Showing papers in "Annual Review of Biophysics and Biomolecular Structure in 2001"

Probing the Relation Between Force—Lifetime—and Chemistry in Single Molecular Bonds

Abstract: On laboratory time scales, the energy landscape of a weak bond along a dissociation pathway is fully explored through Brownian-thermal excitations, and energy barriers become encoded in a dissociation time that varies with applied force. Probed with ramps of force over an enormous range of rates (force/time), this kinetic profile is transformed into a dynamic spectrum of bond rupture force as a function of loading rate. On a logarithmic scale in loading rate, the force spectrum provides an easy-to-read map of the prominent energy barriers traversed along the force-driven pathway and exposes the differences in energy between barriers. In this way, the method of dynamic force spectroscopy (DFS) is being used to probe the complex relation between force-lifetime-and chemistry in single molecular bonds. Most important, DFS probes the inner world of molecular interactions to reveal barriers that are difficult or impossible to detect in assays of near equilibrium dissociation but that determine bond lifetime and strength under rapid detachment. To use an ultrasensitive force probe as a spectroscopic tool, we need to understand the physics of bond dissociation under force, the impact of experimental technique on the measurement of detachment force (bond strength), the consequences of complex interactions in macromolecular bonds, and effects of multiply-bonded attachments.

Biomolecular Simulations: Recent Developments in Force Fields, Simulations of Enzyme Catalysis, Protein-Ligand, Protein-Protein, and Protein-Nucleic Acid Noncovalent Interactions

Abstract: ▪ 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.

Hydrogen bonding, base stacking, and steric effects in dna replication.

Abstract: ■ Abstract Understanding the mechanisms by which genetic information is replicated is important both to basic knowledge of biological organisms and to many useful applications in biomedical research and biotechnology. One of the main functions of a DNA polymerase enzyme is to help DNA recognize itself with high specificity when a strand is being copied. Recent studies have shed new light on the question of what physical forces cause a polymerase enzyme to insert a nucleotide into a strand of DNA and to choose the correct nucleotide over the incorrect ones. This is discussed in the light of three main forces that govern DNA recognition: base stacking, Watson-Crick hydrogen bonding, and steric interactions. These factors are studied with natural and structurally altered DNA nucleosides.

Chaperonin-mediated protein folding.

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.

Protein folding theory: from lattice to all-atom models.

Abstract: ▪ Abstract This review focuses on recent advances in understanding protein folding kinetics in the context of nucleation theory. We present basic concepts such as nucleation, folding nucleus, and transition state ensemble and then discuss recent advances and challenges in theoretical understanding of several key aspects of protein folding kinetics. We cover recent topology-based approaches as well as evolutionary studies and molecular dynamics approaches to determine protein folding nucleus and analyze other aspects of folding kinetics. Finally, we briefly discuss successful all-atom Monte-Carlo simulations of protein folding and conclude with a brief outlook for the future.

Ab Initio Protein Structure Prediction: Progress and Prospects

Abstract: ■ 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.

Properties and biological activities of thioredoxins.

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.

NMR Probes of Molecular Dynamics: Overview and Comparison with Other Techniques

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.

Structural insight into microtubule function.

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.

Structures and proton-pumping strategies of mitochondrial respiratory enzymes.

Abstract: Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes.

Interpreting the effects of small uncharged solutes on protein-folding equilibria.

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.

A structural view of cre-loxp site-specific recombination.

Abstract: Structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. Members of this family, which include the bacteriophage P1 Cre recombinase, bacteriophage lambda integrase, the yeast Flp recombinase, and the bacterial XerCD recombinases, exchange strands between DNA substrates in a stepwise process. One pair of strands is exchanged to form a Holliday junction intermediate, and the second pair of strands is exchanged during resolution of the junction to products. Crystal structures of reaction intermediates in the Cre-loxP site-specific recombination system, together with recent biochemical studies in the field, support a "strand swapping" model for recombination that does not require branch migration of the Holliday junction intermediate in order to test homology between recombining sites.

Binding of Ligands and Activation of Transcription by Nuclear Receptors

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.

Ribozyme structures and mechanisms.

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.

Structure of proteins involved in synaptic vesicle fusion in neurons.

Abstract: ▪ 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.

Mass Spectrometry as a Tool for Protein Crystallography

Abstract: ▪ Abstract Atomic resolution structure determinations of proteins by X-ray crystallography are formidable multidisciplinary undertakings, requiring protein construct design, expression and purification, crystallization trials, phase determination, and model building. Modern mass spectrometric methods can greatly facilitate these obligate tasks. Thus, mass spectrometry can be used to verify that the desired protein construct has been correctly expressed, to define compact domains in the target protein, to assess the components contained within the protein crystals, and to screen for successful incorporation of seleno-methionine and other heavy metal reagents used for phasing. In addition, mass spectrometry can be used to address issues of modeling, topology, and side-chain proximity. Here, we demonstrate how rational use of mass spectrometry assists and expedites high resolution X-ray structure determination through each stage of the process of protein crystallography.

Structural relationships among regulated and unregulated phosphorylases.

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.

Photosystem II: The Solid Structural Era

Abstract: ▪ Abstract Understanding the precise role of photosystem II as an element of oxygenic photosynthesis requires knowledge of the molecular structure of this membrane protein complex. The past few years have been particularly exciting because the structural era of the plant photosystem II has begun. Although the atomic structure has yet to be determined, the map obtained at 6 A resolution by electron crystallography allows assignment of the key reaction center subunits with their associated pigment molecules. In the following, we first review the structural details that have recently emerged and then discuss the primary and secondary photochemical reaction pathways. Finally, in an attempt to establish the evolutionary link between the oxygenic and the anoxygenic photosynthesis, a framework structure common to all photosynthetic reaction centers has been defined, and the implications have been described.