Showing papers in "Advances in Protein Chemistry in 2002"

Identification and functions of usefully disordered proteins.

Abstract: Publisher Summary This chapter illustrates that protein disorder is encoded by the amino acid sequence and that protein disorder is essential for many important biological functions. An ordered protein contains a single canonical set of Ramachandran angles, whereas a disordered protein or region contains an ensemble of divergent angles at any instant and these angles interconvert over time. Intrinsically disordered protein can be extended (random coil–like) or collapsed (molten globule–like). The latter type of disorder typically includes regions of fluctuating secondary structure, so disorder does not mean absence of helix or sheet. Both types of disorders have been observed in apparently native proteins. Intrinsic disorder might not be encoded by the sequence, but rather might be the result of the absence of suitable tertiary interactions. If this were the general cause of intrinsic disorder, any subset of ordered sequences and any subset of disordered sequences would likely be the same within the statistical uncertainty of the sampling. On the other hand, if intrinsic disorders were encoded by the amino acid sequence, any subset of disordered sequences would likely differ significantly from samples of ordered protein sequences.

Is polyproline II a major backbone conformation in unfolded proteins

Abstract: Publisher Summary Protein folding is a process by which a polypeptide chain acquires its native structure from an unfolded state through a transition state. Recent studies of the unfolded states of proteins are based on a modification of the random coil model, recognizing that in many cases some residual native or non-native structure persists.. Combined evidence from the theoretical study of a blocked alanine peptide in aqueous solution and a variety of spectroscopic studies, including ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), two-dimensional vibrational spectroscopy, vibrational circular dichroism (VCD), and vibrational Raman optical activity (VROA) reveal that the polyproline II (P II ) conformation is the dominant conformation in a variety of short model peptides. This chapter discusses the evidence from short peptides. It reviews the circular dichroism of unfolded proteins and addresses the role of P II in unfolded proteins.

Molecular recognition in antibody-antigen complexes.

Abstract: With the numerous detailed molecular descriptions of antibody-antigen interfaces, the structual study of these molecular interactions has evolved from an attempt to understand to immunological function to their use as model systems for protein-protein interactions. In this chapter, we describe the structual aspects common to antibody-antigen interfaces and discuss the roles they may play in antibody cross-rectivity and molecular mimicry. More detailed analysis of these interfaces has required the marriage of structural studies with extensive mutagenesis and thermodynamic analysis efforts. Here, we discuss the thermodynamic mapping of interfaces for two model antibody-antigen complexes, including the identification of thermodynamic hot spots in binding and the various mechanism used to accommodate interface mutations. We also discuss the functional roles for protein plasticity in antigen recognition, including the entropic control of antibody affinity maturation and the use of induced fit mechanism of different types and to varying degrees by mature antibodies in binding their specific antigens.

Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance.

Abstract: Publisher Summary Nuclear magnetic resonance (NMR) is unique in being able to provide detailed insights into the conformation of unfolded and partly folded proteins This chapter presents the structure and dynamics of unfolded states, that is, equilibrium NMR studies of apomyoglobin; characterization of proteins that are unstructured under nondenaturing conditions; and intrinsically unstructured proteins (coupled folding and binding events) NMR is the method of choice to determine the conformational preferences inherent in these domains NMR is also of the greatest utility in the elucidation of pathways of protein folding by allowing structural characterization of equilibrium and kinetic folding intermediates Through the application of state-of-the-art heteronuclear NMR methods, new insights have been obtained into the nature of the conformational ensemble and the dynamics of denatured proteins These studies show that the behavior of denatured proteins is typically far from that of a statistical random coil This new view of the denatured state provides a basis for understanding the earliest events that occur during protein folding

Toward a taxonomy of the denatured state: small angle scattering studies of unfolded proteins.

Abstract: Publisher Summary This chapter reviews recent small-angle X-ray and neutron scattering (SAXS and SANS) studies of putatively “fully” unfolded states formed at equilibrium. It discusses the taxonomy of unfolded states that is chemically denatured state, thermally denatured state, pressure-denatured state, cold-unfolded states, and intrinsically unfolded proteins. The majority of SAXS and SANS studies of the unfolded state focus on the ensembles of states induced by chemical denaturants such as urea, GuHCl, extremes of pH, and organic cosolvents. As urea and GuHCl dominate spectroscopic studies of protein folding thermodynamics and kinetics, these denaturants have similarly been employed in the vast majority of small-angle scattering studies as well. The extraordinary solubility of unfolded proteins at high levels of urea or GuHCl provides an added technical benefit. Limited SAXS studies suggest that urea and GuHCl produce indistinguishable denatured states. Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. Because the molar volume of an unfolded protein is less than that of the native state, increasing pressure leads to denaturation. High-pressure SAXS was employed to monitor the pressure-induced unfolding of Snase.

Molecular recognition by SH2 domains

Abstract: In this chapter, we have described the biophysical investigations which have dissected the mechanisms of SH2 domain function. Due to nearly a decade and a half of investigation on SH2 domains, much about their binding mechanism has been characterized. SH2 domains have been found to have a positively charged binding cavity, largely conserved between different SH2 domains, which coordinates binding of the pTyr in the target. The ionic interactions between this pocket and the pTyr, in particular, between Arg beta B5 and the phosphate, provide the majority of the binding energy stabilizing SH2 domain-target interactions. The specificity in SH2 domain-target interactions emanates most often from the interactions between the residues C-terminal to the pTyr in the target and the specificity determining residues in the C-terminal half of the SH2 domain. However, the interactions in the specificity determining region of SH2 domains are weak, and hence single SH2 domains show only a modest level of specificity for tyrosine phosphorylated targets. Greater specificity in SH2 domain-containing protein-tyrosine phosphorylated target interactions can be achieved by placing SH2 domains in tandem (as is often found) or possibly through specific localization of SH2 domain-containing proteins within the cell. Although a relatively good understanding of how SH2 domains function in isolation has been obtained, the ways in which SH2 domain binding is coupled to allosteric transmission of signals in larger SH2 domain-containing proteins are still not clear. Hence, the future should bring further investigations of the mechanisms by which SH2 domain ligation alters the enzymatic activity and cellular localization of SH2 domain-containing proteins.

Unfolded proteins studied by Raman optical activity.

Abstract: Publisher Summary To understand the behavior of unfolded proteins it is necessary to employ experimental techniques able to discriminate between the dynamic true random coil state and more static types of disorder, including situations in which some ordered secondary structure might be present One such technique is a novel chiroptical spectroscopy called Raman optical activity (ROA) This chapter reviews the application of ROA to studies of unfolded proteins Because many discrete structure-sensitive bands are present in protein ROA spectra, the technique provides a fresh perspective on the structure and behavior of unfolded proteins and of unfolded sequences in proteins such as A-gliadin and prions that contain distinct structured and unstructured domains It also provides new insight into the complexity of order in molten globule and reduced protein states and of the more mobile sequences in fully folded proteins such as β-lactoglobulin The power of ROA in this area derives from the fact that, like the complementary technique of vibrational circular dichroism (VCD), it is a form of vibrational optical activity and so is sensitive to chirality associated with all the 3N−6 fundamental molecular vibrational transitions, where N is the number of atoms

Determinants of the polyproline II helix from modeling studies.

Abstract: Publisher Summary This chapter considers the determinants of polyproline II (PPII) helix formation considered in the light of computational modeling. More complex modeling is also considered ranging from simple hard-sphere computer simulations to calculations involving more detailed energetics. Studies show that the PPII region of (φ,ψ)-space is highly populated in a model employing only steric interactions and hydrogen bonds to describe intramolecular interactions. Data indicate that this region is favored by a lack of unfavorable steric interactions, which follows from the extended nature of the conformation. Recent experimental work has demonstrated that the polypeptide backbone possesses a significant propensity to adopt the PPII helical conformation. The major determinant of this backbone propensity would appear to be backbone solvation. The calculations and modeling described in the chapter provide data in support of this hypothesis.

Hydration theory for molecular biophysics.

Abstract: Publisher Summary Protein stabilization by the presence of water may in part be a manifestation of hydration water destabilizing the unfolded state. This chapter presents a molecular theory of hydration that highlights the role of water in protein stabilization. The focus is on potential distribution theorem whose physical basis and statistical thermodynamic framework with applications to protein solution thermodynamics and protein folding is discussed. The chapter also presents the derivation of an extension of the potential distribution theorem, the quasi-chemical theory, and proposes its implementation to the hydration of folded and unfolded proteins. The perspective and current optimism are justified by the understanding gained from successful applications of the potential distribution theorem to the hydration of simple solutes. The developments shown in the chapter illustrate a thoroughgoing reconstruction of molecular statistical thermodynamic theory of solutions. There are several motivations for this effort, but particularly, the conventional molecular theories are not compelling for biophysical applications and do not make strong connections to the molecular intuitions involved in consideration of simulation and experiment. Some of the discussion treats basic elements of statistical thermodynamics, known in specialized settings but weightier than is typical for this setting. The chapter includes examples to clarify and reinforce the basic concepts.

The expanded denatured state: an ensemble of conformations trapped in a locally encoded topological space.

Abstract: Publisher Summary With the development of nuclear magnetic resonance (NMR)-based experiments for studying folded proteins, structural analysis of denatured proteins entered a new phase. NMR spectroscopy extracts information about individual residues. This chapter discusses NMR analysis of the denatured state of staphylococcal nuclease. The initial experiments that measure local structural parameters, reported small amounts of persisting helical structure, two turns, and indirect evidence for perhaps a three-strand beta meander. When applied to the denatured state in 6 M urea, the same experiments indicated that most of these features are lost. Recent applications of two types of NMR methodologies that provide long-range structural information have painted a very different picture. From these experiments, the denatured state in 0 M urea and 8 M urea appear to be highly similar, both retaining the same overall spatial positioning and orientation of the chain seen in the folded conformation. Simple computer simulations to estimate the importance of side chain–backbone interactions in encoding the long-range structure in denatured proteins suggest that these simple steric effects are of overriding importance. If this conclusion is correct, it appears that much of the estimated entropy available to a random coil protein chain is lost during its synthesis.

A new perspective on unfolded proteins.

Abstract: Publisher Summary This chapter discusses hydrophobic clusters in urea-denatured proteins, rationale for studying denatured proteins in water, stiffness of the random chain, and preferred backbone conformations. Hydrophobic clusters of nonpolar side chains withstands denaturation in concentrated urea solutions and possibly also in 6 M GdmCl. In urea solutions, hydrophobic clusters can be observed in denatured proteins by various NMR probes, such as the nuclear Overhauser effect chemical shifts of aromatic side chain protons or transverse relaxation rates of amide groups in the polypeptide backbone. There are two main reasons for studying denatured proteins in water. First, simulations of the folding process are providing increasingly valuable insights into the folding process and simulations are made in water not in 6 M GdmCl. The starting point for a simulation is usually an extended β-strand, rather than an attempt at simulating a structureless, random chain. The local structures formed rapidly at the start of folding are accessible to simulation. The second reason is that the kinetics of α-helices and β-hairpins can now be measured by fast-reaction methods. The measurements of chain stiffness of denatured proteins are made in the presence of a strong denaturant, such as 8 M urea or 6 M GdmCl, in which peptide H-bonds are weak and peptide helices unfold and the possible presence of α-helices or β-hairpins is not an issue in these denaturants.

Unfolded peptides and proteins studied with infrared absorption and vibrational circular dichroism spectra.

Abstract: Publisher Summary Unfolded proteins and unstructured peptides form vital thermodynamic states critical to the overall protein folding problem. They represent one of a possible set of initial states in the folding process. This chapter addresses unfolded protein studies with optical spectroscopy, focusing on vibrational infrared (IR) absorption spectroscopy and its chiroptical variant, and vibrational circular dichroism (VCD). Optical spectroscopy is low in resolution, so that site-specific information cannot be obtained without isotopic labeling, yet this failing may offer the best way to study the disordered unfolded state. These techniques are very fast sampling conformations on the picosecond time scale. Thus, they can provide a representation of the equilibrium ensemble of structures, sensed as an average over all the residues or can be used to follow fast kinetic changes in the structures, as might occur in the folding process. As such, optical techniques can be ideal for probing rapid fluctuations in a folding protein or peptide and exploring the distribution of local structures that can occur in an unfolded protein or peptide.

Blue copper-binding domains

Abstract: Publisher Summary This chapter introduces the blue copper-binding (BCB) domain-containing proteins based on the analysis of the genomic and expressed sequence tag (EST) sequence data, and presents the classification system. This classification is based on their ability to bind copper and the specific features of their domain organization. Members of the first three classes harbor single or multiple type 1, blue copper-binding sites, while members of the fourth class do not appear to bind copper. Some analysis of codon usage for conserved amino acids involved in copper binding will be used to trace the evolutionary history of the blue copper-binding (BCB) domains within a single genorne. The chapter also discusses the structural and physical characteristics of each kind of blue copper-binding (BCB) domain protein. The large number of blue copper-binding (BCB) domain proteins in plants may be explained by the phenomenon of genome duplication, believed to occur widely in the plant kingdom, as well as by different lateral gene transfers The blue copper-binding (BCB) domain-containing proteins evolved by gene duplication and fusion with other structural modules, resulting in diverse subcellular localization and the ability to carry out complex reactions that often differ from that of the ancestral protein. They are also modified by extensive amino acid substitutions and insertions.

FET3P, ceruloplasmin, and the role of copper in iron metabolism

Abstract: Publisher Summary The chapter focuses on the copper ferroxidases and discusses the role of copper in iron metabolism. The physiologic linkage between copper and iron is now well understood at the molecular level, in terms of the gene products that metabolically link these two essential metal nutrients. The central component in this linkage is a multicopper ferroxidase: ceruloplasmin, Fet3p, in mammals. However, each of these copper proteins relies on a copper ATPase found in the membrane of a specific vesicular compartment for the copper necessary for each protein's activation. The copper pumping that any one of these ATPases does may be, critical to copper homeostasis as well for copper excretion, for example. These pumps in turn rely on a protein, a copper chaperone that ferries the copper from the plasma membrane copper permease through the cytosol to this vesicular compartment. The permease relies on a plasma membrane cuprireductase to supply it with the Cu(I) as substrate for uptake. Nonetheless, irrespective of whether or how a defect in any one of these enzymes, transporters, chaperones, or pumps may contribute to a dysfunction in copper handling, there most certainly will be a direct impact on the copper incorporation into one or more of these ferroxidases leading to a secondary effect on iron homeostasis. The copper ferroxidases are central to this secondary nutritional, metabolic, essentially epistatic relationship between copper and iron in eukaryotes.

Unfolded state of peptides.

Abstract: Publisher Summary This chapter illustrates the current knowledge on the unfolded state of peptides. Currently, the characterization of the populated microscopic states of a peptide is possible only by simulation methods. The focus is on molecular dynamics simulations of spontaneous (i.e., lacking biasing potentials or directed-sampling algorithms) and reversible folding of peptides in solution (i.e., with explicit solvent molecules), since the results from this type of studies are the least dependent on the methodology. A full characterization of the unfolded state becomes as essential as the determination of the folded conformation in two scenarios. The first is in the study of the physical, chemical, and biological properties of peptides. The folded conformation of a peptide is, in general, only marginally more stable than the lowest-free-energy unfolded conformation. As a result, any macroscopic observable of a peptide is weighted with both the folded and the unfolded states. Interpreting such observables in terms of the folded conformation only is therefore not correct. The second scenario is related to the study of peptide and protein folding. The description of equilibrium requires knowledge about each of the states involved. Therefore, it is fundamental to describe not only the folded state but also the unfolded state accurately in order to draw conclusions on the nature and mechanisms of peptide and protein folding.

Understanding the mechanism and function of copper P-type ATPases.

Abstract: Publisher Summary The chapter discusses the mechanism and function of copper P-TYPE ATPases. The catalytic cycle of P-type ATPases is represented generally by the coupled reaction of ATP hydrolysis, transient phosphorylation of an invariant aspartate residue, and cation translocation across the lipid bilayer. The catalytic cycle of calcium P-type ATPases is regarded as a paradigm for heavy metal P-type ATPases (HMPAs). An important characteristic of P-type ATPases is their ability to be transiently phosphorylated at the invariant aspartate residue by inorganic orthophosphate in the absence of the cation. Copper P-type ATPases have evolved from the largely detoxifying role in unicellular organisms to satisfying the physiological requirements of multicellular differentiated systems. This chapter also reviews the copper homeostasis, with particular emphasis on the role of mammalian copper P-type ATPases. The studies on physiological and biochemical properties of mammalian ATP7A and ATP7B suggest that the combination of copper-translocating activity and copper-stimulated trafficking is a key regulator of intracellular copper homeostasis by these transporters.

Cytochrome c oxidase

Abstract: Cytochrome oxidase is the terminal oxidase of most of aerobic organisms and reduces molecular oxygen (O2) to water (1). The electrons and protons required for the formation of water molecules are transferred from both sides of the mitochondrial inner membranes in eukaryotic cells and of the cell membrane in prokaryotic cells (1). The migration of positive and negative charges from the different sides of the membrane produces electric potential across the membrane. In addition to the O2 reduction, this enzyme pumps protons from the inside to the outside of the membrane to produce a proton gradient across the membrane in addition to the membrane potential produced by the net migration of the positive charges (1,2). This enzyme contains heme iron and copper ions in the catalytic center (1). Because of the intriguing reaction of this copper-containing enzyme in addition to its physiological importance, many articles have been published on its structure and function, isolated from various organisms and tissues, in the last 75 yr or so (1,3,4) since its discovery by Warburg (5). Progress in understanding of the reaction mechanism of cytochrome-c oxidase has been accelerated by X-ray structures of the enzyme isolated from mammalian tissue and bacterial cells, which began to appear as late as in 1995 (6,7).

What fluorescence correlation spectroscopy can tell us about unfolded proteins.

Abstract: Publisher Summary Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. Although the diffusion coefficient is relatively insensitive to protein conformation changes, it can be measured with high accuracy by FCS and so can be useful for monitoring structural changes that occur as the protein passes among partially unfolded states. As the protein takes on more compact or more extended conformations, the hydrodynamic radius and consequently the diffusion coefficient change correspondingly; if these changes are large enough, they can be observed in FCS measurements. Among kinetics approaches used to study dynamics within the unfolded state, FCS is closely related to relaxation methods in which small free energy perturbations displace the equilibrium point of a reaction system. The kinetic parameters are deduced from the rate of relaxation to the new equilibrium.

Bacterial copper transport.

Abstract: Publisher Summary The chapter presents the study of the En. hirae model system, which has shown the modes of copper entry into and out of the cell by the action of copper ATPases, transcriptional control of copper homeostatic genes by a copper-responsive repressor, and intracellular copper routing by a copper chaperone. Entencoccus hirae CopA and CopB are the first copper ATPases to be purified. Studies on the mechanism of copper ATPases require highly enriched membrane preparations or purified enzyme. Naturally enriched membranes can be obtained only from the specialized membrane compartments, such as the sarcoplasmic reticulum or the electric organ of eels. Since copper is a toxic trace element, it is never encountered in large quantities in cells and copper ATPases are expressed at only low levels. However, the purification of the CopA and CopB copper ATPases of En. hirae has recently been reported. Both the enzymes have been shown to form acylphosphate intermediates in purified form, reconstituted into proteoliposomes. Acylphosphate formation has also been demonstrated for the human Menkes ATPase in native membrane vesicles.

Nuclear magnetic resonance spectroscopy studies on copper proteins.

Abstract: Publisher Summary This chapter discusses the nuclear magnetic resonance (NMR) studies on mnnonuclear type I copper proteins, mononuclear type II copper-containing proteins, and proteins containing polynuclear copper centers. The studies revealed that type I copper is present at the active site of blue copper proteins, where it is involved in the transfer of a single electron, as well as in multicopper enzymes. The studies also reveal that type II copper(II) sites are present in mononuclear copper enzymes, such as dioxygenases, monoxygenases, nitrite reductases, and nonblne oxidases. The high molecular weight of the most of these enzymes and the unfavorable electron relaxation time of copper ion in the oxidized form has up to now precluded the application of NMR spectroscopy. The Cu A Center is a binuclear center acting as the primary electron acceptor in terminal oxidases. The electrons are then shuttled to another metal center in the same oxidase. The studies shows that Cu A centers exist in two redox states: [Cu(I1)Cu(I)] and [Cu(I)Cu(I)]. The oxidized species is a fully delocalized mixed-valence pair (formally two Cu + 1.5 ions), as revealed by EPR spectroscopy.

Copper metalloregulation of gene expression.

Abstract: Publisher Summary Bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cu metalloregulation of transcription occurs for ATPase genes in bacteria and certain fungi. The En hirae CopY is a transcriptional repressor that limits the expression of the CopA and CopB P-type ATPases in cells cultured in medium containing minimal Cu(lI) levels. An increase in medium Cu(II) levels results in Cu ion uptake, routing of the Cu(l) ions to CopY by the CopZ metallochaperone, and the subsequent dissociation of CuCopY from the copA and copB genes. In contrast to the En. hirae CopY metalloregulation, Sa. Cerevisiae copper metalloregulation involves two transcriptional activators, Acel and Macl. Both the factors reside within the yeast nucleus and are regulated in their function in a Cu-dependent manner. Addition of Cu salts to the growth medium (at greater than micromolar concentrations of Cu) induces Ace1-dependent transcription within 10 min. Cu binding to Ace1 stabilizes a distinct conformation that enables CuAce1 to bind to DNA in a specific manner. Cu(I) ions activate Acel by formation of a tetracopperthiolate cluster within the DNA-binding domain.

Getting to know u.

Abstract: Publisher Summary This chapter introduces the book that deals with protein folding Protein folding was established early in the last century in publications of Wu, Mirsky, and Pauling The three overall questions arising from the understanding of folding are how to characterize the folded state, the unfolded state, and the transition between these two populations The focus of this book is to characterize the unfolded population The equilibrium population is said to have a structure when a substantial fraction of the molecules adopts similar conformations However, the phrase lacking structure does not imply that individual molecules comprising the ensemble lack a conformation; rather, the population is too heterogeneous to be readily characterized using a coherent, structure based descriptor The unfolded state resists ready characterization because it is so diverse Typical biophysical methods report ensemble averaged properties in which important components of the population may be concealed beneath the background or the intrinsic distributions may be lumped into a misleading average The current model that originated with Flory and Tanford treats unfolded proteins as random chains Specifically, under conditions that favor unfolding, the chain is free to adopt all sterically allowed values of φ, ψ -angles, and when it does so, the population is found to be Gaussian-distributed around the radius of gyration expected for random, freely jointed chains of the same length in good solvent, but with excluded volume constraints