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Showing papers in "Protein Science in 2002"


Journal ArticleDOI
TL;DR: Results of this analysis showed that intrinsically unstructured proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity, and the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globule, premolten globules, and random coils) is discussed.
Abstract: The experimental material accumulated in the literature on the conformational behavior of intrinsically unstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity. Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premolten globules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poor solvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second group are essentially more compact, exhibiting some amount of residual secondary structure, although they are still less dense than native or molten globule proteins. An important feature of the intrinsically unstructured proteins is that they undergo disorder–order transition during or prior to their biological function. In this respect, the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globules, premolten globules, and random coils) and transitions between any two of the states, is discussed.

1,750 citations


Journal ArticleDOI
TL;DR: It is suggested that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus and that the swapped domains are diverse in their primary and secondary structures.
Abstract: Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term '3D domain swapping' was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping.

668 citations


Journal ArticleDOI
TL;DR: A new protocol, which is based on the joint probability of each of the three secondary structural types (β‐strand, α‐helix, and random coil) derived from chemical‐shift data, to identify the secondary structure, shows significant improvements in the accuracy and the confidence of identification.
Abstract: For a long time, NMR chemical shifts have been used to identify protein secondary structures. Currently, this is accomplished through comparing the observed 1Hα, 13Cα, 13Cβ, or 13C′ chemical shifts with the random coil values. Here, we present a new protocol, which is based on the joint probability of each of the three secondary structural types (β-strand, α-helix, and random coil) derived from chemical-shift data, to identify the secondary structure. In combination with empirical smooth filters/functions, this protocol shows significant improvements in the accuracy and the confidence of identification. Updated chemical-shift statistics are reported, on the basis of which the reliability of using chemical shift to identify protein secondary structure is evaluated for each nucleus. The reliability varies greatly among the 20 amino acids, but, on average, is in the order of: 13Cα>13C′>1Hα>13Cβ>15N>1HN to distinguish an α-helix from a random coil; and 1Hα>13Cβ >1HN ∼13Cα∼13C′∼15N for a β-strand from a random coil. Amide 15N and 1HN chemical shifts, which are generally excluded from the application, in fact, were found to be helpful in distinguishing a β-strand from a random coil. In addition, the chemical-shift statistical data are compared with those reported previously, and the results are discussed. A JAVA User Interface program has been developed to make the entire procedure fully automated and is available via http://ccsr3150-p3.stanford.edu.

395 citations


Journal ArticleDOI
TL;DR: The first report of the full‐length structure of the collagen‐like polypeptide [(Pro‐Pro‐Gly)10]3 is given, which suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils.
Abstract: The first report of the full-length structure of the collagen-like polypeptide [(Pro-Pro-Gly)(10)](3) is given. This structure was obtained from crystals grown in a microgravity environment, which diffracted up to 1.3 A, using synchrotron radiation. The final model, which was refined to an R(factor) of 0.18, is the highest-resolution description of a collagen triple helix reported to date. This structure provides clues regarding a series of aspects related to collagen triple helix structure and assembly. The strict dependence of proline puckering on the position inside the Pro-Pro-Gly triplets and the correlation between backbone and side chain dihedral angles support the propensity-based mechanism of triple helix stabilization/destabilization induced by hydroxyproline. Furthermore, the analysis of [(Pro-Pro-Gly)(10)](3) packing, which is governed by electrostatic interactions, suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils.

265 citations


Journal ArticleDOI
TL;DR: The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface‐ and solution‐based biophysical methods and binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone.
Abstract: The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.

254 citations


Journal ArticleDOI
TL;DR: It is shown here that a seven‐residue lysine peptide also adopts the PPII conformation, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.
Abstract: The left-handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven-residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side-chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.

211 citations


Journal ArticleDOI
TL;DR: Structural biologists have embraced high-throughput biology by developing and implementing technologies that will enable the structures of hundreds of protein domains to be solved in a relatively short time.
Abstract: Progress in understanding the organization and sequences of genes in model organisms and humans is rapidly accelerating. Although genome sequences from prokaryotes have been available for some time, only recently have the genome sequences of several eukaryotic organisms been reported, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and humans (Green 2001). A logical continuation of this line of scientific inquiry is to understand the structure and function of all genes in simple and complex organisms, including the pathways leading to the organization and biochemical function of macromolecular assemblies, organelles, cells, organs, and whole life forms. Such investigations have been variously called integrative or systems biology and -omics or high-throughput biology (Ideker et al. 2001, Greenbaum et al. 2001, Vidal 2001). These studies have blossomed because of advances in technologies that allow highly parallel examination of multiple genes and gene products as well as a vision of biology that is not purely reductionist. Although a unified understanding of biological organisms is still far in the future, new high-throughput biological approaches are having a drastic impact on the scientific mainstream. One offshoot of the high-throughput approach, which directly leverages the accumulating gene sequence information, involves mining the sequence data to detect important evolutionary relationships, to identify the basic set of genes necessary for independent life, and to reveal important metabolic processes in humans and clinically relevant pathogens. Programs such as MAGPIE (www.genomes.rockefeller.edu/magpie/magpie.html) compare organisms at a whole genome level (Gaasterland and Sensen 1996; Gaasterland and Ragan 1998) and ask what functions are conferred by the new genes that have evolved in higher organisms (Gaasterland and Oprea 2001). Concurrent with computational annotations of gene structure and function, thousands of full-length ORFs from yeast and higher eukaryotes have become available because of advances in cloning and other molecular biology techniques (Walhout et al. 2000a). Structural biologists have embraced high-throughput biology by developing and implementing technologies that will enable the structures of hundreds of protein domains to be solved in a relatively short time. Although thousands of structures are deposited annually in the Protein Data Bank (PDB), most are identical or very similar in sequence to a structure previously existing in the data bank, representing structures of mutants or different ligand bound states (Brenner et al. 1997). Providing structural information for a broader range of sequences requires a focused effort on determining structure for sequences that are divergent from those already in the database. Although structure does not always elucidate function, in many instances (including the structures of two proteins reported here) the atomic structure readily provides insight into the function of a protein whose function was previously unknown. Typically, such functional annotations are based on homologies that are not recognizable at the sequence level but that are clearly revealed on inspection of the protein fold, identification of a conserved constellation of side-chain functionalities, or by the observation of cofactors associated with function (Burley et al. 1999; Shi et al. 2001; Bonanno et al. 2002).

164 citations


Journal ArticleDOI
TL;DR: The contrasting aggregation processes in the two models suggest that there are several distinct mechanisms for polymerization that depend not only on the polypeptide sequence but also on external conditions (such as C, T, pH, and salt concentration).
Abstract: Protein aggregation (Mitraki and King 1989; Sipe 1992; DeYoung et al. 1993; Jaenicke 1995; Wetzel 1996; Fink 1998), resulting in the formation of propagating oligomers, plays a key role in the cause of a number of seemingly unrelated diseases (Cohen and Prusiner 1998). Neurodegenerative diseases (scrapie, Kuru, CJD, FFI, and BSE), which are associated with the abnormal isoform of prion proteins, are among the most well-known examples (Prusiner 1998). In the disease-causing prions, there is a post-translational conformational change from the predominantly α-helical state in the normal isoform of prion protein to a β-sheet conformation in PrPSc, which is the aggregated state of the pathogenic scrapie form (Jarrett and Lansbury 1993; Prusiner 1998). Similarly, the oligomeric form of Aβ-peptides (implicated in Alzheimer's disease) has a predominantly β-sheet architecture even though, in its monomeric form, it is a random coil over a wide range of external conditions (Kelly 1996; Harper and Lansbury 1997). Neither the mechanism of the conformational change nor the propagation leading to oligomers are fully understood at the molecular level. Although the study of the aggregated form of disease-causing proteins has been the object of intense study, only recently has it been appreciated that almost any protein can form aggregates under appropriate conditions (Booth et al. 1997). More surprisingly, Dobson et al. (Chiti et al. 1999; Jimenez et al. 1999) found that even the structures of the aggregates of "normal" proteins (formed under nonphysiological conditions) are similar to the fibrils that are implicated in the neurodegenerative diseases. These findings suggest that there may be generic mechanisms by which aggregation takes place. Because there are a number of distinct folding mechanisms of monomeric proteins, it is likely that, at the molecular level, there may also be several distinct scenarios for protein aggregation. A goal of this paper is to explore the generic mechanism by which oligomerization of proteins takes place using lattice models. An obvious variable, besides the polypeptide sequence, that controls aggregation is protein concentration, C. Interactions between the polypeptide chains become important if C exceeds the overlap concentration C* ⋍ N/V, where λ is a constant, N is approximately the number of residues in the polypeptide chain, and V is the volume associated with one polypeptide chain. If electrostatic interactions are important, V can exceed 4/3; πR3g, where Rg is the radius of gyration of the polypeptide chain (see Materials and Methods). Besides C, other factors can also influence aggregation. The equilibrium between the unfolded (U) and the native (N) states of a monomeric protein, which folds in a single step, depends on the sequence of the polypeptide chain as well as on the external conditions (pH, temperature, and salt concentration). The response of U and N to external conditions can be different (e.g., denaturants can stabilize U, whereas N is destabilized). Such opposing tendencies as well as sequence-dependent variations of folding of the monomer make the study of protein aggregation difficult. Our goal is to decipher general scenarios for protein association using lattice models. We consider two different models. The first one, introduced recently by Harrison, Chan, Prusiner, and Cohen (HCPC) (Harrison et al. 2001), is used to probe a number of aspects of self-propagation that involves conformational change from a compact state in the monomer to an extended "β-sheet" conformation in the oligomer phase. This model was designed to be a toy model for probing prion-like behavior. Aggregation and chain propagation in this system occur from a conformation other than U or N. This is in accord with the popular proposal that aggregation in amyloid forming peptides and proteins takes place by populating (at least transiently) partially folded intermediates (Kelly 1996). In contrast to the previous examples in which oligomer assembly is accompanied by a large conformational transition, several studies (Silow and Oliveberg 1997; Silow et al. 1999) suggest that proteins, which fold by two-state kinetics at infinite dilution (C → 0), can undergo reversible aggregation directly from the U state. To decipher the general principles that govern aggregation in these two-state proteins, we use three-dimensional lattice models with side chains (LMSC). Using LMSC and temperature (T) and C as variables, we address the following questions: What are the "phases" in the (T,C) plane? How does the assembly into some of these states occur starting from U? Because of the simplicity of the models used, only general questions about principles can be addressed. Nevertheless, we show that questions of experimental interest, such as the role of partially folded intermediates in facilitating aggregation, can be addressed using simple models. The present work, which expands on previous studies of aggregation using ON AND OFF-lattice models (Broglia et al. 1998; Gupta et al. 1998; Istrail et al. 1999; Giugliarelli et al. 2000; Smith and Hall 2001), provides a framework for obtaining insights into the scenarios for oligomerization.

162 citations


Journal ArticleDOI
TL;DR: Analytical centrifugation, as well as column chromatography coupled with multiangle light scatter, is used to show that in solution SecA undergoes at least two monomer‐dimer equilibrium reactions that are sensitive to temperature and to concentration of salt.
Abstract: SecA, a homodimeric protein involved in protein export in Escherichia coli, exists in the cell both associated with the membrane translocation apparatus and free in the cytosol. SecA is a multifunctional protein involved in protein localization and regulation of its own expression. To carry out these functions, SecA interacts with a variety of proteins, phospholipids, nucleotides, and nucleic acid and shows two enzymic activities. It is an ATPase and a helicase. Its role during protein localization involves interaction with the precursor polypeptides to be exported, the cytosolic chaperone SecB, and the SecY subunit of the membrane-associated translocase, as well as with acidic phospholipids. At the membrane, SecA undergoes a cycle of binding and hydrolysis of ATP coupled to conformational changes that result in translocation of precursors through the cytoplasmic membrane. The helicase activity of SecA and its affinity for its mRNA are involved in regulation of its own expression. SecA has been reported to exist in at least two conformational states during its functional cycle. Here we have used analytical centrifugation, as well as column chromatography coupled with multiangle light scatter, to show that in solution SecA undergoes at least two monomer-dimer equilibrium reactions that are sensitive to temperature and to concentration of salt.

146 citations


Journal ArticleDOI
TL;DR: The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry and turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.
Abstract: The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.

132 citations


Journal ArticleDOI
TL;DR: The subunit composition of a leukocidin pore is deduced by two independent methods: gel shift electrophoresis and site‐specific chemical modification during single‐channel recording.
Abstract: Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homo-oligomeric pores; for example, the staphylococcal α-hemolysin (αHL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the αHL pore. It is also pertinent to the mechanism of assembly of β-PFT pores and suggests new possibilities for engineering these proteins.

Journal ArticleDOI
TL;DR: These findings highlight common misconceptions about hydrogen bonding in proteins, and provide explanations for why the explicit hydrogen bonding terms of some popular force field sets are often best switched off.
Abstract: A new semi-empirical force field has been developed to describe hydrogen-bonding interactions with a directional component. The hydrogen bond potential supports two alternative target angles, motivated by the observation that carbonyl hydrogen bond acceptor angles have a bimodal distribution. It has been implemented as a module for a macromolecular refinement package to be combined with other force field terms in the stereochemically restrained refinement of macromolecules. The parameters for the hydrogen bond potential were optimized to best fit crystallographic data from a number of protein structures. Refinement of medium-resolution structures with this additional restraint leads to improved structure, reducing both the free R-factor and over-fitting. However, the improvement is seen only when stringent hydrogen bond selection criteria are used. These findings highlight common misconceptions about hydrogen bonding in proteins, and provide explanations for why the explicit hydrogen bonding terms of some popular force field sets are often best switched off.

Journal ArticleDOI
TL;DR: The quality of the modeling approach is estimated by comparing the models with the experimentally determined structures and it is suggested that the general architecture, curvature, “interior/exterior” orientations of side chains, and backbone conformation of the LRR structures can be predicted correctly.
Abstract: The three-dimensional structures of leucine-rich repeat (LRR)-containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor, using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general, cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, "interior/exterior" orientations of side chains, and backbone conformation of the LRR structures can be predicted correctly. On the other hand, the analysis revealed that, in some cases, it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

Journal ArticleDOI
TL;DR: It was revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment‐based methods.
Abstract: We have developed an alignment-independent method for classification of G-protein coupled receptors (GPCRs) according to the principal chemical properties of their amino acid sequences. The method relies on a multivariate approach where the primary amino acid sequences are translated into vectors based on the principal physicochemical properties of the amino acids and transformation of the data into a uniform matrix by applying a modified autocross-covariance transform. The application of principal component analysis to a data set of 929 class A GPCRs showed a clear separation of the major classes of GPCRs. The application of partial least squares projection to latent structures created a highly valid model (cross-validated correlation coefficient, Q2 = 0.895) that gave unambiguous classification of the GPCRs in the training set according to their ligand binding class. The model was further validated by external prediction of 535 novel GPCRs not included in the training set. Of the latter, only 14 sequences, confined in rapidly expanding GPCR classes, were mispredicted. Moreover, 90 orphan GPCRs out of 165 were tentatively identified to GPCR ligand binding class. The alignment-independent method could be used to assess the importance of the principal chemical properties of every single amino acid in the protein sequences for their contributions in explaining GPCR family membership. It was then revealed that all amino acids in the unaligned sequences contributed to the classifications, albeit to varying extent; the most important amino acids being those that could also be determined to be conserved by using traditional alignment-based methods.

Journal ArticleDOI
TL;DR: It is suggested that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide.
Abstract: The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns. The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution. We report here the crystal structures of the streptavidin mutants '1' and '2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II. Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 A. Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket--comprising residues 45 through 52--adopts an 'open' conformation in all four subunits within the asymmetric unit. The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin. Our findings suggest that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide. Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism.

Journal ArticleDOI
TL;DR: The relationship between protein folding rates and the contact order (CO) of the native structure has implications for ab initio protein structure prediction, and lack of simulation convergence can indicate the need for improved sampling of high CO conformations.
Abstract: Although much of the motivation for experimental studies of protein folding is to obtain insights for improving protein structure prediction, there has been relatively little connection between experimental protein folding studies and computational structural prediction work in recent years. In the present study, we show that the relationship between protein folding rates and the contact order (CO) of the native structure has implications for ab initio protein structure prediction. Rosetta ab initio folding simulations produce a dearth of high CO structures and an excess of low CO structures, as expected if the computer simulations mimic to some extent the actual folding process. Consistent with this, the majority of failures in ab initio prediction in the CASP4 (critical assessment of structure prediction) experiment involved high CO structures likely to fold much more slowly than the lower CO structures for which reasonable predictions were made. This bias against high CO structures can be partially alleviated by performing large numbers of additional simulations, selecting out the higher CO structures, and eliminating the very low CO structures; this leads to a modest improvement in prediction quality. More significant improvements in predictions for proteins with complex topologies may be possible following significant increases in high–performance computing power, which will be required for thoroughly sampling high CO conformations (high CO proteins can take six orders of magnitude longer to fold than low CO proteins). Importantly for such a strategy, simulations performed for high CO structures converge much less strongly than those for low CO structures, and hence, lack of simulation convergence can indicate the need for improved sampling of high CO conformations. The parallels between Rosetta simulations and folding in vivo may extend to misfolding: The very low CO structures that accumulate in Rosetta simulations consist primarily of local up–down β–sheets that may resemble precursors to amyloid formation.

Journal ArticleDOI
TL;DR: Histidine pKa values were measured in charge‐reversal and charge‐neutralization mutants of staphylococcal nuclease by 1H‐NMR spectroscopy to describe the distance dependence and salt sensitivity of pairwise coulombic interactions.
Abstract: Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions. Calculations with a continuum electrostatics method captured the experimental DeltaG(ij) when static structures were used and when the protein interior was treated empirically with a dielectric constant of 20. The DeltaG(ij) when r(ij) < or = 10 A were exaggerated slightly in the calculations. Coulomb's law with a dielectric constant near 80 and a Debye-Huckel term to account for screening by the ionic strength reproduced the salt sensitivity and distance dependence of DeltaG(ij) as well as the structure-based method. In their interactions with each other, surface charges behave as if immersed in water; the Debye length describes realistically the distance where interactions become negligible at a given ionic strength. On average, charges separated by distances (r(ij)) approximately 5 A interacted with DeltaG(ij) approximately 0.6 kcal/mole in 0.01 M KCl, but DeltaG(ij) decayed to < or =0.10 kcal/mole when r(ij) = 20 A. In 0.10 M KCl, DeltaG(ij) approximately 0.10 kcal/mole when r(ij) = 10 A. In 1.5 M KCl, only short-range interactions with r(ij) < or = 5 A persisted. Although at physiological ionic strengths the interactions between charges separated by more than 10 A are extremely weak, in situations where charge imbalance exists many weak interactions can cumulatively produce substantial effects.

Journal ArticleDOI
TL;DR: Analysis of the data for TMC‐126 and KNI‐764, another second‐generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.
Abstract: Amprenavir is one of six protease inhibitors presently approved for clinical use in the therapeutic treatment of AIDS. Biochemical and clinical studies have shown that, unlike other inhibitors, Amprenavir is severely affected by the protease mutation I50V, located in the flap region of the enzyme. TMC-126 is a second-generation inhibitor, chemically related to Amprenavir, with a reported extremely low susceptibility to existing resistant mutations including I50V. In this paper, we have studied the thermodynamic and molecular origin of the response of these two inhibitors to the I50V mutation and the double active-site mutation V82F/I84V that affects all existing clinical inhibitors. Amprenavir binds to the wild-type HIV-1 protease with high affinity (5.0 × 109 M−1 or 200 pM) in a process equally favored by enthalpic and entropic contributions. The mutations I50V and V82F/I84V lower the binding affinity of Amprenavir by a factor of 147 and 104, respectively. TMC-126, on the other hand, binds to the wild-type protease with extremely high binding affinity (2.6 × 1011 M−1 or 3.9 pM) in a process in which enthalpic contributions overpower entropic contributions by almost a factor of 4. The mutations I50V and V82F/I84V lower the binding affinity of TMC-126 by only a factor of 16 and 11, respectively, indicating that the binding affinity of TMC-126 to the drug-resistant mutants is still higher than the affinity of Amprenavir to the wild-type protease. Analysis of the data for TMC-126 and KNI-764, another second-generation inhibitor, indicates that their low susceptibility to mutations is caused by their ability to compensate for the loss of interactions with the mutated target by a more favorable entropy of binding.

Journal ArticleDOI
TL;DR: In this paper, a set of 475 cocrystallized protein-protein complexes was used to build an array of models of each protein in the set, and a sophisticated procedure was created to generate the models with RMS deviations of 1, 2, 3,..., 10 Å from the crystal structure.
Abstract: An adequate description of entire genomes has to include information on the three-dimensional (3D) structure of proteins. Most of these protein structures will be determined by high-throughput modeling procedures. Thus, a structure-based analysis of the network of protein–protein interactions in genomes requires docking methodologies that are capable of dealing with significant structural inaccuracies in the modeled structures of proteins. We present a systematic study of the applicability of our low-resolution docking method to protein models of different accuracies. A representative nonredundant set of 475 cocrystallized protein–protein complexes was used to build an array of models of each protein in the set. A sophisticated procedure was created to generate the models with RMS deviations of 1, 2, 3, . . ., 10 Å from the crystal structure. The docking was performed for all the models, and the predictions were compared with the configurations of the original cocrystallized complexes. Statistical analysis showed that the low-resolution docking can determine the gross structural features of protein–protein interactions for a significant percent of complexes of highly inaccurate protein models. Such predictions may serve as starting points for a more detailed structural analysis, as well as complement experimental and computational data on protein–protein interactions obtained by other techniques.

Journal ArticleDOI
TL;DR: Structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications to develop inhibitors that bind sufficiently tightly to drug‐resistant variants of HIV‐1 protease to potentially become the next generation of therapeutic agents.
Abstract: The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to ∼2.2 Aresolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

Journal ArticleDOI
John W. Crabb1, June O'Neil1, Masaru Miyagi1, K.A. West1, Henry F. Hoff1 
TL;DR: The hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE is supported.
Abstract: Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 μM HNE lost ∼76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

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TL;DR: The enthalpic favorability of folding in these designed β‐hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.
Abstract: Autonomously folding β-hairpins (two-strand antiparallel β-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a β-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in β-hairpin stability by comparing 1 with DP→ N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the DPG segment decreases the entropic cost of β-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of DPG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed β-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.

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TL;DR: The organization of these subunits within cytoplasmic dynein was examined by separating the molecule into two distinct subcomplexes that were competent to reassemble into a molecule with dyne in‐like properties and suggest that individual dyneIn molecules have distinct molecular compositions in vivo.
Abstract: Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo.

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TL;DR: It is shown that the formation of the β‐sheet state of SC3 proceeds via two intermediates, the first intermediate has an infrared spectrum indistinguishable from that of the α‐helical state ofSC3, and the end state has the same secondary structure, but is characterized by the familiar 10‐nm‐wide rodlets.
Abstract: Hydrophobins self assemble into amphipathic films at hydrophobic–hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the α-helical state that is formed upon binding to a hydrophobic solid, and the β-sheet state, which is formed at the air–water interface. Here, we show that the formation of the β-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the α-helical state of SC3. The second intermediate is rich in β-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets.

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TL;DR: The redesigned hGH computationally to improve its thermostability was redesigned using a previously developed combinatorial optimization algorithm based on the dead‐end elimination theorem and shows the utility of the free energy function in automated protein design.
Abstract: Recombinant human growth hormone (hGH) is used worldwide for the treatment of pediatric hypopituitary dwarfism and in children suffering from low levels of hGH. It has limited stability in solution, and because of poor oral absorption, is administered by injection, typically several times a week. Development has therefore focused on more stable or sustained-release formulations and alternatives to injectable delivery that would increase bioavailability and make it easier for patients to use. We redesigned hGH computationally to improve its thermostability. A more stable variant of hGH could have improved pharmacokinetics or enhanced shelf-life, or be more amenable to use in alternate delivery systems and formulations. The computational design was performed using a previously developed combinatorial optimization algorithm based on the dead-end elimination theorem. The algorithm uses an empirical free energy function for scoring designed sequences. This function was augmented with a term that accounts for the loss of backbone and side-chain conformational entropy. The weighting factors for this term, the electrostatic interaction term, and the polar hydrogen burial term were optimized by minimizing the number of mutations designed by the algorithm relative to wild-type. Forty-five residues in the core of the protein were selected for optimization with the modified potential function. The proteins designed using the developed scoring function contained six to 10 mutations, showed enhancement in the melting temperature of up to 16°C, and were biologically active in cell proliferation studies. These results show the utility of our free energy function in automated protein design.

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TL;DR: This study finds that the present microscopic model is indeed consistent with HIs and transition states, but such states occur in parallel, rather than along the single pathway predicted by the sequential stabilization model.
Abstract: Do two-state proteins fold by pathways or funnels? Native-state hydrogen exchange experiments show discrete nonnative structures in equilibrium with the native state. These could be called hidden intermediates (HI) because their populations are small at equilibrium, and they are not detected in kinetic experiments. HIs have been invoked as disproof of funnel models, because funnel pictures appear to indicate (1) no specific sequences of events in folding; (2) a continuum, rather than a discrete ladder, of structures; and (3) smooth landscapes. In the present study, we solve the exact dynamics of a simple model. We find, instead, that the present microscopic model is indeed consistent with HIs and transition states, but such states occur in parallel, rather than along the single pathway predicted by the sequential stabilization model. At the microscopic level, we observe a huge multiplicity of trajectories. But at the macroscopic level, we observe two pathways of specific sequences of events that are relatively traditional except that they are in parallel, so there is not a single reaction coordinate. Using singular value decomposition, we show an accurate representation of the shapes of the model energy landscapes. They are highly complex funnels.

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TL;DR: The structure of the bacterial α‐amylase from the psychrophilic micro‐organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved and it is shown that a localized negative charge such as that of Cl−, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation.
Abstract: To further investigate the mechanism and function of allosteric activation by chloride in some alpha-amylases, the structure of the bacterial alpha-amylase from the psychrophilic micro-organism Pseudoalteromonas haloplanktis in complex with nitrate has been solved at 2.1 A degrees, as well as the structure of the mutants Lys300Gln (2.5 A degrees ) and Lys300Arg (2.25 A degrees ). Nitrate binds strongly to alpha-amylase but is a weak activator. Mutation of the critical chloride ligand Lys300 into Gln results in a chloride-independent enzyme, whereas the mutation into Arg mimics the binding site as is found in animal alpha-amylases with, however, a lower affinity for chloride. These structures reveal that the triangular conformation of the chloride ligands and the nearly equatorial coordination allow the perfect accommodation of planar trigonal monovalent anions such as NO3-, explaining their unusual strong binding. It is also shown that a localized negative charge such as that of Cl-, rather than a delocalized charge as in the case of nitrate, is essential for maximal activation. The chloride-free mutant Lys300Gln indicates that chloride is not mandatory for the catalytic mechanism but strongly increases the reactivity at the active site. Disappearance of the putative catalytic water molecule in this weakly active mutant supports the view that chloride helps to polarize the hydrolytic water molecule and enhances the rate of the second step in the catalytic reaction.

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TL;DR: The data suggest that the variations observed in amino acid frequencies in different phi/psi/chi1 environments in folded proteins reflect energetically important local side-chain-backbone interactions, interactions that may severely restrict the ensemble of conformations populated in the denatured state to a relatively small subset with nativelike structure.
Abstract: To estimate how extensively the ensemble of denatured-state conformations is constrained by local side-chain-backbone interactions, propensities of each of the 20 amino acids to occur in mono- and dipeptides mapped to discrete regions of the Ramachandran map are computed from proteins of known structure. In addition, propensities are computed for the trans, gauche-, and gauche+ rotamers, with or without consideration of the values of phi and psi. These propensities are used in scoring functions for fragment threading, which estimates the energetic favorability of fragments of protein sequence to adopt the native conformation as opposed to hundreds of thousands of incorrect conformations. As finer subdivisions of the Ramachandran plot, neighboring residue phi/psi angles, and rotamers are incorporated, scoring functions become better at ranking the native conformation as the most favorable. With the best composite propensity function, the native structure can be distinguished from 300,000 incorrect structures for 71% of the 2130 arbitrary protein segments of length 40, 48% of 2247 segments of length 30, and 20% of 2368 segments of length 20. A majority of fragments of length 30-40 are estimated to be folded into the native conformation a substantial fraction of the time. These data suggest that the variations observed in amino acid frequencies in different phi/psi/chi1 environments in folded proteins reflect energetically important local side-chain-backbone interactions, interactions that may severely restrict the ensemble of conformations populated in the denatured state to a relatively small subset with nativelike structure.

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TL;DR: The results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.
Abstract: The contribution of solvent-exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site-directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys-amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (ΔG = 18 kJ/mol). These results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.

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TL;DR: A clinically observed human mutation was used to narrow the search for potential catalytic groups within the fumarase active site, and kinetic and X‐ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.
Abstract: Fumarase catalyzes the reversible conversion of fumarate to S- malate during the operation of the ubiquitous Kreb's cycle. Previous studies have shown that the active site includes side chains from three of the four subunits within the tetrameric enzyme. We used a clinically observed human mutation to narrow our search for potential catalytic groups within the fumarase active site. Offspring homozygous for the missense mutation, a G-955–C transversion in the fumarase gene, results in the substitution of a glutamine at amino acid 319 for the normal glutamic acid. To more fully understand the implications of this mutation, a single-step site-directed mutagenesis method was used to generate the homologous substitution at position 315 within fumarase C from Escherichia coli. Subsequent kinetic and X-ray crystal structure analyses show changes in the turnover number and the cocrystal structure with bound citrate.