NMR analysis of the monomeric form of a mutant unliganded bovine neurophysin: comparison with the crystal structure of a neurophysin dimer

Determination of the structure of the unliganded monomeric state of neurophysin is central to an understanding of the allosteric relationship between neurophysin peptide-binding and dimerization. We examined this state by NMR, using the weakly dimerizing H80E mutant of bovine neurophysin-I. The derived structure, to which more than one conformer appeared to contribute, was compared with the crystal structure of the unliganded des 1-6 bovine neurophysin-II dimer. Significant conformational differences between the two proteins were evident in the orientation of the 3,10 helix, in the 50-58 loop, in beta-turns, and in specific intrachain contacts between amino- and carboxyl domains. However, both had similar secondary structures, in independent confirmation of earlier circular dichroism studies. Previously suggested interactions between the amino terminus and the 50-58 loop in the monomer were also confirmed. Comparison of the observed differences between the two proteins with demonstrated effects of dimerization on the NMR spectrum of bovine neurophysin-I, and preliminary investigation of the effects of dimerization on H80E spectra, allowed tentative distinction between the contributions of sequence and self-association differences to the difference in conformation. Regions altered by dimerization encompass most binding site residues, providing a potential explanation of differences in binding affinity between the unliganded monomeric and dimeric states. Differences between monomer and dimer states in turns, interdomain contacts, and within the interdomain segment of the 50-58 loop suggest that the effects of dimerization on intrasubunit conformation reflect the need to adjust the relative positions of the interface segments of the two domains for optimal interaction with the adjacent subunit and/or reflect the dual role of some residues as participants both at the interface and in interdomain contacts.     

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms

The structures of des 1-6 bovine neurophysin-II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 A and 2.3 A, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full-length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding-induced conformational changes that were the largest in the loop region comprising residues 50-58 and in the 7-10 region. A subtle binding-induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of beta-sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone alpha-amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.     

NMR investigation of main-chain dynamics of the H80E mutant of bovine neurophysin-I: demonstration of dimerization-induced changes at the hormone-binding site

Neurophysins are hormone-binding proteins composed of two partially homologous domains. Ligand-binding (localized to the amino domain) and dimerization (involves both domains) are cooperatively linked by an as yet undefined allosteric mechanism. To help define this mechanism, we investigated the backbone dynamics of the unliganded monomeric state of the H80E mutant of bovine neurophysin-I by (15)N NMR. Model-free analysis of the NMR relaxation parameters indicated significantly greater flexibility in the carboxyl domain than in the amino domain, particularly at their dimerization interface segments. Amino domain residues critical to hormone binding were highly structured, constraining potential allosteric mechanisms. Model-free analysis additionally demonstrated chemical exchange effects, manifest as R(ex) terms, in 16 residues, 14 of which are located in the amino domain at, or immediately adjacent to, either the dimerization interface or the hormone-binding site. The chemical exchange process was further characterized using relaxation-compensated CPMG measurements, the results allowing assignment of the process to monomer-dimer exchange and calculation of the exchange kinetics, which were slow on the NMR time scale. An apparently different concentration-dependent process, distinguished from normal dimerization by its fast exchange behavior and pH-independence, also principally involved a subset of residues at and immediately adjacent to either the hormone-binding site or the amino domain dimerization interface. The data represent the first direct demonstration of an effect of dimerization in the unliganded state on neurophysin's hormone-binding site, the effect particularly involving residues that interact with hormone residue 2, and specifically identify Ser25 and Ile26 as likely intermediaries between the sites of dimerization and of hormone binding. Consistent with recent views of the role of anchor residues in protein interactions, we propose that dimerization proceeds by a fast pH-independent association of the well-structured amino domain interface that is rapidly communicated to the binding site for hormone residue 2, followed by a rate-determining pH-dependent interaction of the less structured carboxyl domain interface.     

Amino acid sequence of bovine neurophysin-II: a reinvestigation.

The entire amino acid sequence of bovine neurophysin-II has been redetermined by manual Edman degradation of tryptic peptides obtained from performic acid-oxidized neurophysin. Electrophoretically homogeneous bovine neurophysin-II was found to be a mixture of two species of protein molecules both containing 95 amino acid residues. The only difference between the two species of the neurophysin molecules is a single amino acid substitution at residue 89. Of the bovine neurophysin-II used in this work 70% of the protein material contained valine and 30% contained isoleucine at residue 89 in their sequences. The redetermined sequences of bovine neurophysin-II shown in Fig. 2 differ substantially from the reported sequence of bovine neurophysin-II but resemble closely that of porcine neurophysin-I and ovine neurophysin-III (Fig. 3).     

Binding and fluorescence studies of the relationship between neurophysin-peptide interaction and neurophysin self-association: an allosteric system exhibiting minimal cooperativity

The mechanism of peptide-enhanced neurophysin self-association was investigated to address questions raised by the crystal structure of a neurophysin-dipeptide complex. The dependence on protein concentration of the binding of a broad range of peptides to the principal hormone-binding site confirmed that occupancy of this site alone, and not a site that bridges the monomer-monomer interface, is the trigger for enhanced dimerization. For the binding of most peptides to the principal hormone-binding site on bovine neurophysin I, the affinity of each dimer site was at least 10 times that of monomer under the conditions used. No interactions between the two sites of the dimer were evident. Fluorescence polarization studies of pressure-induced dimer dissociation indicated that the volume change for this reaction was almost 4 times greater in the liganded than in the unliganded state, pointing to a significant alteration of the monomer-monomer interface upon peptide binding. Novel conformational changes in the vicinity of the single neurophysin tyrosine, Tyr-49, induced by pressures lower than required for subunit dissociation, were also observed. The bovine neurophysin I dimer therefore appears to represent an allosteric system in which there is thermodynamic and functional communication between each binding site and the monomer-monomer interface, but no communication across the interface to the binding site of the other subunit. A model for the peptide-enhanced dimerization is proposed in which intersubunit contacts between monomers reduce the large unfavorable free energy associated with binding-induced intrasubunit conformational change. Structural origins of the lack of communication across the interface are suggested on the basis of the low volume change associated with dimerization in the unliganded state and monomer-monomer contacts in the crystal structure. Potential roles for the peptide alpha-amino group and position 2 phenyl ring in triggering conformational change are discussed.     

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.     

Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: relationships to the intermolecular oxytocin-neurophysin complex.

Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.     

Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.     

Dimerization of native and proteolytically modified neurophysins as monitored by proton magnetic resonance spectroscopy: proximity of tyrosine-49 to the subunit interface

Neurophysin is a self-associating protein in which peptide-hormone binding and dimerization are thermodynamically linked. The structural basis of the linkage is unknown. We have studied the dimerization of bovine neurophysin I and two proteolytically modified derivatives by proton nuclear magnetic resonance spectroscopy in order to identify residues at the intersubunit contact regions and to evaluate the origin of the reported loss of dimerization associated with tryptic excision of residues 1-8. The concentration dependence at neural pH of the spectra of native neurophysin and des-90-92-neurophysin demonstrated a finite set of dimerization-sensitive resonances that included the ring protons of Tyr-49. Using these to monitor dimerization, we confirmed predictions of a large increase in the dimerization constant associated with carboxyl protonation. By the same criteria, dimerization of the des-1-8 protein, in disagreement with earlier reports, was found to be undiminished relative to that of the native protein. However, spectral changes in the Tyr-49 ring ortho proton region associated with dimerization of the des-1-8 protein differed significantly from those in the native protein and indicated an altered conformation of the des-1-8 dimer apparently restricted to the vicinity of Tyr-49. The results are shown to place Tyr-49 adjacent to both the intersubunit contact region and the 1-8 sequence in the native protein, loss of stabilizing interactions with 1-8 leading to altered interactions of Tyr-49 with the subunit interface. Because Tyr-49 is also close to the peptide-binding site, this arrangement spatially links the peptide-binding and dimerization sites of neurophysin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon-13 nuclear magnetic resonance studies of the binding of selectively 13C-enriched oxytocins to the neurophypophyseal protein, bovine neurophysin II

Complex formation between bovine neurophysin II and oxytocin molecules containing 85% 13C enrichment in specific amino acid residues was studied using 13C nuclear magnetic resonance spectroscopy. Chemical shift and relaxation time values of the analogue [13C-Leu3]oxytocin, [13C-Gly9]oxytocin, and the doubly labeled [13C-Ile3 Gly9]oxytocin were obtained for the hormones in the absence and presence of neurophysin. The results showed that certain 13C nuclear magnetic resonance parameters of residue 3 but not of residue 9 of oxytocin are altered upon binding to neurophysin. These observations suggest that residue 3 but not residue 9 is involved in the protein-hormone interaction and they demonstrate the general applicability of selective 13C enrichment for the study of peptide-protein interactions.     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.     

Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor

An oxytocin/bovine neurophysin I biosynthetic precursor, [N epsilon-diacetimidyl-30,71, des-His106]pro-OT/BNPI, was synthesized from a synthetic oxytocinyl peptide, 1/2Cys-Tyr-Ile-Gln-Asn-1/2Cys-Pro-Leu-Gly-Gly-Lys-Arg, and native neurophysin by chemical semisynthesis. The semisynthetic precursor contains the entire sequence of the biosynthetic precursor deduced from the complementary DNA structure except for omission of the carboxyl-terminal histidine residue. The covalent structure of the semisynthetic product was verified by amino acid analysis and amino-terminal analysis. Analytical affinity chromatography was employed to evaluate noncovalent binding properties of the precursor. The precursor does not bind significantly to immobilized Met-Tyr-Phe, a hormone binding site ligand. In contrast, the acetimidated precursor binds to immobilized bovine neurophysin II, with a 13-fold higher affinity than does acetimidated neurophysin itself. When a hormonal ligand, [Lys8]vasopressin, was added to the elution buffer at the concentration of 0.1 mM so that a major portion of the immobilized BNPII was liganded, the affinity between the immobilized liganded BNPII and the precursor was enhanced 8-fold and approached the affinity for the liganded (bovine neurophysin I-immobilized BNPII) interaction. The data imply that the precursor can self-associate and that this self-association is closely related to that of liganded neurophysin. The tripeptide affinity matrix data argue that, in the precursor, the ligand binding site of the neurophysin domain is occupied intramolecularly by the hormone domain. The data verify the view that both the self-association surface and hormone binding site are established upon precursor folding. A disulfide stability analysis showed the resistance, to disulfide interchange by dithiothreitol, of semisynthetic precursor but not of neurophysin, as judged by protein association and peptide ligand binding activities, respectively. The results argue that the molecular structure of the precursor is established upon precursor folding and before enzymatic processing that produces mature hormone and neurophysin.     

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.     

Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue

Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.     

Molecular dissection,tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans

We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability. A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues.     

The occurrence of C--H...O hydrogen bonds in alpha-helices and helix termini in globular proteins

A comprehensive database analysis of C--H...O hydrogen bonds in 3124 alpha-helices and their corresponding helix termini has been carried out from a nonredundant data set of high-resolution globular protein structures resolved at better than 2.0 A in order to investigate their role in the helix, the important protein secondary structural element. The possible occurrence of 5 --> 1 C--H...O hydrogen bond between the ith residue CH group and (i - 4)th residue C==O with C...O < or = 3.8 A is studied, considering as potential donors the main-chain Calpha and the side-chain carbon atoms Cbeta, Cgamma, Cdelta and Cepsilon. Similar analysis has been carried out for 4 --> 1 C--H...O hydrogen bonds, since the C--H...O hydrogen bonds found in helices are predominantly of type 5 --> 1 or 4 --> 1. A total of 17,367 (9310 of type 5 --> 1 and 8057 of type 4 --> 1) C--H...O hydrogen bonds are found to satisfy the selected criteria. The average stereochemical parameters for the data set suggest that the observed C--H...O hydrogen bonds are attractive interactions. Our analysis reveals that the Cgamma and Cbeta hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic beta-branched residue Ile to participate in 5 --> 1 C--H...O hydrogen bonds involving methylene Cgamma 1 atom as donor in alpha-helices. This may be an enthalpic compensation for the greater loss of side-chain conformational entropy for beta-branched amino acids due to the constraint on side-chain torsion angle, namely, chi1, when they occur in helices. The preference of amino acids for 4 --> 1 C--H...O hydrogen bonds is found to be more for Asp, Cys, and for aromatic residues Trp, Phe, and His. Interestingly, overall propensity for C--H...O hydrogen bonds shows that a majority of the helix favoring residues such as Met, Glu, Arg, Lys, Leu, and Gln, which also have large side-chains, prefer to be involved in such types of weak attractive interactions in helices. The amino acid side-chains that participate in C--H...O interactions are found to shield the acceptor carbonyl oxygen atom from the solvent. In addition, C--H...O hydrogen bonds are present along with helix stabilizing salt bridges. A novel helix terminating interaction motif, X-Gly with Gly at C(cap) position having 5 --> 1 Calpha--H...O, and a chain reversal structural motif having 1 --> 5 Calpha-H...O have been identified and discussed. Our analysis highlights that a multitude of local C--H...O hydrogen bonds formed by a variety of amino acid side-chains and Calpha hydrogen atoms occur in helices and more so at the helix termini. It may be surmised that the main-chain Calpha and the side-chain CH that participate in C--H...O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N--H...O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins.     

Enthalpies of ligand binding to bovine neurophysins

Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligand binding by bovine neurophysins I and II. Calorimetry studies were supplemented by van't Hoff analyses of binding constants determined by circular dichroism. Free energies of binding of a series of di- and tripeptides that bind to the strong hormone binding site of neurophysin were partitioned into their enthalpic and entropic components. The results indicate that, at 25 degrees C, the binding of most peptides is an enthalpy-driven reaction associated with negative entropy and heat capacity changes. Studies elsewhere, supported by evidence here, indicate that the principal component of the negative enthalpy change does not arise from the increase in neurophysin dimerization associated with peptide binding. Accordingly, the negative enthalpy change is attributed to direct bonding interactions with peptide and possibly also to peptide-induced changes in tertiary or quaternary organization. Comparison of the binding enthalpies of different peptides indicated two types of bonding interactions that contribute to the negative enthalpy change of peptide ligation. Substitution of an aromatic- or sulfur-containing side chain for an aliphatic side chain in position 1 of bound peptides led to increases in negative enthalpy of from 1 to 6 kcal/mol, demonstrating that interactions typically classified as hydrophobic can have a significant exothermic component at 25 degrees C. Similarly, loss of hydrogen bonding potential in the peptide decreased the enthalpy change upon binding, in keeping with the expected enthalpic contribution of hydrogen bonds. In particular, the data suggested that the peptide backbone between residues 2 and 3 and the phenolic hydroxyl group in position 2 participate in hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutron diffraction studies of the structure of filamentous bacteriophage Pf1. Demonstration that the coat protein consists of a pair of alpha-helices with an intervening, non-helical surface loop

The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and specifically deuterated phage. These methods have been used to determine the positions of the two methionine, two tyrosine and six isoleucine residues of the coat protein. Combined with the positions of the five valine residues previously determined, they represent one third (15 of 46) of the residues of the coat protein. These 15 amino acid residue positions have been used as the basis for constructing a model for the protein consisting of two alpha-helices with an intervening surface loop. The first helix extends from near the amino terminus to Ile12. The second helix extends from Lys20 to at least Met42, and may contain a bend between Ile32 and Val35. The two helices are tilted by about 15 degrees relative to one another, and are positioned in such a way that they appear to be bound end-to-end by main-chain hydrogen bonds. The intervening, non-helical loop, made up of Thr13 to Met19, connects the two helices without disrupting the pattern of main-chain hydrogen bonding, but does not result in a bend in the otherwise continuous helical structure. This model is used to predict the approximate positions of all amino acid residues in the Pf1 protein coat, providing a basis for further understanding of a number of viral properties including the symmetry transitions, the non-isomorphism of heavy-atom derivatives, and the protein-protein and protein-DNA interactions in the virion.     

Proton magnetic resonance and binding studies of proteolytically modified neurophysins

The proton NMR spectra and role in peptide binding of carboxyl-terminal and NH2-terminal neurophysin residues were studied by preparation of bovine neurophysin-I derivatives from which residues 90-92 had been cleaved by carboxypeptidase or residues 1-8 excised by trypsin. The carboxypeptidase-treated protein showed normal peptide-binding behavior. NMR comparisons of this derivative and the native protein allowed identification of proton resonances associated with residues 89-92, confirmed a lack of functional role for this region of the protein, and permitted new observations on the behavior of neurophysin's aromatic residues. The trypsin-treated protein bound peptide with an affinity only 1/50 that of the native protein at pH 6 but evinced the same binding specificity and pH dependence of binding as the native protein. These results argued against direct interaction of residues in the 1-8 sequence with bound peptide and for a role for these residues, particularly Arg-8, in conformational stabilization of the active site; this role is held to be additional to the reported influence of 1-8 on dimerization. NMR comparisons of the trypsin product and native protein allowed preliminary assignment of a set of alkyl proton resonances to residues within the 1-8 sequence and were compatible with a restricted environment for Arg-8. Conformational differences between native and trypsin-treated proteins were manifest particularly by differences in the NMR spectra of Phe and Tyr-49 ring protons. The behavior of Phe ring protons was consistent with the reported decreased dimerization constant of the trypsin product and suggested participation of Phe-22 or -35 in dimerization. The behavior of Tyr-49 provided the first direct evidence of a change in secondary or tertiary structure associated with excision of residues 1-8. Suggested mechanisms by which this conformational change reduces binding include a direct effect on Tyr-49 and/or a conformational rearrangement of active site residues near Tyr-49.