Hydroxyl radical-induced hydrogen/deuterium exchange in amino acid carbon-hydrogen bonds

BB rats are used as models of autoimmune human IDDM. Genetic control of IDDM in both species is complex, including both major histocompatibility complex (MHC)-linked and non-MHC-linked genes. DP-BB rats develop IDDM spontaneously. Expression of disease in these animals requires homozygosity at the lyp locus, which causes lymphopenia. All genetic analyses of BB rat diabetes to date have backcrossed to the DP-BB strain or used (DP-BB x non-BB)F2 animals to ensure that a fraction of progeny are homozygous for lyp. Here we report the analysis of a backcross of the DP-BB rat to the histocompatible WF rat. Neither WF nor (WF x DP-BB)F1 animals develop spontaneous IDDM. However, 95% of (WF x DP-BB)F1 rats and a fraction of (WF x DP-BB) x WF backcross animals readily develop IDDM after treatment with polyinosinic:polycytidylic acid and a cytotoxic anti-RT6.1 monoclonal antibody. Using simple sequence length polymorphism analysis, we have mapped loci on chromosomes 4 and 13 that show significant linkage to IDDM expression and insulitis. The susceptibility locus on chromosome 4 is linked to, but not identical to, lyp. We propose a disease model for the BB rat that requires 1) the RT1u MHC haplotype for disease susceptibility, 2) a new locus on chromosome 4 for disease initiation (as measured by insulitis), 3) a new locus on chromosome 13 for disease progression in response to environmental perturbation, and 4) lyp for spontaneous expression of disease.     

H NMR probes for inter-segmental hydrogen bonds in myoglobins

NMR signals arising from the HisB5 N delta H and HisEF5 N epsilon H protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.     

Electromigration of hydrogen and deuterium in vanadium and niobium by a resistance method

The electric mobility of hydrogen and deuterium has been measured at 30°C in niobium (Cb) and vanadium by a resistance method. The electric mobility was found to be 5.7 × 10⁻⁴ cm²/V-s for hydrogen and 2.8 × 10⁻⁴ for deuterium in niobium. In vanadium the electric mobilities were 2.3 × 10⁻³ and 1.3 × 10⁻³ cm²/V-s for hydrogen and deuterium respectively. The effective charges calculated using reported diffusion coefficients are positive and are slightly greater for deuterium than for hydrogen in both vanadium and niobium. The resistivity increase due to the hydrogen isotopes in vanadium and niobium was also measured. Hydrogen was found to contribute 0.65 μ ohm-cm/at, pct and deuter-ium 0.58 μ ohm−cm/at, pct to the resistivity of niobium. In vanadium, the solute resistivi-ties were found to be 0.98 μ. ohm-cm/at, pct and 0.90 μ ohm−cm/at, pct for hydrogen and deuterium respectively. C. L. JENSEN, formerly a Graduate Student, Department of Materials Science and Engineering, Iowa State University     

A diminished role for hydrogen bonds in antifreeze protein binding to ice

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.     

Differential deuterium isotope shifts and one-bond 1H-13C scalar couplings in the conformational analysis of protein glycine residues

The one-bond deuterium isotope shift effect for glycine C alpha resonances exhibits a conformational dependence comparable to that of the corresponding 1JHC scalar coupling in both magnitude (approximately 11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the 1JHC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (phi, psi) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the 1JHC scalar couplings can determine conformations in the left- and right-handed helical-to-bridge regions of the (phi, psi) plane to an accuracy of approximately 13 degrees. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the 1JHC scalar couplings can be combined with limited independent structural information (e.g., the sign of phi) to determine the chirality of the deuterium substitution.     

Distribution of deuterium and hydrogen in Zr and Ti foil assemblies under the action of a pulsed deuterium high-temperature plasma

Deuteron and proton elastic recoil detection analysis is used to study the accumulation and redistribution of deuterium and hydrogen in assemblies of two high-pure zirconium or titanium foils upon pulsed action of high-temperature deuterium plasma (PHTDP) in a plasma-focus installation PF-4. It is noted that, under the action of PHTDP, an implanted deuterium and hydrogen gas impurity are redistributed in the irradiated foils in large depths, which are significantly larger than the deuterium ion free paths (at their maximum velocity to ~10⁸ cm/s). The observed phenomenon is attributed to the carrying out of implanted deuterium and hydrogen under the action of powerful shock waves formed in the metallic foils under the action of PHTDP and/or the acceleration of diffusion of deuterium and hydrogen atoms under the action of a compression–rarefaction shock wave at the shock wave front with the redistribution of deuterium and hydrogen to large depths.     

Thermal denaturation of Escherichia coli thioredoxin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry: monitoring a two-state protein unfolding transition

Thermally denatured oxidized Escherichia coli thioredoxin (TRX) in 2% acetic acid was examined by electrospray ionization mass spectrometry (ESI-MS) and circular dichroism. Conformational dynamics during thermal unfolding were probed by hydrogen/deuterium (H/D) exchange-in experiments. ESI-MS was used to determine the H/D ratios. TRX shows only a marginal change in negative ellipticity at 222 nm during thermal unfolding, but in the near-UV circular dichroism (240-350 nm) a clear transition is observed (Tm = 61 degrees C), and unfolding goes to completion. ESI mass spectra were recorded as a function of temperature, and the observed bimodal charge state distributions were analyzed assuming a two-state unfolding mechanism which allowed an estimation of the midpoint temperature, Tm = 64 degrees C. Under conditions at which the compact, folded conformational state is only marginally stable (80 degrees C, 2% acetic acid-d1), H/D exchange-in experiments in combination with ESI-MS resulted in mass spectra differing in the number of incorporated deuteriums which indicates the presence of two distinct populations of molecules after short incubation periods. As the exchange-in time increases, the population representing the unfolded state increases and the population which is protected against exchange decreases. The rate of conversion was used to estimate the rate constant of unfolding which was 2.1 +/- 0.2 min-1. The results presented here indicate that thermally denatured TRX under the conditions used may represent a collapsed unfolded state with properties often attributed to molten globule-like states, such as pronounced secondary structure but absence of rigid tertiary structure and, hence, lack of protection against H/D exchange.     

Electromigration of hydrogen and deuterium in tantalum: Isotope effect

Electromigration experiments at steady-state were conducted to determine the effective valence of hydrogen isotopes in tantalum specimens at room temperature (25 °C). Varying solute concentrations were obtained by gas-phase and electrolytic charging of the specimens. The transient concentration profiles of several specimens were monitored by a resistance technique allowing a controlled current through the samples. Steady-state concentration profiles were obtained by direct hot-vacuum extraction measurements. The results indicate a strong concentration dependence for the effective valence and evidence of a possible reversal in the direction of solute migration. While the reversal in the case of deuterium migration is not certain, an isotope effect on the effective valence as a whole is substantial. Deuterium has a 30 pct higher effective valence. The hypothesis that solute segregation at lattice defects and differing electronic structure of the defects and the perfect lattice are responsible for the concentration dependence of effective valence is found invalid as both cold-worked and annealed specimens did not indicate any microstructure effect on Z*. The concentration dependence of diffusion coefficient does not justify the strong dependence of Z* on concentration observed experimentally.     

Direct NMR measurement of folding kinetics of a trimeric peptide

Direct NMR measurements of the folding kinetics are performed on a collagen-like triple helical peptide. The triple helical peptide was designed to model a biologically important region of collagen and has the sequence (POG)3ITGARGLAG(POG)4. Triple helical peptides were synthesized with specifically labeled 15N amino acid residues in key positions, and the kinetics of folding of the individual residues were monitored directly by measuring the loss of monomer intensity and the increase in trimer intensity. The residues at the terminal ends and central region could be followed independently and quantitated directly. Residues located at the terminal ends have rates and kinetics of folding that are distinct from residues in the central region of the peptide. This allows the monitoring of different steps in the folding mechanism and the postulation of the existence of a kinetic intermediate. The NMR data are consistent with a mechanism of association/nucleation and propagation. Hereditary connective tissue diseases are associated with mutations that result in abnormal folding of collagen, and the NMR folding experiments on a collagen-like peptide provide a basis for characterizing the molecular defect in folding mutations.     

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity

The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine chi1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 A for the backbone atom, and 1.68 A for all heavy atoms. For the well-defined region encompassing residues 2-12 and 18-27, the corresponding values were 0.71 and 1.66 A, respectively. Circulin A adopts a compact structure consisting of beta-turns and a distorted segment of triple-stranded beta-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a "cystine-knot" motif. This motif is present in a range of other biologically active proteins, including omega-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.