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At least 19 recordsLinked to original sources
PubMed1979

Amide hydrogen exchange rates of peptides in H2O solution by 1H nuclear magnetic resonance transfer of solvent saturation method. Conformations of oxytocin and lysine vasopressin in aqueous solution.

The NH exchange rates in aqueous media of oxytocin and 8-lysine vasopressin (LVP) have been measured by using transfer of solvent saturation method. The data are consistent with a "highly motile" dynamic equilibrium between folded and highly solvated conformations. The highly-motility limit applies to the exchange of NH hydrogens of oxytocin and LVP. Folded structures are more prevalent in oxytocin than in LVP. Partial shielding is indicated for peptide hydrogens of Asn5 and perhaps also Cys6 of oxytocin and for Cys6 of LVP. It is tentatively proposed that the folded conformation of oxytocin in aqueous media may contain a parallel beta-structure in the tocinamide ring consisting of two hydrogen bonds: one between the Tyr2 C = O and Asn5 peptide NH as originally proposed for the preferred conformation of oxytocin in dimethyl sulfoxide (D. W. Urry and R. Walter), and the second between he Cys1 C = O and the Cys6 NH. In LVP the hydrogen bond between the Tyr2 C = O and Asn5 peptide NH appears to be absent. The acylic tripeptide sequences (-Pro-X-Gly-NH2) of both hormones appear to be predominantly solvated. The second-order rate constants for acid catalyzed exchange of the primary amide hydrogens of Gln4, Asn5, and Gly9 of oxytocin are consistently greater for the trans NH than for the corresponding cis NH. This observation can be rationalized in terms of mechanisms involving protonation of either the amide oxygen, or the amide nitrogen, but with limited rotation about the C - N bond.

Lypressin↗
PubMed1978

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.

Amino Acid Sequence↗
PubMed1977

Total body water and the exchangeable hydrogen. I. Theoretical calculation of nonaqueous exchangeable hydrogen in man.

A theoretical calculation of the total nonaqueous exchangeable hydrogen in protein, carbohydrates, and fat in man has been made. It shows that of the total exchangeable hydrogen in the body 5.22% is located in biochemical components, soluble in body water, containing hydrogen that is exchangeable with the isotope. This value represents a maximum upward distortion of total body water measurements by isotope dilution, due to the maximum possible exchangeability in these molecular conformations. From comparative measurements reported in the literature it is clear that this maximum is not achieved during the short period of time during which tritium-dilution studies are performed. It is the authors' belief that the hard-to-exchange amide hydrogens described by Blout in the protein conformations account for this failure of the isotope to achieve complete exchange in the short time allowed.

Body Water↗
PubMed1976

Hydrogen exchange at the amide group of reduced pyridine nucleotides and the inhibition of that reaction by dehydrogenases.

Stopped flow ultraviolet spectroscopy has been used to measure the rate of hydrogen exchange with solvent at the amide group of reduced nicotinamide nucleotide coenzymes. Several mechanisms for the exchange reaction are considered in the light of the kinetic data. Complex formation between the coenzyme and any of four dehydrogenases markedly slows the rate of hydrogen exchange. Hydrogen bond formation and/or hydrophobic interactions within these complexes are thought to be the reasons for the decreased rate of exchange.

Alcohol Oxidoreductases↗
PubMed1978

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.

Amino Acid Sequence↗
PubMed2025

NumSimEX: A method using EXX hydrogen exchange mass spectrometry to map the energetics of protein folding landscapes.

Hydrogen exchange mass spectrometry (HXMS) is a powerful tool to understand protein folding pathways and energetics. However, HXMS experiments to date have used exchange conditions termed EX1 or EX2 which limit the information that can be gained compared to the more general EXX exchange regime. If EXX behavior could be understood and analyzed, a single HXMS timecourse on an intact protein could fully map its folding landscape without requiring denaturation. To address this challenge, we developed a numerical simulation method called NumSimEX that models EXX exchange for arbitrarily complex folding pathways. NumSimEx fits protein folding dynamics to experimental HXMS data by iteratively comparing the simulated and experimental timecourses, allowing for determination of both kinetic and thermodynamic protein folding parameters. After analytically verifying NumSimEX's accuracy, we demonstrated its power on HXMS data from beta-2 microglobulin (β2M), a protein involved in dialysis-related amyloidosis. In particular, using NumSimEX, we identified three-state kinetics that near-perfectly matched experimental observation. This proof-of-principle application of NumSimEX sets the stage for harnessing HXMS to expand our understanding of proteins currently excluded from traditional protein folding methods. NumSimEX is freely available at https://github.com/JaswalLab/NumSimEX_Public.

Protein Folding↗
PubMed1979

Structural characterization by nuclear magnetic resonance of a reactive-site 13carbon-labelled basic pancreatic trypsin inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed.

With the use of an enzymatic replacement method, 90%-enriched [1-13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive-site peptide bond Lys-15--Ala-16 was properly resynthesized, the polypeptide chain was cleaved at the peptide bond Arg-39--Ala-40 and Arg-39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi-state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the [1-13C]lys-15-labelled inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase-inhibitor interactions.

Alanine↗
PubMed1977

Hydrogen exchange study of membrane-bound rhodopsin. I. Protein structure.

Structural parameters of rhodopsin in disc membrane preparations from frog and cattle were studied by hydrogen exchange methods. The method measures the exchange of protein amide hydrogens with water and can distinguish protons which are internally bonded from those which are hydrogen-bonded to water. The results show that about 70% of rhodopsin's peptide group protons are exposed to water. The identification of these groups as free peptides was made initially on the usual basis of the identity of their exchange rate with the well characterized free peptide rate; other experiments specifically excluded contributions from lipids, protein side chains, adventitious mucopolysaccharides, and intradisc water. In contrast to rhodopsin, other proteins generally have only 20 to 40% free peptide groups. Apparently rhodopsin has some unusual structural feature. Our results together with available information on rhodopsin suggest that a considerable length of its polypeptide chain is arranged at the surface of a channel of water penetrating into the membrane. Physicochemical considerations indicate that such a channel would have to be quite wide, 10 to 12 A or more, to explain the hydrogen exchange results.

Animals↗
PubMed1977

Isolation and characterization of four peptide hydrolases from the cytosol of rat intestinal mucosa.

The high speed supernatant fluid prepared from rat intestinal mucosa was subjected to ion-exchange chromatography on diethlaminoethyl-cellulose eluted with a linear gradient of sodium chloride (0 to 0.27 M). Assay of eluted fractions for Phe-Gly hydrolase activity revealed four distinct peaks of enzyme activity. These cytosol enzymes have been designated I, II, III, and IV in order of their elution from the column. Examination of the substrate specificity of the four enzymes by use of 20 mM peptide concentrations indicated the most discriminating substrates for the four enzymes were Leu-Gly-Gly, His-Met, Ser-Phe, and leucine amide, respectively. The mean distribution of the recovered peptide hydrolase activities against these substrates among the four enzymes I, II, III, and IV was 96.1, 1.4, 1.7, and 0.8%, respectively, for Leu-Gly-Gly; 0.6, 96.4, 2.4, and 0.6% for His-Met; 0, 0, 95.8, and 4.2% for Ser-Phe; and 20.8, 19.8, 5.6, and 53.8% for leucine amide. Ion-exchange chromatography resulted in increases in specific activity of 19-, 19-, 46-, and 3.5-fold for enzymes I, II, III, and IV, respectively. The activity of all four enzymes, but especially III and IV, were stabilized by the presence of 150 muM dithioerythritol. Activity of each of the four enzymes was decreased 79 to 100% by 1mM ethylenediaminetetraacetate, HgCl2, 1, 10-phenanthroline, or 0.5 mM p-hydroxymercuribenzoate, except that the activity of enzyme I was decreased only 15% by ethylenediaminetetraacetate. No significant activation of the partially purified enzymes occurred in the presence of 500 muM Zn++, Co++, or Mg++. The four enzymes exhibited distinct pH profiles with optima at 7.5, 7.5, 8.5, and 8.0 for enzymes I, II, III, and IV, respectively. Molecular weights of the four enzymes determined by gel filtration on Sephadex G-200 were 58,500, 74,000, 97,500, and 113,000, respectively. All four enzymes lost more than 85% of their activity after 1 hr at temperatures of 50 degrees C or higher in sodium phosphate buffer, pH 7.0. The Km values determined with the most specific substrates for each enzyme were 0.76, 0.44, 3.82, and 8.3 mM for enzymes I, II, III, and IV, respectively. Recent evidence suggests that a significant amount of some small peptides are absorbed intact and hydrolyzed by cytosol peptide hydrolases. Adequate understanding of the function and control of these intracellular enzymes requires knowledge of the characteristics and substrates specificity of individual enzymes. The study described here demonstrates the presence of at least four cytosol peptide hydrolases with distinct substrate specificities. Substrates almost exclusively hydrolyzed by each of three of the enzymes, and therefore suitable for assay of each of these enzymes in the presence of the others, have been identified.

Animals↗
PubMed1975

Solvent accessibility in folded proteins. Studies of hydrogen exchange in trypsin.

In a native protein, the exchange of a peptide amide proton with solvent occurs by one of two pathways, either directly from the folded protein, or via unfolding, exchange taking place from the unfolded protein. From the thermal unfolding rate constants, the contribution of unfolding to the over-all kinetics as a function of solvent and temperature has been determined. Exchange involving unfolding of the protein is characterized by a high activation energy, in the range of 50 to 60 Cal per mol. The activiation energy (Eapp) of the rates of exchange directly from the folded protein is approximately 20 to 25 Cal per mol. Because for the proton transfer step, Eapp approximately equal to 20 Cal per mol, the activation energy for any contributing protein conformational process(es) is approximately equal to 0 to 5 Cal per mol. Most, if not all, of the peptide amide protons in a folded protein can exchange directly with solvent without the protein unfolding. The number of "slowly" exchanging protons at a given condition of pH and temperature is not related to a discrete structural unit, but rather to the distribution of observed rates within the broader distribution of actual rates. The large attenuation of hydrogen exchange rates in folded proteins, resulting in a distribution of first order rates over 6 orders of magnitude, is primarily due to the effects of restricted solvent accessibility of labile protons in the three-dimensional structure. Any protein conformational process, such as protein fluctuations, invoked to explain the solvent accessibility must be of low activation energy and attenuated by ethanol and other co-solvents (Woodward, C. K., Ellis, L. M., and Rosenberg, A. (1974) J. Biol. Chem. 250, 440-444).

Binding Sites↗
PubMed1977

The synthesis and chromatography of peptine nitriles.

Di- and tripeptide nitriles, glycylaminoacetonitrile (Gly-AAN), diglycylaminoacetonitrile (Gly-Gly-AAN), alany-alpha-aminopropionitrile (Ala-APN), and dialanyl-alpha-aminopropionitrile (Ala-Ala-APN) were synthesized first. These peptide nitriles and related peptides and peptide amides were analyzed by means of ion-exchange chromatography. The every two diastereomers of dialanine, dialanine amide, and Ala-APN were separated into two peaks by using a pH 3.25 buffer as an eluent. The four isomers of trialanine, trialanine amide, and Ala--Ala APN gave four, two, and one peak, respectively under the same conditions. The trimethylsilyl derivatives of alanyl peptides and related compounds were analyzed by means of gas chromatogrpahy combined with mass-spectrometry. The parent (M+ and/or M+-15) and other mass numbers observed in their mass-spectra supported the introduction of various numbers of trimethylsilyl groups.

Chromatography, Gas↗
PubMed1978

The conformation of membrane-bound and detergent-solubilised bovine rhodopsin. A comparative hydrogen-isotope exchange study.

The conformations of the intrinsic membrane protein, rhodopsin, in its membrane-bound and detergent-solubilised states have been compared by hydrogen isotope exchange measurements. The infrared peptide exchange data show that the highly hydrophobic nature of rhodopsin is conserved in the presence of the two detergents used: Cemulsol LA 90 and Ammonyx LO. Only about 50% of the peptide hydrogens exchange under conditions where about 80% would exchange in most soluble proteins. The conformational stability of rhodopsin in these two detergents is also demonstrated by the similarity of the tritium exchange-out kinetics and the infrared amide I band frequencies for both membrane-bound and detergent-solubilised rhodopsin. Upon illumination of rhodopsin (bleaching) in the presence of detergents, the hydrogen exchange rates are greatly increased and shifts in the amide I band frequencies are observed, indicative of a large conformation change. No such change occurs upon bleaching membrane-bound rhodopsin. We conclude that the conformation of rhodopsin is not altered by solubilisation in non-ionic detergents. However, in agreement with previously published results, bleached rhodopsin is stabilised by the membrane but does not retain a native conformation in these detergents.

Animals↗