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F R Gurd

Publications and source records attributed to F R Gurd.

At least 19 recordsLinked to original sources
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↗
PubMed1979

Electrostatic stabilization in myoglobin. Interactive free energies between individual sites.

The pattern of electrostatic interactions between pairs of charge sites in sperm whale ferrimyoglobin was examined as a function of pH in terms of proton site occupancy, static solvent accessibility, and distance of separation. By grouping all examples of the most stabilizing interactions and all examples of the most destabilizing interactions, we can easily show that at pH 7.50 the former is much stronger; that is, the negative contributions to electrostatic free energy far outweigh the positive contributions. Much of the electrostatic energy of stabilization in native myoglobin is provided by specific charge-pair partners that are very highly conserved among 53 mammalian myoglobin species and is invariant substantially from pH 8.5 to 3.5. Destablizing interactions that become most significant, but not actually dominant, near the acid unfolding pH range can be recognized in emerging clusters of uncompensated positive charges. Binding of azide ion by the heme iron effectively reduces the most prominent destabilizing set of such interactions. In general, thoe charged residues that experience the largest summed stabilizing interactions with other groups are the most conserved between species. The histidine residues, however, show their best correlation of conservation with low values of static accessibility. Although histidine residue 64 has an effective pK corresponding to the midpoint of the unfolding transition near pH 4.2 at an ionic strength of 0.10 M and so might be called a "trigger group", its interactions contribute only a modest fraction of the overall pH-dependent free energy change. An examination of the primary stabilizing interactions represented by the charge-pair partners indicates a probably major role of electrostatic interactions in the nucleation and docking stages of the condensation of the polypeptide chain into the compact native structure.

Amino Acids↗
PubMed1979

Electrostatic effects in hemoglobin: hydrogen ion equilibria in human deoxy- and oxyhemoglobin A.

The modified Tanford-Kirkwood theory of Shire et al. [Shire, S. J., Hanania, G.I.H., & Gurd, F.R.N. (1974) Biochemistry 13, 2967] for electrostatic interactions was applied to the hydrogen ion equilibria of human deoxyhemoglobin and oxyhemoglobin. Atomic coordinates for oxyhemoglobin were generated by the application of the appropriate rigid rotation function to alpha and beta chains of the deoxyhemoglobin structure [Fermi, G. (1975) J. Mol. Biol. 97, 237]. The model employs two sets of parameters derived from the crystalline protein structures, the atomic coordinates of charged amino acid residues and static solvent accessibility factors to reflect their individual degrees of exposure to solvent. Theoretical titration curves based on a consistent set of pKint values compared closely with experimental potentiometric curves. Theoretical pK values at half-titration for individual protein sites corresponded to available observed values for both quaternary states. The results bring out the cumulative effects of numerous electrostatic interactions in the tetrameric structures and the major effects of the quaternary transition that result from changes in static solvent accessibility of certain ionizable groups.

Amino Acids↗
PubMed1979

Electrostatic effects in hemoglobin: Bohr effect and ionic strength dependence of individual groups.

The electrostatic treatment applied in the preceding paper in this issue [Matthew, J. B., Hanania, G.I.H., & Gurd, F.R.N. (1979) Biochemistry (preceding paper in this issue)] to the titration behavior of individual groups in human deoxyhemoglobin and oxyhemoglobin was applied to the computation of the alkaline Bohr effect at various values of ionic strength. The enhanced proton binding of deoxyhemoglobin in the pH range of 6--9 was accounted for at ionic strength 0.01 M by the effects of the unique charge distributions of ionizable groups in the two quaternary states. At ionic strength 0.10 M the effects of 2--4 bound anions had to be considered in addition in the deoxyhemoglobin charge configuration. At the higher ionic strength 10 groups per tetramer contributed to the Bohr effect, whereas 28 groups were contributory at the lower ionic strength. The ionic strength dependence of individual groups in the two tetrameric structures as well as in the alpha-chain monomer was explained in terms of the electrostatic treatment. This examination showed that the differences in electrostatic behavior of deoxy- and oxyhemoglobin follow from particular dissymmetries in their configurations with respect to charge and static solvent accessibility.

Electrochemistry↗
PubMed1979

Complete amino acid sequence of the myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani.

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Amino Acid Sequence↗
PubMed1979

Complete amino acid sequence of the myoglobin from the Pacific sei whale, Balaenoptera borealis.

The complete amino acid sequence of the major component myoglobin from Pacific sei whale, Balaenoptera borealis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apomyoglobin, over 75% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by the sequence analysis of peptides that resulted from further digestion of the amino-terminal and central cyanogen bromide fragments. The amino-terminal fragment was specifically cleaved at its two tryptophanyl residues with N-chlorosuccinimide and the central cyanogen bromide fragment was cleaved at its glutamyl residues with staphylococcal protease and at its single tyrosyl residue with N-bromosuccinimide. The primary structure of this myoglobin proved identical with that from the gray whale but differs from that of the finback whale at four positions, from that of the minke whale at three positions and from the myoglobin of the humpback whale at one position. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Amino Acid Sequence↗
PubMed1979

Motions in proteins.

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Allosteric Regulation↗
PubMed1979

Aliphatic groups of sperm whale myoglobin: 13C NMR study.

The aliphatic region of the 13C NMR spectrum of sperm whale cyanoferrimyoglobin has been examined at 67.9 MHz. Fifty partially resolved or well-resolved resonances, representing at least half of the aliphatic carbons in the molecule, are observed in the spectral region from 9 to 29 ppm downfield of tetramethylsilane. Analyses of the spin lattice relaxation times (T1) and nuclear Overhauser enhancements for these resonances reveal considerable motion freedom of the aliphatic side chains. In the spectral region from 9 to 15 ppm, eight single carbon resonances are observed and tentatively assigned to Cdelta 1 of eight of the nine isoleucine residues. In at least five cases the reorientational motion of the isoleucine side chains could not be characterized solely by rotation of the Cdelta 1 methyl groups. The simplest model consistent with the data is a restricted diffusion model with two degrees of internal rotation [Wittenbort, R. J. & Szabo, A. (1978) J. Chem. Phys. 69, 1722--1736]. In light of the packing densities within the myoglobin molecule these results are taken to imply concerted motions of the buried aliphatic residues.

Animals↗
PubMed1979

Use of thiocyanic acid to form 2-thiohydantoins at the carboxyl terminus of proteins.

The chemistry of the formation of 2-thiohydantoins on the carboxyl terminal of peptides or proteins was investigated. It was found that thiocyanic acid was much more reactive for the formation of 2-thiohydantoins than were the thiocyanate salts. The physical reasons for this observation are explained. The kinetics of the reaction of a number of proteins, and some of their fragments, with thiocyanic acid were also determined. Simple and safe procedures for the preparation of anhydrous thiocyanic acid solutions were devised. The prospective application of these procedures to sequencing from the carboxyl terminal of a polypeptide is discussed.

Amines↗
PubMed1978

Complete amino acid sequence of the major component myoglobin from the humpback whale, Megaptera novaeangliae.

The complete primary structure of the major component myoglobin from the humpback whale, Megaptera novaeangliae, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the acetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determination of the remainder of the covalent structure. This myoglobin differs from that of sperm whale, Physeter catodon, at 12 positions, and dwarf sperm whale, Kogia simus, at 14 positions, finback whale Balaenoptera physalus at 3 positions, minke whale, Balaenoptera acutorostrata at 2 positions, and California gray whal Eschrichtius gibbosus, at 1 position. All of the substitutions observed in this sequence fit readily into the three-dimensional structure of sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1978

Characterization of the reaction of methyl acetimidate with sperm whale myoglobin.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.

Amino Acid Sequence↗
PubMed1978

Complete amino acid sequence of the major component myoglobin of finback whale (Balaenoptera physalus).

The complete amino acid sequence of the major component myoglobin from finback whale, Balaenoptera physalus, was determined by the automated Edman degradation of several large peptides obtained by specific cleavages of the protein. Three easily separable peptides were obtained by cleaving with cyanogen bromide at the two methionine residues and one large peptide was isolated after cleavage with (2-p-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. More than 60% of the covalent structure was established by the sequential degradation of three of these peptides and the apomyoglobin. An additional 30% of the primary sequence was established with peptides obtained from tryptic digestion of both the apomyoglobin and the acetimidoapomyoglobin, and the final 10% of the sequence was completed after digestion of the two larger cyanogen bromide peptides with S. aureus strain V8 protease. This myoglobin differs from that of the sperm whale, Physeter catodon, at 15 positions, from that of the arctic minke whale, Balaenoptera acutorostrata, at 3 positions, and from that of the California gray whale, Eschrichtius gibbosus, at 4 positions. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.

Amino Acid Sequence↗
PubMed1978

Complete amino acid sequence of myoglobin from the pilot whale, Globicephala melaena.

The complete amino acid sequence of the major component myoglobin from the pilot whale, Globicephala melaena, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The apomyoglobin was selectively cleaved at the two methionyl residues with cyanogen bromide and the acetimidated apomyoglobin was cleaved at the three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apoprotein, over 90% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by sequence analysis of three of the tryptic peptides isolated from the central cyanogen bromide fragment after modification of its single arginyl residue with 1,2-cyclohexanedione. This myoglobin differs from that of the Black Sea dolphin at four positions and from the myoglobin of the killer whale, Pacific common dolphin, and Atlantic bottlenosed dolphin at two positions. The above differences reflect the close taxonomic relationship of these five species of Cetacea. This sequence determination was aided by the use of a Texas Instruments 980A minicomputer system which performed peak integrations for all samples subjected to amino acid analysis.

Amino Acid Sequence↗