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F R Gurd

Publications and source records attributed to F R Gurd.

At least 19 recordsLinked to original sources
PubMed1988

Site-specific semisynthetic variant of human hemoglobin.

A single round of Edman degradation was employed to remove the NH2-terminal valine from isolated alpha chains of human hemoglobin. Reconstitution of normal beta chains with truncated or substituted alpha chains was used to form truncated (des-Val1-alpha 1) and substituted ([[1-13C]Gly1]alpha 1) tetrameric hemoglobin analogs. Structural homology of the analogs with untreated native hemoglobin was established by using several spectroscopic and physical methods. Functional studies indicate that the reconstituted tetrameric protein containing des-Val1-alpha chains has a higher affinity for oxygen, is less influenced by chloride ions or 2,3-bisphosphoglycerate, and shows lower cooperativity than native hemoglobin. These results confirm the key functional role of the alpha-chain NH2 terminus in mediating cooperative oxygen binding across the dimer interface. The NH2-terminal pK1/2 value was determined for the [13C]glycine-substituted analog to be 7.46 +/- 0.09 at 15 degrees C in the carbon monoxide-liganded form. This value, measured directly by 13C NMR, agrees with the determination made by the less-direct 13CO2 method and confirms the role of this residue as a contributor to the alkaline Bohr effect; however, it is inconsistent with the presence of an NH2-terminal salt bridge to the carboxylate of Arg-141 of the alpha chain in the liganded form.

Hemoglobins↗
PubMed1987

Semisynthetic D-His1,N epsilon-acetimidoglucagon: structure-function relationships.

The histidine residue at the amino terminus of lysine-12 protected glucagon was replaced by its D-isomer by an established semisynthetic strategy to extend a stepwise series of replacements at this position. The product was examined for its secondary structure and its function. Circular dichroism spectra obtained at concentrations from 0.25 to 1.09 mg/ml at pH 10.2 in 0.2 M phosphate buffer were similar to those obtained with native hormone. Competitive binding assays and adenylate cyclase activation assays with partially purified rat liver plasma membranes show this D-His1 analog of glucagon to be a full agonist, causing the same maximum activation of adenylate cyclase as native hormone; but both binding and activation assays show the binding affinity to be diminished about 10-fold. The data suggest that the adjustment of the bonding of the imidazole group to the receptor to bring about transduction results in constraints on the conformation along the peptide sequence which interfere with the peptide adopting the same binding conformation achieved by the native hormone.

Adenylyl Cyclases↗
PubMed1985

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions.

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.

Amino Acids↗
PubMed1985

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with 13C and determination and interpretation of terminal pK values.

The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.

Amino Acid Sequence↗
PubMed1985

Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

Phospholamban, the putative protein regulator of the Ca2+ pump of cardiac sarcoplasmic reticulum, was purified to apparent homogeneity from canine cardiac sarcoplasmic reticulum vesicles by selective extraction with sodium cholate, followed by adsorption to calcium oxalate, solubilization in Zwittergent 3-14, and specific elution from p-hydroxymercuribenzoate-agarose. Phospholamban, isolated in the dephosphorylated state, was purified 80-fold in 15% yield (approximately 2 mg of phospholamban/g of sarcoplasmic reticulum protein). Nondissociated phospholamban exhibited an apparent Mr = 25,000 in sodium dodecyl sulfate-polyacrylamide gels. Partially dissociated phospholamban, induced by boiling in sodium dodecyl sulfate, exhibited five distinct mobility forms in sodium dodecyl sulfate-polyacrylamide gels, of apparent molecular weights between 5,000-6,000 and 25,000. Phospholamban was phosphorylated to a level of 190 nmol of Pi/mg of protein by cAMP-dependent protein kinase, consistent by minimum stoichiometry with a subunit molecular weight of approximately 5,000. Phospholamban prepared by the present method was different in several respects from the proteins that have been isolated in other laboratories. Pure phospholamban was cysteine rich, containing 6 residues/100 amino acid residues. Dephosphorylated phospholamban was strongly basic with a pI = 10; phosphorylation decreased the pI to approximately 6.7. Pure phospholamban (and phospholamban present in sarcoplasmic reticulum vesicles) was not readily extracted into acidified chloroform/methanol, suggesting that the protein does not behave as an acidic proteolipid. The purified protein was highly antigenic. Phospholamban was localized by immunochemical methods to cardiac membranes enriched in sarcoplasmic reticulum, but was absent from sarcoplasmic reticulum membranes prepared from fast skeletal muscle. The method described for isolation of cardiac phospholamban is highly reproducible and relatively simple, and should be useful for further detailed studies designed to probe the molecular structure of the protein.

Adenosine Triphosphatases↗
PubMed1985

pH-dependent processes in proteins.

Recent improvements in the understanding of electrostatic interactions in proteins serve as a focus for the general topic of pH-dependent processes in proteins. The general importance of pH-dependent processes is first set out in terms of hydrogen ion equilibria, stability, ligand interactions, assembly, dynamics, and events in related molecular systems. The development of various theoretical treatments includes various formalisms in addition to the solvent interface model developed by Shire et al. as an extension of the Tanford-Kirkwood treatment. A number of detailed applications of the model are presented and future potentialities are sketched.

Animals↗
PubMed1984

A major anti-myoglobin idiotype. Influence of H-2-linked Ir genes on idiotype expression.

A rabbit antiidiotypic antiserum raised against an A.SW IgG1K monoclonal anti-sperm whale myoglobin (Mb) antibody, HAL19, and extensively absorbed with normal mouse immunoglobulin and MOPC 21 (IgG1K), was found to detect a common or major anti-Mb idiotype expressed by some but not all anti-Mb monoclonal antibodies, regardless of immunoglobulin G (IgG) subclass, and by 40-50% of the anti-Mb antibodies in immune serum from five high responder strains of mice representing five different Igh allotypes. It did not inhibit antibodies to three unrelated protein antigens. The fraction of antibodies expressing this idiotype, denoted IdHAL19, was regulated by H-2-linked genes that correlated exactly in four independent haplotypes and an F1 with the known Mb immune response (Ir) genes and may be identical to these. Whereas less than 50% of antibodies from high responder mice were inhibitable by anti-IdHAL19, greater than 80% of antibodies from low responder mice, tested at comparable final antibody concentration, were inhibitable. This result was true for both low responder haplotypes, H-2b (B10) and H-2k (B10.BR). The idiotype was found to be present on antibodies that bound to native Mb but not fragments 1-55 or 132-153 of Mb or a denatured form, S-methyl Mb. This specificity for native Mb paralleled that of the monoclonal idiotype HAL19 itself. Therefore, the production of antibodies specific for native in contrast to denatured Mb was studied in H-2-congenic high and low responder strains. Strikingly, low responders produced antibodies that reacted almost exclusively with the native conformation, whereas a larger proportion of antibodies from high responder mice also reacted with the denatured form, S-methyl Mb. Bypassing of the Ir gene defect by immunization with Mb attached to a carrier, F gamma G, resulted in low responder antisera resembling higher responder sera in both idiotype expression and conformational specificity. The simplest explanation of these results is that H-2-linked Ir genes control antibody fine specificity, which is reflected in the idiotypes of the variable regions expressed. We suggest that low responder mice produce a more limited repertoire of antibodies consisting primarily of IdHAL19-positive antibodies specific for the native conformation of Mb. High responder mice produce a greater diversity of antibodies to Mb, so that the IdHAL19-positive, conformation-specific population represents a smaller proportion of the total. Similarly, the use of carrier-specific helper T cells in low responder mice results in a greater diversity of antibodies, which dilutes out the IdHAL19 subset.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Antigen conformation determines processing requirements for T-cell activation.

We studied the difference in requirements for processing and presentation to a single T-cell clone of four different forms of the same epitope of sperm whale myoglobin--namely, on the native protein, on two conformationally altered forms of the protein, or as a 22-residue antigenic peptide fragment. The T-cell clone was I-Ed-restricted and specific for an epitope on the CNBr fragment 132-153 involving Lys-140. As inhibitors of macrophage processing of antigen, we used several agents that inhibit lysosomal function: the weak bases chloroquine and NH4Cl, the cationic ionophore monensin, and the competitive protease inhibitor leupeptin. When these agents were used to inhibit processing of antigen by presenting cells and then washed out before T cells were added to culture, they inhibited the presentation of native antigen but not of fragment 132-153. To our surprise, the intact but denatured form, S-methylmyoglobin, behaved like the fragment not like the native protein. Apomyoglobin was intermediate in susceptibility to inhibition. Thus, native myoglobin requires a processing step that appears to involve lysosomal proteolysis, which is not required by fragment 132-153 or the denatured unfolded forms. For an antigen the size of myoglobin (Mr 17,800), it appears that unfolding of the native conformation, rather than further reduction in size, is the critical parameter determining the need for processing. Since a major difference between native myoglobin and the other forms is the greater accessibility in the latter of sites, such as hydrophobic residues, buried in the native protein, we propose that processing may be necessary to expose these sites, perhaps for interaction with the cell membrane or the Ia of the antigen-presenting cell.

Ammonium Chloride↗
PubMed1984

Identification of distinct predominant epitopes recognized by myoglobin-specific T cells under the control of different Ir genes and characterization of representative T cell clones.

We characterized the fine specificity of antigen recognition of myoglobin-immune T cells from B10.D2, B10.GD, and B10.A(5R) mice. Polyclonal H-2d T cells show a predominant response to an epitope centering on Glu 109 and His 116. These residues localize a dominant epitope to one segment of the myoglobin surface, but probably are not the only amino acid residues involved. This response pattern maps genetically to I-Ad (or Kd) based on the B10.GD recombinant strain. A different epitope specificity was detected in B10.A(5R) T cells mapping to I-E beta b E alpha k or I-Cd, but no difference was detected between strains differing at the Igh locus. Thus, epitope specificity varied with Ia haplotype but not Igh allotype. Myoglobin-specific B10.D2 T cell lines were established, and five clones specific for the Glu 109, His 116 epitope were isolated; these were all restricted to I-Ad antigen-presenting cells. These clones represent the dominant specificity in the polyclonal T cell response to myoglobin and will be useful in characterizing the structure and function of T cell receptors for antigen and Ia. The differences in number and nature of T cell epitopes compared to B cell epitopes of myoglobin are discussed.

Animals↗
PubMed1983

Contributions of individual amino acid residues to the structural stability of cetacean myoglobins.

The acid-denaturation behavior of eleven cetacean myoglobins has been studied at two ionic strengths, 0.01 and 0.10 M, at 25.0 degrees C. The myoglobulins studied fall into four phylogenetic suborders, representing the sperm whales, dolphins, baleen whales, and beaked whales. The differences in response to acid denaturation among these closely related myoglobins are small but statistically significant. In three cases, free-energy differences between myoglobins can be ascribed to one amino acid difference and in three others to two differences. The differences in response were analyzed in terms of the changes in noncovalent interactions occurring in the native structure. The effects of changes in electrostatic interactions over the whole charge array were calculated for each myoglobin species by using the modified Tanford-Kirkwood theory. The predicted changes in stability correlated well with the experimental observations in most cases. When differences in hydrogen-bonding capability were considered at a first approximation, substantial effects were predicted. When these effects were taken in conjunction with the electrostatic interactions, the correlation with experiment was improved. Additionally, restrictions in motional freedom and packing constraints appeared to be significant in the single-site analysis. The detectable differences in stability due to single amino acid substitutions along with the small differences in stability between the cetacean suborders indicate that compensatory interactions provide the mechanism for the conservation of stability among the myoglobins studied.

Amino Acid Sequence↗
PubMed1982

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor.

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.

Animals↗
PubMed1982

Stepwise sequence determination from the carboxyl terminus of peptides.

The thiocyanate method for stepwise degradation of peptides from their COOH termini [Stark, G. R. (1968) Biochemistry 7, 1796] has been investigated. The method involves first the reaction of the COOH-terminal residue with thiocyanate in an activation solvent of acetic acid and acetic anhydride and then cleavage of the COOH-terminal residue as its 2-thiohydantoin by acetohydroxamate in aqueous solution. The two steps of the degradation have been studied by using model peptides, and conditions have been developed for the rapid efficient removal and identification of the COOH-terminal residue of short peptides. The methods have been applied to peptides that have been covalently attached to insoluble supports. In this solid phase version of the degradation, a highly substituted porous glass activated with N,N'-carbonyldiimidazole has been prepared for use as the insoluble support. A number of peptides have been coupled to the porous glass, and several rounds of the degradation have been performed on immobilized peptides. High-pressure liquid chromatography provides a rapid, sensitive identification method for the 2-thiohydantoins. In addition, gas-liquid chromatography of the amino acid 2-thiohydantoins and reconversion to the parent amino acid have been used to identify the cleaved residues. The method of sequential degradation has been applied to a number of short model peptides such as Gly-Leu-Tyr, Met-enkephalin, and Val-Leu-Ser-Glu-Gly and has been used to determine the COOH-terminal sequence of 4 residues of a 22-residue cyanogen bromide fragment of pygmy sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1982

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin.

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.

Amino Acid Sequence↗
PubMed1982

A possible immunodominant epitope recognized by murine T lymphocytes immune to different myoglobins.

We find that a single region on the surface of different species of myoglobin appears to be immunodominant for T lymphocytes, even though the residues in that region vary sufficiently that the T cells immune to one myoglobin do not crossreact with other myoglobins bearing substitutions at that site. Immunization of B10.S mice with sperm whale myoglobin elicits T-lymphocyte populations capable of recognizing sperm whale myoglobin but not horse myoglobin, whereas the converse is true when these mice are immunized with horse myoglobin. Using a series of myoglobin variants, we tested the effect of changes in primary sequence on the T-lymphocyte proliferative response. We were able to divide the myoglobin variants into two groups, depending on whether they cross stimulate sperm whale immune or horse immune T lymphocytes. The patterns of cross stimulation of both populations of myoglobin immune T lymphocytes were explained by amino acid substitutions at position 109. However, because sperm whale and horse myoglobin differ at this residue (glutamate vs. aspartate, respectively), T lymphocytes immune to each myoglobin do not crossreact with the other myoglobin. Additional data suggest that this immunodominant epitope also includes other residues nearby on the surface of the native molecule. Mixing experiments showed that the specificity was that of T lymphocytes and not antigen-presenting cells. Monoclonal anti-I-A blocking studies showed that both myoglobins are presented in association with the same Ia antigen. Possible explanations for the apparent immunodominance of this antigenic epitope, consisting of residue 109 and nearby residues on the surface of both myoglobins, include a peculiar immunogenicity of the surface topography of this site of a preferred orientation of the molecule imposed by antigen-presenting cells when T cells first encounter the antigen. The latter explanation is related to but distinct from "determinant selection." T-cell recognition of conformation is discussed.

Amino Acid Sequence↗
PubMed1982

Semisynthesis of sperm whale myoglobin by fragment condensation.

Reconstruction of the sperm whale myoglobin structure was accomplished by a series of aqueous condensations of suitably protected synthetic myoglobin fragments to a large fragment prepared from the native protein. Reaction of N alpha,N epsilon 19-acetimidomyoglobin with 3-bromo-2-(2-nitrophenylsulfenyl)skatole (BNPS-skatole) yielded the fragment corresponding to residues 15-153. The covalent structure was reformed by sequential coupling of the N-hydroxysuccinimide esters of o-nitrophenylsulfenyl-L-tryptophan (residue 14) and selectively protected peptides corresponding to residues 1-5 and 6-13, which were synthesized by the solid-phase method and removed from the resin by methoxide-catalyzed methanolysis. A mixed aqueous solvent system containing methanol and N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine/trifluoroacetic acid buffer (Quadrol) solubilized the peptide and protein fragments during the condensations. Replacement of the heme moiety and immunoaffinity chromatography made possible the isolation and purification of the reconstructed native molecule. The development of this nondestructive synthetic procedure allows investigation of the structural and functional significance of individual residues by isotopic enrichment or selective amino acid substitutions.

Animals↗