Assignment of resonances in the 1H NMR spectrum of human lysozyme

Assignments in the 1H NMR spectrum for more than 120 resonances arising from 38 of the 130 amino acid residues of human lysozyme are presented. Assignments have been achieved using a combination of one and two-dimensional NMR techniques. Two-dimensional double-quantum correlated spectroscopy and relayed coherence transfer spectroscopy were found to be particularly useful for the identification of spin systems in the aromatic and methyl regions of the spectrum. These spin systems were assigned to specific residues in human lysozyme with reference to the X-ray crystal structure using one-dimensional nuclear Overhauser enhancement (NOE) data and a computer-based search procedure. Unique assignments were found for resonances of 27 amino acid residues even when a distance constraint on NOE effects of 0.7 nm was used in the search procedure; for the remaining residues closer constraints or additional information were required. The assignments include all but one of the resonances in the aromatic region of the spectrum and all the methyl group resonances in the region upfield of 0.6 ppm. The assignments presented here provide a basis for a comparison of the NMR spectra of human lysozyme and the more widely studied hen lysozyme.     

Assignment of resonances in the Escherichia coli 5 S RNA fragment proton NMR spectrum using uniform nitrogen-15 enrichment

The downfield proton NMR spectrum of aqueous uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 chemical shifts of fragment imino nitrogens. Nitrogen chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges. Nitrogen-15 and proton chemical shift correlation by the heteronuclear decoupling permitted the identification of the base type of some previously unassigned imino proton resonances in the 5 S RNA fragment spectrum. Corresponding resonances in the natural isotopic abundance 5 S RNA fragment spectrum are assigned to base types by comparison with the enriched sample spectrum.     

Quaternary interactions in hemoglobin beta subunit tetramers. Kinetics of ligand binding and self-assembly

We have investigated the rates of monomer in equilibrium with tetramer self-association of oxygenated beta SH subunits of human hemoglobin A as well as the influence of self-association on the binding kinetics for O2 and CO. A 4 beta in equilibrium with 2 beta 2 in equilibrium with beta 4 assembly pathway can be used to describe the association equilibria and kinetics. We have determined all four elementary rate constants for this assembly pathway at 15 degrees C in 0.1 M Tris-HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. These data imply that a significant amount (approximately 17%) of beta 2 can be present. Laser photolysis kinetic studies of O2 binding indicate that the O2 association rate constant is unaffected by the degree of self-association. In contrast, photolysis of beta CO solutions shows an overall rate of CO binding that increases at higher protein concentrations. These data are consistent with a concentration-dependent equilibrium between two protein species with CO association rates differing by a factor of 2.5, but they do not appear to be compatible with a direct assignment of different CO binding rates to the different assembly states. Rather, we believe the data imply that CO binding to beta oligomers is heterogeneous, with both a fast binding and a slow binding form being present in single association states. The fast binding form predominates (approximately equal to 87%) in beta 4, while the beta monomer has very little or none of the fast binding form. We propose that the slow binding component within beta 4 may be those subunits with rotationally disordered hemes (La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) Biochemistry 24, 3826-3831). The implications of these findings for the use of isolated subunits as models for the subunits within "R state" hemoglobin tetramers are discussed.     

Resonance assignments in the proton-NMR spectrum of carbonmonoxy hemoglobin by two-dimensional methods

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr = 64,500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heme substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in beta subunits which, in solution, appears to have stronger contacts with the heme groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr less than or equal to 65,000).     

Individual assignments of amide proton resonances in the proton NMR spectrum of the basic pancreatic trypsin inhibitor.

Studies of proton-proton nuclear Overhauser effects were used to obtain individual assignments of 17 amide proton resonances in the 360 MHz proton nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor. First, optimizing the conditions for obtaining selective nuclear Overhauser effects in the presence of spin diffusion in macromolecules is discussed. Truncated driven nuclear Overhauser experiments were used to assing the amide proton resonances of the beta-sheet in the inhibitor. It is suggested that these techniques could serve quite generally to obtain individual resonance assignments in beta-sheet secondary structures of proteins. Combination of nuclear Overhauser studies with spin decoupling further resulted in individual assignments of the gamma-methyl resonances of the two isoleucines and numerous Calpha and Cbeta protons.     

Assignment of paramagnetically shifted resonances in the 1H NMR spectrum of horse ferricytochrome c.

The proton resonances of the heme, the axial ligands, and other hyperfine-shifted resonances in the 1H nuclear magnetic resonance spectrum of horse ferricytochrome c have been investigated by means of one- and two-dimensional nuclear Overhauser and magnetization transfer methods. Conditions for saturation transfer experiments in mixtures of ferro- and ferricytochrome c were optimized for the cross assignment of corresponding resonances in the two oxidation states. New resonance assignments were obtained for the methine protons of both thioether bridges, the beta and gamma meso protons, the propionate six heme substituent, the N pi H of His-18, and the Tyr-67 OH. In addition, several recently reported assignments were confirmed. All of the resolved hyperfine-shifted resonances in the spectrum of ferricytochrome c are now identified. The Fermi contact shifts experienced by the heme and ligand protons are discussed.     

Assignment of resonances for 'acute-phase' glycoproteins in high resolution proton NMR spectra of human blood plasma

Broad resonances at 2.04 and 2.08 ppm in 500 MHz Hahn spin-echo 1H NMR spectra of human blood plasma are assigned to the N-acetyl groups of mobile carbohydrate side-chains (largely N-acetylglucosamine and N-acetylneuraminic acid) of glycoproteins such as alpha 1-acid glycoprotein. Their intensities in spin-echo spectra correlate with clinical conditions in which an elevation of the level of 'acute-phase' glycoproteins is expected, and so may be of value in the study of certain diseases.     

Assignment of methylene proton resonances in NMR spectra of embryonic and transformed cells to plasma membrane triglyceride

Some biological characteristics of cancer cells and solid tumors are identifiable by the high resolution NMR relaxation behavior of their nonaqueous components. Chemical analysis and two-dimensional scalar correlated (COSY) NMR spectroscopy show these resonances arise from neutral lipid in the plasma membrane. Triglyceride is shown to be the main plasma membrane component giving rise to the NMR spectrum, while soluble nonmembrane components account for 90% of the remaining resonances in the spectrum of intact cells. The presence of triglyceride has been detected by chemical analysis in highly purified plasma membranes from two different cell lines. The COSY spectra of cancer cells are comparable with that obtained for the triglyceride-rich very low density human lipoprotein.     

Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of human hemoglobin alpha-chains

Assignments are reported for a substantial number of heme and amino acid proton resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of isolated hemoglobin alpha-chains. These resonances provide information on the solution conformation of the protein, particularly in the vicinity of the heme. The heme pocket structure is generally similar to that of carbonmonoxymyoglobin; several conserved residues adopt virtually identical positions relative to the heme in the two proteins. The largest conformational differences involve residues surrounding the ligand-binding site, notably Val62 (E11) and His58 (E7). The chemical shifts of the proximal His87 (F8) resonances are very similar in spectra of the two proteins, indicating a highly conserved coordination geometry and similar hydrogen bonding to the backbone carbonyl of Leu83 (F4).     

13C and proton NMR studies of horse cytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of the 55 methyl groups of horse cytochrome c have been determined over a range of temperatures both in the diamagnetic ferrocytochrome and in the paramagnetic ferricytochrome. Specific assignments of many proton resonances have been published previously and all of the remaining methyl proton resonances are now specifically assigned. The corresponding 13C assignments follow directly, including those of contact shifted 13C resonances which are reported for the first time.     

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.     

Assignment of resonances in the 31P NMR spectrum of d(GGAATTCC) by regiospecific labeling with oxygen-17

The chemical synthesis of the octanucleotide d(GGAATTCC) in which each of the phosphate groups is sequentially replaced by an 17O-containing phosphate group using a polymer-supported phosphoramidite method is described. All seven phosphorus resonances in the 31P spectrum of d(GGAATTCC) can be resolved. Assignment of these resonances to a particular phosphate group in the chain is possible because labeling of a phosphate with 17O causes its particular signal to disappear from the spectrum. Phosphate residues toward the middle of the octamer have 31P NMR shifts similar to those found in polydeoxynucleotides, whereas those toward the ends resemble those of dinucleoside phosphates. These data are interpreted in terms of less flexibility of the phosphate groups in the center of the molecule as compared to those at the ends.     

The dissociation of carbon monoxide from the alpha and the beta subunits of human carbonmonoxy hemoglobin

We have measured the intrinsic CO dissociation rates from the subunits of the human hemoglobin tetramers (alpha CO beta NO)2 and (alpha NO beta CO)2 using microperoxidase and a stopped-flow spectrophotometer. The dissociation of NO is negligible. The rate constants for the and the subunits are similar (0.014 s-1 vs. 0.011 s-1, respectively, at pH 7, 20 C; and 0.016 s-1 for both in the presence of inositol hexaphosphate), indicating that they are equivalent in the first step of the CO dissociation. Therefore, the chain unequality observed in the third and fourth steps (Samaja, M., Rovida, E., Niggeler, M., Perrella, M., and Rossi-Bernardi, L. (1987). J. Biol. Chem.: 262, 4528-4533) are not due to the intrinsic properties of the subunits, but to the conformational state of the molecule.     

Assignment of selected hyperfine proton NMR resonances in the met forms of Glycera dibranchiata monomer hemoglobins and comparisons with sperm whale metmyoglobin

This work indicates a high degree of purity for our preparations of all three of the primary Glycera dibranchiata monomer hemoglobins and details assignments of the heme methyl and vinyl protons in the hyperfine shift region of the ferric (aquo?) protein forms. The assignments were carried out by reconstituting the apoproteins of each component with selectively deuteriated hemes. The results indicate that even though the individual component preparations consist of essentially a single protein, the proton NMR spectra indicate spectroscopic heterogeneity. Evidence is presented for identification and classification of major and minor protein forms that are present in solutions of each component. Finally, in contrast to previous results, a detailed analysis of the proton hyperfine shift patterns of the major and minor forms of each component, in comparison to the major and minor forms of metmyoglobin, leads to the conclusion that the corresponding forms of the proteins from each species have strikingly similar heme-globin contacts and display nearly identical heme electronic structures and coordination numbers.     

Assignment of the histidine 12-threonine 45 hydrogen-bonded proton in the NMR spectrum of ribonuclease A in H2O.

Described herein are proton nmr experiments on chemically modified derivatives of ribonuclease A designed to elucidate the origin of an exchangeable resonance, assigned previously to a histidine ring N proton that titrates between 11 to 13 ppm with a pK_a of 6.1 in H₂O solution. Histidines 48 and 105, which are distant from the active site, are eliminated as candidates for this resonance from inhibitor binding studies on the enzyme in acetate–water solutions. This exchangeable resonance titrates with modified pK_a's and constant area over the above pH range in His-119-N₁-carboxymethylated-RNase A and des-(121–124)-RNase A, thus eliminating the imidazole N₃ proton in the His 119-Asp 121 hydrogen bond. In His-12-N₁-carboxymethylated-RNase A, this resonance is also observable, but broadens on raising the pH above 7 and at elevated temperatures above neutrality. It exhibits a pH-independent chemical shift characteristic of the protonated state of histidine. On the basis of these findings, this exchangeable resonance, designated a, is assigned to the imidazole N₁ proton of His 12, which is hydrogen-bonded to the carbonyl oxygen of Thr 45 in the crystal.