellar mass function are in hand, although all current masses depend on models. It appears that numerically, brown dwarfs may well be almost as common as stars (though they appear not to contain a dynamically interesting amount of mass).

Notes: Abstract This review deals with the winds from "normal" hot stars such as O-stars, B- and A-supergiants, and Central Stars of Planetary Nebulae with O-type spectra. The advanced diagnostic methods of stellar winds, including an assessment of the accuracy of the determinations of global stellar wind parameters (terminal velocities, mass-loss rates, wind momenta, and energies), are introduced and scaling relations as a function of stellar parameters are provided. Observational results are interpreted in the framework of the stationary, one-dimensional (1-D) theory of line-driven winds. Systematic effects caused by nonhomogeneous structures, time dependence, and deviations from spherical symmetry are discussed. The review finishes with a brief description of the role of stellar winds as extragalactic distance indicators and as tracers of the chemical composition of galaxies at high redshift.

Notes: Abstract We focus on new observational capabilities (Yohkoh, SoHO, TRACE), observations, modeling approaches, and insights into physical processes of the solar corona. The most impressive new results and problems discussed in this article can be appreciated from the movies available on the Annual Reviews website and at http://www.lmsal.com/pub/araa/araa.html . "The Sun is new each day." Heraclites (ca 530-475 BC) "Everything flows." Heraclites (ca 530-475 BC)

Notes: Abstract This review takes a critical look at the cosmological scenario at the turn of the century by examining the available cosmological models in the light of the present observational evidence. The center stage is held by the big bang models, which are collectively referred to here as standard cosmology (SC) and its extensions. SC itself is characterized by a seven parameter set of models based on Einstein's general theory of relativity. The seven parameters are H0, OmegaB, OmegaDM, OmegaLambda, OmegaR (describing the background universe, and A, n (specifying the amplitude and power law index of initial fluctuation spectrum). The extended SC includes extrapolations of the SC to earlier epochs when the mean energies of the particles were greater than about 100 GeV. The strength of the SC is seen to lie in its successful prediction of the expansion of the universe, the abundance of light nuclei, and the spectrum and anisotropies of the cosmic microwave background (CMBR). The SC has led to a whole class of theories of structure formation, which are, in principle, testable observationally. The subject of twentieth century cosmology gained considerably from occasional ideas different from the SC; some of these are briefly outlined and placed in historical perspective. Currently there is only one alternative cosmology, the quasi steady state cosmology (QSSC), that has been developed to a stage where it can be compared with observations and also with the SC. Although the SC does appear quite successful, there are still many unresolved issues that keep the cosmological scene fairly open.

Notes: Abstract The Kuiper Belt consists of a large number of small, solid bodies in heliocentric orbit beyond Neptune. Discovered as recently as 1992, the Kuiper Belt objects (KBOs) are thought to hold the keys to understanding the early solar system, as well as the origin of outer solar system objects, such as the short-period comets and the Pluto-Charon binary. The KBOs are probably best viewed as aged relics of the Sun's accretion disk. Dynamical structures in the Kuiper Belt provide evidence for processes operative in the earliest days of the solar system, including a phase of planetary migration and a clearing phase, in which substantial mass was lost from the disk. Dust is produced to this day by collisions between KBOs. In its youth, the Kuiper Belt may have compared to the dust rings observed now around such stars as GG Tau and HR 4796A. This review presents the basic physical parameters of the KBOs and makes connections with the disks observed around nearby stars.

Notes: Abstract Cosmic microwave background (CMB) temperature anisotropies have and will continue to revolutionize our understanding of cosmology. The recent discovery of the previously predicted acoustic peaks in the power spectrum has established a working cosmological model: a critical density universe consisting of mainly dark matter and dark energy, which formed its structure through gravitational instability from quantum fluctuations during an inflationary epoch. Future observations should test this model and measure its key cosmological parameters with unprecedented precision. The phenomenology and cosmological implications of the acoustic peaks are developed in detail. Beyond the peaks, the yet to be detected secondary anisotropies and polarization present opportunities to study the physics of inflation and the dark energy. The analysis techniques devised to extract cosmological information from voluminous CMB data sets are outlined, given their increasing importance in experimental cosmology as a whole.

Notes: Abstract Considerable progress has been made over the past decade in the study of the evolutionary trends of the population of galaxy clusters in the Universe. In this review we focus on observations in the X-ray band. X-ray surveys with the ROSAT satellite, supplemented by follow-up studies with ASCA and Beppo-SAX, have allowed an assessment of the evolution of the space density of clusters out to z= 1 and the evolution of the physical properties of the intracluster medium out to z= 0.5. With the advent of Chandra and Newton-XMM and their unprecedented sensitivity and angular resolution, these studies have been extended beyond redshift unity and have revealed the complexity of the thermodynamical structure of clusters. The properties of the intracluster gas are significantly affected by nongravitational processes including star formation and Active Galactic Nuclei (AGN) activity. Convincing evidence has emerged for modest evolution of both the bulk of the X-ray cluster population and their thermodynamical properties since redshift unity. Such an observational scenario is consistent with hierarchical models of structure formation in a flat low-density universe with Omegam= 0.3 and sigma8= 0.7-0.8 for the normalization of the power spectrum. Basic methodologies for construction of X-ray-selected cluster samples are reviewed, and implications of cluster evolution for cosmological models are discussed.

Notes: Abstract Giant planet research has moved from the study of a handful of solar system objects to that of a class of bodies with dozens of known members. Since the original 1995 discovery of the first extrasolar giant planets (EGPs), the total number of known examples has increased to ~80 (circa November 2001). Current theoretical studies of giant planets emphasize predicted observable properties, such as luminosity, effective temperature, radius, external gravity field, atmospheric composition, and emergent spectra as a function of mass and age. This review focuses on the general theory of hydrogen-rich giant planets; smaller giant planets with the mass and composition of Uranus and Neptune are not covered. We discuss the status of the theory of the nonideal thermodynamics of hydrogen and hydrogen-helium mixtures under the conditions found in giant-planet interiors, and the experimental constraints on it. We provide an overview of observations of extrasolar giant planets and our own giant planets by which the theory can be validated.

Notes: Abstract Magnetic fields in the intercluster medium have been measured using a variety of techniques, including studies of synchrotron relic and halo radio sources within clusters, studies of inverse Compton X-ray emission from clusters, surveys of Faraday rotation measures of polarized radio sources both within and behind clusters, and studies of cluster cold fronts in X-ray images. These measurements imply that most cluster atmospheres are substantially magnetized, with typical field strengths of order 1 muGauss with high areal filling factors out to Mpc radii. There is likely to be considerable variation in field strengths and topologies both within and between clusters, especially when comparing dynamically relaxed clusters to those that have recently undergone a merger. In some locations, such as the cores of cooling flow clusters, the magnetic fields reach levels of 10-40 muG and may be dynamically important. In all clusters the magnetic fields have a significant effect on energy transport in the intracluster medium. We also review current theories on the origin of cluster magnetic fields.

Notes: Turbulence affects the structure and motions of nearly all temperature and density regimes in the interstellar gas. This two-part review summarizes the observations, theory, and simulations of interstellar turbulence and their implications for many fields of astrophysics. The first part begins with diagnostics for turbulence that have been applied to the cool interstellar medium and highlights their main results. The energy sources for interstellar turbulence are then summarized along with numerical estimates for their power input. Supernovae and superbubbles dominate the total power, but many other sources spanning a large range of scales, from swing-amplified gravitational instabilities to cosmic ray streaming, all contribute in some way. Turbulence theory is considered in detail, including the basic fluid equations, solenoidal and compressible modes, global inviscid quadratic invariants, scaling arguments for the power spectrum, phenomenological models for the scaling of higher-order structure functions, the direction and locality of energy transfer and cascade, velocity probability distributions, and turbulent pressure. We emphasize expected differences between incompressible and compressible turbulence. Theories of magnetic turbulence on scales smaller than the collision mean free path are included, as are theories of magnetohydrodynamic turbulence and their various proposals for power spectra. Numerical simulations of interstellar turbulence are reviewed. Models have reproduced the basic features of the observed scaling relations, predicted fast decay rates for supersonic MHD turbulence, and derived probability distribution functions for density. Thermal instabilities and thermal phases have a new interpretation in a supersonically turbulent medium. Large-scale models with various combinations of self-gravity, magnetic fields, supernovae, and star formation are beginning to resemble the observed interstellar medium in morphology and statistical properties. The role of self-gravity in turbulent gas evolution is clarified, leading to new paradigms for the formation of star clusters, the stellar mass function, the origin of stellar rotation and binary stars, and the effects of magnetic fields. The review ends with a reflection on the progress that has been made in our understanding of the interstellar medium and offers a list of outstanding problems.

Notes: Observation of cooling neutron stars can potentially provide information about the states of matter at supernuclear densities. We review physical properties important for cooling such as neutrino emission processes and superfluidity in the stellar interior, surface envelopes of light elements owing to accretion of matter, and strong surface magnetic fields. The neutrino processes include the modified Urca process and the direct Urca process for nucleons and exotic states of matter, such as a pion condensate, kaon condensate, or quark matter. The dependence of theoretical cooling curves on physical input and observations of thermal radiation from isolated neutron stars are described. The comparison of observation and theory leads to a unified interpretation in terms of three characteristic types of neutron stars: high-mass stars, which cool primarily by some version of the direct Urca process; low-mass stars, which cool via slower processes; and medium-mass stars, which have an intermediate behavior. The related problem of thermal states of transiently accreting neutron stars with deep crustal burning of accreted matter is discussed in connection with observations of soft X-ray transients. Observations imply that some stars cool more rapidly than can be explained on the basis of nonsuperfluid neutron star models cooling via the modified Urca process, whereas other star cool less rapidly. We describe possible theoretical models that are consistent with observations.

Notes: We review recent theoretical results on the formation of the first stars in the universe, and emphasize related open questions. In particular, we discuss the initial conditions for Population III star formation, as given by variants of the cold dark matter cosmology. Numerical simulations have investigated the collapse and the fragmentation of metal-free gas, showing that the first stars were predominantly very massive. The exact determination of the stellar masses, and the precise form of the primordial initial mass function, is still hampered by our limited understanding of the accretion physics and the protostellar feedback effects. We address the importance of heavy elements in bringing about the transition from an early star formation mode dominated by massive stars to the familiar mode dominated by low-mass stars at later times. We show how complementary observations, both at high redshifts and in our local cosmic neighborhood, can be utilized to probe the first epoch of star formation.

Notes: Until the late 1990s the rich Hyades and the sparse UMa clusters were the only coeval, comoving concentrations of stars known within 60 pc of Earth. Both are hundreds of millions of years old. Then beginning in the late 1990s the TW Hydrae Association, the Tucana/Horologium Association, the beta Pictoris Moving Group, and the AB Doradus Moving Group were identified within ~60 pc of Earth, and the eta Chamaeleontis cluster was found at 97 pc. These young groups (ages 8-50 Myr), along with other nearby, young stars, will enable imaging and spectroscopic studies of the origin and early evolution of planetary systems.

Notes: GRS 1915+105-the first stellar-scale, highly relativistic jet source identified-is a key system for our understanding of the disc-jet coupling in accreting black hole systems. Comprehending the coupling between inflow and outflow in this source not only is important for X-ray binary systems but has a broader relevance for studies of active galactic nuclei and gamma-ray bursts. In this paper, we present a detailed review of the observational properties of the system, as established in the decade since its discovery. We attempt to place it in context by a detailed comparison with other sources, and construct a simple model for the disc-jet coupling, which may be more widely applicable to accreting black hole systems.