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E Breslow

Publications and source records attributed to E Breslow.

At least 19 recordsLinked to original sources
PubMed2001

Effects of diabetes insipidus mutations on neurophysin folding and function.

Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, DeltaE47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.

Amino Acid Sequence↗
PubMed2001

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.

Allosteric Regulation↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1999

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.

Amino Acids↗
PubMed1997

Modulation of dimerization by residues distant from the interface in bovine neurophysin-II.

The crystal structure of bovine neurophysin-II in its liganded state (Chen et al. [1991] Proc. Natl. Acad. Sci. USA 88, 4240-4244) indicates that the 1-6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer-monomer interface. Cleavage of the 1-6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1-6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des-1-6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1-6 sequence differed in liganded and unliganded states. Additionally, the unliganded des-1-6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des-1-6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des-1-6 protein, as evidenced by signals from an internal dimerization-sensitive alpha-proton. Thus, the 1-6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1-6 protein with an internal clip after binding site residue Glu-47, the clip presumably breaking the short 3,10 helix that most directly connects the interface to the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des-1-6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1-6 sequence. A model is proposed in which the 1-6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self-association.

Amino Acid Sequence↗
PubMed1996

Differential binding of desmopressin and vasopressin to neurophysin-II.

Desmopressin is a synthetic analog of the peptide hormone vasopressin in which the N-terminal alpha-amino group has been removed and L-arginine in position 8 has been replaced by D-arginine. Using 1H-NMR spectroscopy, we show that desmopressin binds to neurophysin-II, whereas deamino-vasopressin does not bind. Thus, the change in configuration at Arg8 causes a significant difference in the binding of these hormones to neurophysin-II. We have determined the structure of desmopressin bound to neurophysin-II using two-dimensional 1H nuclear magnetic resonance-transferred nuclear Overhauser effect techniques. A common binding motif for vasopressin and desmopressin is proposed that includes a positive charge group along with the hydrophobic surface formed by the side chains of Tyr2 and a beta-methylene provided by Phe-3. In vasopressin, the positive charge is provided by the N-terminal NH3+, whereas in desmopressin, the side chain of Arg-8 contributes the positive charge. The type II beta-turn found in residues Cys6-Pro7-D-Arg8-Gly9 of the bound structure of desmopressin folds the Arg8 side chain back toward the disulfide-bond loop, which allows the positive charged side chain of Arg8 to participate in binding. Such a type II beta-turn is not found in deamino-vasopressin in the presence of neurophysin-II.

Animals↗
PubMed1996

The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties.

The active site of liganded neurophysin contains a salt bridge that involves the side chains of Arg-8 and Glu-47 of the protein and the alpha-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or to which the environment of Arg-8 in the unliganded state differs in monomers and dimers, is relevant to an understanding of allosteric mechanism in this system. In the present study, the behavior of the salt bridge was investigated by 15N NMR and chemical replacement of Arg-8. Bovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by 15N-substituted Arg or by other residues using chemical semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord with the view of the salt bridge in the liganded state obtained from crystals of bovine neurophysin-II complexes. In the unliganded state, comparison of the 15N and proton NMR signals from Arg-8 with those in smaller arginine systems suggested the absence of significant interactions between the guanidinium of Arg-8 and Glu-47 or between the amino terminal region of Arg-8 and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and dimers was found. Marked spectral changes accompanying the binding of oxytocin indicated changes in the environment of both the side chain and amino terminal region of Arg-8. The NMR results were in good agreement with a recently emerging comparison of bovine neurophysin-II derivatives in the liganded and unliganded states, with the notable exception of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, significant differences between the two bovine neurophysins in the susceptibility to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential allosteric mechanisms underlying neurophysin ligand-facilitated dimerization.

Allosteric Regulation↗
PubMed1996

Crystals of ligand-free bovine neurophysin II.

A modified neurophysin, des 1-6 bovine neurophysin II, has been crystallized in the absence of bound hormone or hormone analogue. These crystals represent the first crystals of ligand-free neurophysin, and are essential for understanding neurophysin-hormone recognition as well as hormone-induced neurophysin dimerization. The crystals diffract to beyond 1.8 A resolution, belong to space group P3(1)21 (or P3(2)21) with a = 48.86, c = 78.61 A, and contain one molecule per asymmetric unit.

Journal Article↗
PubMed1996

Thermodynamic role of the pro region of the neurophysin precursor in neurophysin folding: evidence from the effects of ligand peptides on folding.

Attention has focused recently on the role of amino-terminal precursor pro regions in protein folding, with particular emphasis on their effects on folding kinetics. We examined the kinetic and thermodynamic effects of ligand peptides on the folding of neurophysin from the reduced state; these peptides serve as analogs of the pro regions of the common precursors of the neurophysins and the hormones oxytocin and vasopressin. Folding of reduced, mononitrated bovine neurophysin-II was monitored by circular dichroism in a glutathione redox buffer. The results confirmed the ability of neurophysin to fold to a limited extent (20-25% in this system) in the absence of ligand peptides. Ligand peptides increased the efficiency of folding to 100%, the exact efficiency being dependent on peptide identity and concentration. However, the rate of folding was peptide-independent. Analysis of the folding reaction demonstrated relatively rapid conversion of the reduced state to a disulfide-scrambled state, which slowly converted (half-life of 5 h at pH 7.3) to the folded state. Native unliganded neurophysin also equilibrated with the disulfide-scrambled state in the same redox buffers. For each peptide, an equilibrium constant for the folding reaction, representing the amount of peptide bound in the folding system as a function of peptide concentration, was calculated. Comparison of this constant with the intrinsic binding constants of the native protein allowed the derivation, under conditions at or approaching thermodynamic reversibility, of the relative stability of the native and disulfide-scrambled states. The results indicate that the scrambled state, which probably represents the presence of incorrect disulfide pairs in both protein domains, is more stable than the native unliganded state by approximately 1 kcal/mol in this system. The role of ligand peptide therefore is to stabilize the folded protein after it is formed, i.e., it provides a thermodynamic sink. The results contrast with the putative behavior of exogenous peptides representative of the pro regions of subtilisin and alpha-lytic protease, which are generally considered to facilitate folding by reaction with folding intermediates. A potential alternative view of the role of propeptides in protease folding is suggested.

Animals↗
PubMed1996

Crystal structure of the neurophysin-oxytocin complex.

The first crystal structure of the pituitary hormone oxytocin complexed with its carrier protein neurophysin has been determined and refined to 3.0 A resolution. The hormone-binding site is located at the end of a 3(10)-helix and involves residues from both domains of each monomer. Hormone residues Tyr 2, which is buried deep in the binding pocket, and Cys 1 have been confirmed as the key residues involved in neurophysin-hormone recognition. We have compared the bound oxytocin observed in the neurophysin-oxytocin complex, the X-ray structures of unbound oxytocin analogues and the NMR-derived structure for bound oxytocin. We find that while our structure is in agreement with the previous crystallographic findings, it differs from the NMR result with regard to how Tyr 2 of the hormone is recognized by neurophysin.

Amino Acid Sequence↗
PubMed1995

NMR behavior of the aromatic protons of bovine neurophysin-I and its peptide complexes: implications for solution structure and for function.

The NMR behavior of the aromatic protons of bovine neurophysin-I and its complexes was interpreted with reference to the 2.8 A crystal structure of the dipeptide complex of bovine neurophysin-II and to mechanisms underlying the thermodynamic linkage between neurophysin dimerization and peptide binding. Large binding-induced shifts in the ring proton signals of Tyr-2 of ligand peptides (approximately 0.5 ppm upfield and approximately 0.35 ppm downfield at 25 degrees C for the 3,5- and 2,6-ring protons, respectively) were demonstrated. Consistent with the crystal structure, and in disagreement with conclusions by other investigators, evidence is presented indicating the absence of dipolar contact between Tyr-2 ring protons and protein Phe ring protons. The large binding-induced shifts are attributed to a strong influence of proximal neurophysin carbonyl and disulfide groups on the bound Tyr-2 ring, of potential importance in binding specificity. Resolution of the behavior of neurophysin Phe residues -22 and -35 and of their proton NOE contacts provided insights into the conformational changes associated with peptide binding and with dimerization. Within the amino domain of the protein, as evidenced by the behavior of interface residue Phe-35 and its NOE contacts, binding-induced changes in the subunit interface appeared to involve principally the junction between this interface region and the 3,10-helix that connects it to the binding site in the liganded state. By contrast, as judged by the NOE contacts of His-80, the corresponding interface participant of the carboxyl domain, peptide binding induced a marked decrease in side-chain mobility within the carboxyl domain segment of the interface. Interactions of Phe-22 with protons assigned to Ala-68, neither of which is an interface participant, were demonstrated to be markedly altered both by dimerization in the unliganded state and by peptide binding to the dimer. Since Phe-22 is adjacent to the peptide-binding site, the results collectively support a model in which conformational differences between unliganded monomer and dimer are important contributors to the preferential binding of peptide to the dimer and indicate that the amino and carboxyl domain segments of the interface, which are homologous, are affected differently by peptide binding.

Animals↗
PubMed1994

Amino acid sequence and properties of vasopressin-associated elephant neurophysin.

The primary structure of an elephant neurophysin, homologous to vasopressin-associated neurophysins, is reported. The protein contains a Tyr for Asn substitution at position 75, a position in direct contact with residues 77 and 78 of the monomer-monomer interface. This Tyr residue therefore serves as a potential reporter of the path involved in the long-range linkage between peptide binding and dimerization in this system. NMR studies of the protein in unliganded and liganded states demonstrated normal dimerization properties and the expected increase in dimerization associated with binding peptide. In keeping with an elevated pKa of 11.1 assigned to Tyr-75 by UV spectrophotometric titration, the NMR signals from the 3,5 and 2,6 ring protons of Tyr-75 were shifted 0.3 and 0.2 ppm upfield, respectively, relative to their positions in small peptides, indicating significant shielding and/or hydrogen bonding. The Tyr-75 ring proton signals narrowed slightly, with no discernible change in chemical shift, on conversion from dimer to monomer in the unliganded state. Ring protons of Tyr-49, distant from the monomer-monomer interface, but adjacent to the peptide-binding site, were markedly perturbed by dimerization, in accord with their behavior in bovine neurophysins. The results suggest that the secondary and tertiary structure of the region 75-78 is largely unchanged by dimerization, and argue against an important role for this region in dimerization-mediated conformational changes that alter the binding site in the unliganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Modulation of allosteric interactions in neurophysin induced by succinylation of serine-56 or cleavage of residues 1-8.

Neurophysin is an allosteric protein in which peptide binding and self-association are positively linked. Reaction of neurophysin with succinic anhydride led to a large decrease in peptide affinity assignable to succinylation of a serine or threonine hydroxyl group. To identify the residue involved, acetimidated protein was reacted with [14C]succinic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp-N mapping of the two components, followed by automated Edman degradation, allowed identification of the critical residue as Ser-56. This residue is not a direct participant in peptide binding and is distant from the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial diabetes insipidus. Examination in solution of the peptide affinity of neurophysin succinylated at Ser-56 indicated a binding affinity approximately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinity on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of residues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affinity of the des-1-8 protein were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Site↗
PubMed1992

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)

Amino Acid Sequence↗
PubMed1992

Identification of the unstable neurophysin disulfide and localization to the hormone-binding site. Relationship to folding-unfolding pathways.

For unliganded neurophysin, the effects of reduction of a single disulfide and limited regeneration of activity following reduction have suggested metastable disulfide pairing relative to that of the neurophysin precursor. This metastability was confirmed in the present study by the demonstration of almost complete regeneration of activity from the reduced state in the presence of ligand peptides, conditions mimicking precursor folding. To assign the source of the metastability of the unliganded mature protein, the disulfide(s) most susceptible to reduction and the last to be reoxidized following complete reduction were identified. Partial reduction of the first disulfide followed by trapping of the generated thiols with [14C]iodoacetate gave a distribution of label consistent with identification of the unstable disulfide as the 10-54 bridge and rapid interchange of the Cys-10 thiol with other disulfides in the amino-terminal disulfide domain. The same thiol distribution was seen at the terminal stage of reoxidation following complete reduction, providing evidence that unfolding and folding pathways are the same at this stage. The results indicate that, in the absence of bound peptide, the state with correct pairing of the 10-54 bridge has no significant thermodynamic advantage over interchanged states of the amino-terminal domain. However, since the 10-54 bridge is located at the peptide-binding site, the correct pairing is directly stabilized by ligand peptides. Moreover, since the other three bridges of the amino domain are homologous to bridges in the carboxyl-terminal domain that do not appear to be unstable, the results allow the possibility that the 10-54 bridge, which is unique to the amino domain, destabilizes other disulfides in that domain.

Amino Acid Sequence↗
PubMed1992

Binding and spectroscopic properties of ostrich neurophysins. Probing the role of residue 35 at the monomer-monomer interface.

Binding and spectroscopic properties of ostrich neurophysins were examined with emphasis on the behavior of Tyr-35, a residue that provides a potential probe of the monomer-monomer interface and of allosteric interrelationships between this region and the binding site. Mesotocin-associated ostrich neurophysin was found to bind oxytocin and related peptides with affinities comparable to the mammalian proteins, but induced a significantly different optical activity in bound peptides than the mammalian proteins. Gel-filtration studies indicated higher dimerization constants for the ostrich neurophysins than for the bovine neurophysins. Consistent with this, Tyr-35 was found to be largely buried, as monitored by tyrosine titration and lack of reactivity towards tetranitromethane under non-denaturing conditions. Reaction of Tyr-35 of the mesotocin-associated protein with tetranitromethane under denaturing conditions, followed by refolding, allowed isolation of an active product with an altered interface region as partially evidenced by its titration properties and consistent with its markedly altered CD spectrum. Comparison of the CD spectra of the modified and native proteins and analysis of pH effects indicated the contribution of Tyr-35 to an unusual 237 nm band in the mesotocin-associated protein. Small shifts in the 350 nm CD band of nitrated Tyr-35 on binding peptide and apparent effects of nitration on the induced optical activity in bound peptide provided evidence of at least weak structural communication between Tyr-35 and the binding site. However, no significant effect of nitration on binding affinity was observed, suggesting that, in the mesotocin-associated protein, the region around residue 35 is not a stringent modulator of the thermodynamic behavior of the binding site.

Amino Acid Sequence↗