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H J Dyson

Publications and source records attributed to H J Dyson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Complete 13C assignments for recombinant Cu(I) rusticyanin. Prediction of secondary structure from patterns of chemical shifts.

Complete resonance assignments for the 13C spectrum of reduced (Cu(I)) rusticyanin have been made using 13C, 15N doubly labeled recombinant material. The reported assignments include those for the carboxyl and carbonyl carbon atoms and protonated aromatic ring carbons, and were obtained using a variety of 2- and 3D inverse-detected NMR experiments, including 13C, 15N, 1H triple resonance experiments and HCCH-COSY and -TOCSY. Backbone carbonyl assignments were obtained using 3D HNCO and HCACO spectra, and modified versions of 2D H(CA)CO and HMBC spectra were used to obtain side-chain carboxyl carbon and methionine epsilon-methyl carbon assignments, respectively. A comparison of the 13C alpha, 13C beta and 13CO chemical shifts with published 'random coil' values confirms the conclusion reached from a consideration of the 3JHN alpha coupling constants and the pattern of sequential NOEs, that the protein consists largely of beta-structure.

Amino Acid Sequence↗
PubMed1995

Detection of a catalytic antibody species acylated at the active site by electrospray mass spectrometry.

The chemical interactions between a catalytic antibody Fv fragment and ester substrates were examined using pneumatically assisted electrospray (ion spray) mass spectrometry. Upon addition of the p-nitrophenyl ester substrate to the antibody fragment, an antibody fragment species that represents approximately 8% of the total Fv concentration is clearly observed in the electrospray spectrum. The observed increase in molecular weight of the Fv fragment corresponds to the mass of the acyl group of the substrate. Formation of the acyl-Fv species is blocked by preincubation of the antibody fragment with hapten inhibitor, suggesting that the acyl linkage involves a residue in the active site of the antibody. The acyl-Fv species is not observed when the corresponding p-chlorophenyl ester substrate is used, indicating that the level of this species is dependent on the leaving group of the substrate. The acylated species is not observed for a site-directed mutant lacking catalytic activity, His L91 Gln. The present results are consistent with modeling studies of the structure of the Fv fragment and provide strong confirmatory evidence for the multistep kinetic mechanism previously proposed for this antibody.

Acylation↗
PubMed1995

Comparison of the hydrogen-exchange behavior of reduced and oxidized Escherichia coli thioredoxin.

Hydrogen-deuterium exchange rates for the amide protons in oxidized (disulfide) and reduced (dithiol) thioredoxin have been measured using a series of 15N-1H HSQC spectra at various times after buffer exchange into 99% 2H2O. Information on exchange rates and protection factors was obtained for both forms of thioredoxin for 68 amide protons using this method; in general, the rates obtained by this method were for amide protons of residues in the hydrogen-bonded beta-sheet and alpha-helix secondary structure of thioredoxin. Estimates of the exchange rate for those amide protons that exchanged with rates too fast to measure by hydrogen--deuterium exchange were made by saturation-transfer measurements, which were particularly useful in defining the hydrogen exchange behavior of the active site Cys-Gly-Pro-Cys sequence and of the loops adjacent to it (residues 73-75 and 91-98). Amide proton exchange rates provide a qualitative estimate of the backbone mobility, and the differences in hydrogen exchange behavior between the two forms of thioredoxin are consistent with those observed in calculations of polypeptide chain dynamics obtained from 15N relaxation measurements [Stone, M. J., et al. (1993) Biochemistry 32, 426-435]. For most of the protein, the exchange rates are close to identical in the two forms, consistent with their very close similarity in structure and backbone dynamics. Significant differences in behavior are observed in the active site sequence and in the regions of the protein that are close to this sequence in the three-dimensional structure, including portions of the beta-strand and alpha-helical sequences immediately adjacent to the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

1H, 13C and 15N chemical shift referencing in biomolecular NMR.

A considerable degree of variability exists in the way that 1H, 13C and 15N chemical shifts are reported and referenced for biomolecules. In this article we explore some of the reasons for this situation and propose guidelines for future chemical shift referencing and for conversion from many common 1H, 13C and 15N chemical shift standards, now used in biomolecular NMR, to those proposed here.

Carbon Isotopes↗
PubMed1995

'Random coil' 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG.

Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D 1H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain 'random coil' amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these 'random coil' peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K., (1979) Biopolymers, 18, 285-297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH2 [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67-81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.

Amides↗
PubMed1995

Antigenic peptides.

Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.

Animals↗
PubMed1994

Nuclear magnetic resonance 15N and 1H resonance assignments and global fold of rusticyanin. Insights into the ligation and acid stability of the blue copper site.

Nuclear magnetic resonance assignments are reported at pH approximately 3 for a type 1 ("blue") copper protein, rusticyanin, obtained from the acidophilic organism Thiobacillus ferrooxidans. A combination of homonuclear proton and heteronuclear 15N-edited NMR spectra has been used to assign most of the 1H and 15N resonances of reduced rusticyanin. The copper-binding site is shown by analogy with other blue copper proteins to contain the side-chains of Cys138, His143 and Met148 at the C-terminal end of the sequence and a fourth ligand that is most likely a histidine, His85, consistent with the constitution of other type 1 copper sites. The global fold of the molecule is a compact beta-barrel or beta-sandwich, which contains a high proportion of beta-sheet secondary structure and a hydrophobic core particularly rich in aromatic residues. The copper-binding active site is surrounded by aromatic residues, and many of the resonances of the residues flanking the active site are shifted to unusual values, consistent with the effects of ring currents. The protected nature of the copper site is demonstrated by the large number of amide protons that are persistent in this region in 99% 2H2O solution at pH 3.4. We suggest that the unusual acid stability, both of the protein itself and of the blue copper active site, is a direct result of the protected and highly hydrophobic nature of the active site sequence and contacting loops and the high proportion of secondary structure in the protein.

Amino Acid Sequence↗
PubMed1994

Stabilization of a type VI turn in a family of linear peptides in water solution.

The sources of the stability of a type VI turn formed with high population in the cis isomeric form of an unblocked six residue peptide, Ser1-Tyr2-Pro3-Tyr4-Asp5-Val6 (SYPYDV), were investigated by making extensive amino acid substitutions at residues 2, 4 and 5. Several NMR parameters indicate the presence of the turn, including significant upfield shifts of the proton resonances of the cis proline, a small 3JHN alpha coupling constant for residue 2, a cross-turn d alpha N(i,i+2) from residue 2 to residue 4 and in increased mole fraction of the cis form in the conformational ensemble. By these criteria, a number of peptides were found to contain significant populations of type VI turn conformers in the cis form of the peptide. The NMR parameters are highly dependent on the sequence of the peptide, and are strongly correlated with each other and with the population of type VI turn. The greatest populations of turn conformations were observed for peptides of the general form AA-Ar-Pro-Ar-Hp, where AA represents any amino acid, Ar an aromatic residue and Hp a small hydrophilic residue. There is no evidence in the form of lowered amide proton temperature coefficients for direct hydrogen bonding as a primary source of turn stability. Instead, the major stabilizing factor, indicated by the strong dependence of the turn population on the presence of aromatic (not hydrophobic) residues at positions 2 and 4, is the stacking of the aromatic and proline rings. A measurable preference for deprotonated aspartate at position 5, which is not part of the turn itself, and the destabilization of the turn at high and low pH, indicate that electrostatic interactions between the unblocked N terminus and the aspartate carboxyl group also act to stabilize the turn conformation when the Ar-Pro-Ar sequence is present. Implications for stabilization of local elements of secondary structure during the earliest events in protein folding are discussed.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure of a type VI turn in a linear peptide in water solution. Evidence for stacking of aromatic rings as a major stabilizing factor.

Structures have been calculated for the folded conformation found at high population in the cis isomeric form of the peptide NH3(+)-Ser-Tyr-Pro-Phe-Asp-Val-COO- (SYPFDV) in aqueous solution, using distance geometry and restrained molecular dynamics. A number of NMR parameters, including NOE distance restraints and phi and chi 1 dihedral angle restraints derived from coupling constants were used in the calculation. The restraints were carefully selected to exclude those that might have a significant contribution from the unfolded states of the peptide, so that the calculated structure represents only the folded form (present at about 70% population) and contains no information on the other members of the conformational ensemble. The calculations give a tight family of structures in the region Tyr2 to Phe4, all containing a type VI turn with an unusual disposition of the aromatic rings of Tyr and Phe, which stack against the proline ring. Both backbone and side-chain conformations are well defined in this region. At the ends of the molecule the polypeptide chain is disordered. The structures are consistent with all of the available NMR information, including upfield chemical shifts observed for the proline ring protons in the cis isomer, and with the independently determined proline ring pucker. There is no evidence for cross-turn hydrogen bonding. According to the calculated structures, the major source of stabilization of the turn conformation appears to be the stacking of the aromatic and proline rings.

Amino Acid Sequence↗
PubMed1994

High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin.

BACKGROUND: Thioredoxin participates in thiol-disulfide exchange reactions and both oxidized thioredoxin (disulfide form) and reduced thioredoxin (dithiol form) are found under physiological conditions. Previous structural studies suggested that the two forms were extremely similar, although significant functional and spectroscopic differences exist. We therefore undertook high-resolution solution structural studies of the two forms of Escherichia coli thioredoxin in order to detect subtle conformational differences. RESULTS: The solution structures of reduced and oxidized thioredoxin are extremely similar. Backbone structure is largely identical in the two forms, with slight differences in the region of the active site, which includes Cys32 and Cys35. The side chain sulfur atom of Cys32 is tilted away from that of Cys35 in the reduced form of the protein to accommodate the increase in S-S distance that occurs upon reduction of the disulfide, but the chi 1 angles of the two cysteines remain the same in the two forms. CONCLUSIONS: Only subtle conformational changes occur upon changing the oxidation state of the active site cysteines, including the positions of some side chains and in hydrogen bonding patterns in the active site region. Functional differences between the two forms are probably therefore related to differences in local conformational flexibility in and near the active site loop.

Amino Acid Sequence↗
PubMed1994

Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein.

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.

Amino Acid Sequence↗
PubMed1994

Effect of disulfide bridge formation on the NMR spectrum of a protein: studies on oxidized and reduced Escherichia coli thioredoxin.

As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherichia coli thioredoxin (M(r) 11,700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic 13C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between 13C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C beta and C gamma shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys32-Gly-Pro-Cys35 sequence causes changes in the 1H, 15N and 13C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp28 and Trp31). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.

Amino Acid Sequence↗
PubMed1993

Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization.

The unfolding and refolding transitions of French bean apoplastocyanin (apo-Pc), a beta-sandwich protein, have been characterized. The apoprotein is stabilized by sodium sulfate and can be reversibly unfolded by guanidine hydrochloride (GuHCl). However, in contrast to holo-Pc, apo-Pc is unstable at low ionic strength, suggesting that the copper ion stabilizes the holoprotein. The equilibrium unfolding transition monitored by peptide circular dichroism (CD) and tyrosine fluorescence is described by a two-state model. The kinetics of the unfolding transition were monitored using a manual mixing technique and are consistent with a single two-state transition. In contrast, the kinetics of the refolding reaction measured by fluorescence and CD show two transitions with different rates. The relaxation time of the slower phase (800-1000 s) is almost independent of GuHCl concentration. The faster phase was observed only under strongly native conditions, and its relaxation time is GuHCl-dependent. Double-jump experiments and acceleration by cyclophilin demonstrate that both phases involve cis-trans isomerization of proline residues. The changes in fluorescence associated with the two phases are more than 150% of the total change expected from equilibrium experiments, indicating the presence of intermediate(s) with fluorescence greater than the unfolded state. Amide hydrogen-exchange experiments coupled with two-dimensional NMR spectroscopy demonstrate the formation of an intermediate in the very low refolding reaction in which amide protons in the beta-sheets are weakly protected from exchange. No CD evidence for nativelike beta-sheet formation was found for this intermediate. The NMR experiments suggest that the intermediate is compact with flexible beta-sheets and altered packing of the hydrophobic core. It has many of the characteristics of a molten globule. However, the 1H NMR spectrum of the intermediate exhibits a small number of shifted resonances that indicate the presence of specific tertiary interactions in a localized region. A mechanism for refolding of apoplastocyanin is proposed that includes two slow steps corresponding to trans-->cis isomerization of two prolines.

Amides↗
PubMed1993

Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the G- and H-helices of myoglobin.

Myoglobin has been extensively studied as a model system for protein folding in vitro. As part of an ongoing study of myoglobin folding, we have synthesized a series of peptide fragments corresponding to portions of the sequence of the sperm whale protein. The conformational preferences of these peptides have been investigated by circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution. In this paper we describe the folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence. The Mb-G peptide shows evidence of a very small population of helical conformations in aqueous solution, both by CD and NMR. By contrast, the monomeric Mb-H peptide is found by CD to adopt a significant population (ca. 30%) of ordered helix and by NMR to populate helical conformations in rapid dynamic equilibrium with unfolded states. The Mb-H peptide undergoes a well-characterized, concentration-dependent monomer-tetramer equilibrium. At peptide concentrations greater than 1 mM there is an increase in the population of helix, to approximately 85% according to the CD spectrum, through self-association to form a tetramer. Both medium-range NOE connectivities and a CD spectrum characteristic of ordered helix are observed at low peptide concentrations, establishing that helical conformations are present in the monomeric state of Mb-H. The relative helicity at various sites throughout the Mb-H peptide has been estimated using a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities. The population of ordered helix is seen to be highest in the center of the peptide sequence; the ends of the peptide show evidence of pronounced fraying.

Amino Acid Sequence↗
PubMed1993

Peptide models of protein folding initiation sites. 2. The G-H turn region of myoglobin acts as a helix stop signal.

A series of peptide fragments of sperm whale myoglobin, corresponding to segments of the region between the G- and H-helices of the protein, have been synthesized and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The smallest fragment, Mb-GH5, a five-residue peptide with the sequence HPGDF corresponding to the connecting loop between the two helices in the folded protein, adopts highly populated turn conformations in aqueous solution. A 25-residue peptide, Mb-GH25, containing the same sequence flanked by contiguous segments of the G- and H-helix sequences, was also found to contain a high proportion of conformers with a turn in this region. No helix formation was observed in the flanking sequences in water solution, either in Mb-GH25 or in control 10-residue peptides (Mb-G10 and Mb-H10) with sequences corresponding to the G- and H-helix segments. No additional helicity above that of the sum of the components was observed for Mb-GH25, indicating that a helical hairpin structure is not formed in the monomeric peptide in aqueous solution. In the presence of TFE, ordered helix is formed in Mb-GH25 according to the CD spectrum, and NMR spectra indicate that this is localized in the N-terminal portion of the peptide. NOESY spectra clearly show that the turn conformation is retained under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin.

As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized, and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The behavior of short fragments corresponding to the sequences of the G- and H-helices of myoglobin and to the turn region between these helices has been described in accompanying papers. At the next level of complexity, peptide model compounds have been synthesized to explore the longer-range interactions which may take place in protein folding after initial secondary structure formation has occurred. A series of disulfide-bridged dimeric peptides containing the complete sequences of the G- and H-helices of myoglobin were synthesized and their conformational preferences examined. CD spectra indicate that disulfide-bridged peptides consisting of two H-helix sequences (Mb-HssH) and of one G- and one H-helix (Mb-GssH) are highly helical in water solution, as a result of intermolecular association. A 51-residue peptide, Mb-GH51, encompassing the entire G-H helical hairpin of myoglobin, including the turn sequence between the two helices, has been successfully synthesized by standard methods. This peptide was designed to be monomeric in aqueous solution. Mb-GH51 does not appear from CD spectra to contain any additional helix in water solution above what would be expected from an equimolar mixture of the G- and H-helix peptides. NMR spectra indicate that the turn conformation observed in shorter peptide fragments is retained in Mb-GH51 in high population.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗