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.     

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.     

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.     

Protein thermal stability: hydrogen bonds or internal packing?

Thermally stable proteins are of interest for several reasons. They can be used to improve the efficiency of many industrial processes and provide insight into the general mechanisms of protein folding and stabilization. Comparison of tertiary structural properties of several protein families with members of different thermostability should help to delineate the role of individual factors in achieving stability at high temperature. In this work, 16 protein families with at least one known thermophilic and one known mesophilic tertiary structure were examined for the number and type of hydrogen bonds and salt links, polar surface composition, internal cavities and packing densities, and secondary structural composition. The results show a consistent increase in the number of hydrogen bonds and in polar surface area fraction with increased thermostability.     

1H-NMR study of inter-segmental hydrogen bonds in sperm whale and horse apomyoglobins

NMR signals for HisB5 N(delta)H and HisEF5 N(epsilon)H protons of sperm whale and horse apomyoglobins were assigned and compared with the corresponding signals of the holoproteins in terms of pH and temperature dependence behaviors of their shifts and line widths in order to gain insight into structural difference between the apoproteins and the holoproteins. Since these protons are involved in internal hydrogen bonds at the interfaces between the B helix and the GH corner and between the EF corner and the H helix, local structures of the interfaces in these proteins have been inferred from the analyses of these signals. A large difference in the line width of HisEF5 N(epsilon)H proton signal between the apoproteins and the holoproteins strongly suggested that a sizable structural alteration is induced in the EF-H interface by the removal of heme. However, the results for HisB5 N(delta)H proton resonance indicated the absence of a significant structural alteration in the B-GH interface by heme extraction. These results are consistent with the data obtained from mutation [Hughson, F. M. & Baldwin, R. L. (1989) Biochemistry 28, 4415-4422] and amide-proton-exchange kinetic [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548] studies, which indicated that the A, B, G and H helices in apomyoglobin maintain the same packing as they do in holoprotein.     

Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex

The high-affinity streptavidin-biotin complex is characterized by an extensive hydrogen-bonding network. A study of hydrogen-bonding energetics at the ureido oxygen of biotin has been conducted with site-directed mutations at Asn 23, Ser 27, and Tyr 43. A new competitive biotin binding assay was developed to provide direct equilibrium measurements of the alterations in Kd. S27A, Y43F, Y43A, N23A, and N23E mutants display DeltaDeltaG degrees at 37 degrees C relative to wild-type streptavidin of 2.9, 1.2, 2.6, 3.5, and 2.6 kcal/mol, respectively. The equilibrium-binding enthalpies for all of the mutants were measured by isothermal titration calorimetry, and the Y43A and N23A mutants display large decreases in the equilibrium binding enthalpy at 25 degrees C of 8.9 and 6.9 kcal/mol, respectively. The S27A and N23E mutants displayed small decreases in binding enthalpy of 1.6 and 0.9 kcal/mol relative to wild-type, while the Y43F mutant displayed a -2.6 kcal/mol increase in the binding enthalpy at 25 degrees C. At 37 degrees C, the Y43A and N23A mutants display decreases of 7.8 and 7.9 kcal/mol, respectively, while the S27A, N23E, and Y43F mutants displayed decreases of 4.9, 3.7, and 1.2 kcal/mol relative to wild-type. Kinetic analyses were also conducted to probe the contributions of the hydrogen bonds to the activation barrier. Wild-type streptavidin at 37 degrees C displays a koff of (4.1 +/- 0.3) x 10(-5) s-1, and the conservative Y43F, S27A, and N23A mutants displayed increases in koff to (20 +/- 1) x 10(-5) s-1, (660 +/- 40) x 10(-5) s-1, and (1030 +/- 220) x 10(-)5 s-1, respectively. The Y43A and N23E mutants displayed 93-fold and 188-fold increases in koff, respectively. Activation energies and enthalpies for each of the mutants were determined by transition-state analysis of the dissociation rate temperature dependence. All of the mutants except Y43F display large reductions in the activation enthalpy. The Y43F mutant has a more positive activation enthalpy, and thus a more favorable activation entropy that underlies the overall reduction in the activation barrier. For the most conservative mutant at each ureido oxygen hydrogen-bonding position, bound-state alterations account for most of the energetic changes in a single transition-state model, suggesting that the ureido oxygen hydrogen-bonding interactions are broken in the dissociation transition state.     

Solution NMR characterization of hydrogen bonds in a protein by indirect measurement of deuterium quadrupole couplings

Hydrogen bonds stabilize protein and nucleic acid structure, but little direct spectroscopic data have been available for characterizing these critical interactions in biological macromolecules. It is demonstrated that the electric field gradient at the nucleus of an amide hydrogen can be determined residue-specific by measurement of 15N NMR relaxation times in proteins dissolved in D2O, and uniformly enriched with 13C and 15N. In D2O, all backbone amide protons can be exchanged with solvent deuterons, and the T1 relaxation rate of a deuteron is dominated by its quadrupole coupling constant (QCC), which is directly proportional to the electric field gradient at the nucleus. 2HN T1 relaxation can be measured quantitatively through its effect on the T2 relaxation of its directly attached 15N. QCC values calculated from 2HN T1 and previously reported spectral densities correlate with the inverse cube of the X-ray crystal structure-derived hydrogen bond lengths: QCC = 228 + Sigmai 130 cos alphai/ri3 kHz, where alpha is the N-H...Oi angle and r is the backbone-backbone (N-)H...Oi(=C) hydrogen bond distance in angstroms.     

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.     

Energetics of drug-DNA interactions

Understanding the thermodynamics of drug binding to DNA is of both practical and fundamental interest. The practical interest lies in the contribution that thermodynamics can make to the rational design process for the development of new DNA targeted drugs. Thermodynamics offer key insights into the molecular forces that drive complex formation that cannot be obtained by structural or computational studies alone. The fundamental interest in these interactions lies in what they can reveal about the general problems of parsing and predicting ligand binding free energies. For these problems, drug-DNA interactions offer several distinct advantages, among them being that the structures of many drug-DNA complexes are known at high resolution and that such structures reveal that in many cases the drug acts as a rigid body, with little conformational change upon binding. Complete thermodynamic profiles (delta G, delta H, delta S, delta Cp) for numerous drug-DNA interactions have been obtained, with the help of high-sensitivity microcalorimetry. The purpose of this article is to offer a perspective on the interpretation of these thermodynamics parameters, and in particular how they might be correlated with known structural features. Obligatory conformational changes in the DNA to accommodate intercalators and the loss of translational and rotational freedom upon complex formation both present unfavorable free energy barriers for binding. Such barriers must be overcome by favorable free energy contributions from the hydrophobic transfer of ligand from solution into the binding site, polyelectrolyte contributions from coupled ion release, and molecular interactions (hydrogen and ionic bonds, van der Waals interactions) that form within the binding site. Theoretical and semiempirical tools that allow estimates of these contributions to be made will be discussed, and their use in dissecting experimental data illustrated. This process, even at the current level of approximation, can shed considerable light on the drug-DNA binding process.     

Energetics of steelmaking processes

The basic energy indices of steelmaking processes, namely, the consumption of energy carriers, the total energy intensity, the full energy efficiency of a process, and the amount carbon dioxide released in atmosphere, are considered. The energy, ecology, and economic efficiencies of the processes are quantitatively analyzed for various alternative energy carriers during steelmaking in an ASF. The problem of sustainable development of an object in the environment is analyzed in relation to its main three parameters, namely, economics, energetics, and ecology.     

电弧放电法合成Mg-Mg2Si-Si复合纳米粉体

在SiH4 Ar的混合气氛下用电弧放电法蒸发纯Mg制备了Mg-Mg2Si-Si复合纳米粉体.用XRD、TEM、DSC-TG、氧含量分析等手段对样品进行了分析.结果表明,样品中Mg2Si含量随气氛中SiH4含量(压力)的增加而增加,平均粒度和氧含量随Mg2Si含量增加而减小.在室温大气条件下,样品中Mg2Si相的含量越高则耐氧化性越好.在空气中,加热到410℃左右复合粉体氧化速率加快,至580℃左右几乎完全氧化.