Amide proton temperature coefficients as hydrogen bond indicators in proteins

Correlations between amide proton temperature coefficients (_HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show _HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3₁₀ helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.     

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: A monitor of intramolecular hydrogen bonds in helical peptides?

Summary 2D ¹H NMR spectroscopy of two -helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from –10 to –6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the -helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in -helices in H₂O/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in -helices. Supplementary Material is available upon request, comprising seven pages with listings of experimental details and the NMR shift data for the two peptides.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Temperature coefficients of peptides dissolved in hexafluoroisopropanol monitor distortions of helices

Temperature coefficients are widely used as an indication of solvent accessibility to amide protons. Low temperature coefficients are related to low accessibility and are often interpreted as evidence for intramolecular hydrogen bonding. Conformational shifts, i.e. the difference between chemical shifts of a particular residue in a structured and in a random-coil conformation, provide information on secondary structure. In particular, negative CH conformational shifts are often used to delineate the extent of helical stretches. NH conformational shifts show large oscillations within a helix that have been interpreted as the result of helix distortions affecting hydrogen bond lengths. In the course of the study of different peptides that adopt a helical structure in the presence of the structure-inducing solvent hexafluoroisopropanol (HFIP), we have found a strong correlation between temperature coefficients and amide conformational shifts. However, contrary to the initial expectations, lower temperature coefficients were associated to amide protons involved in longer, and presumably weaker, hydrogen bonds. The correlation can be explained, however, assuming that, in helical peptides dissolved in HFIP, temperature affects the chemical shift of amide protons mainly by changing the average length of intramolecular hydrogen bonds and changes in solvent accessibility play only a secondary role under these experimental conditions. The pattern of temperature coefficients in helical peptides can therefore be used to identify short or long hydrogen bonds causing bending of the helix axis.     

Linkage studies on α-glycerophosphate and isocitrate dehydrogenases in Aedes albopictus (diptera: Culicidae)

Linkage studies were carried out on -glycerophosphate dehydrogenase (-GPDH) and isocitrate dehydrogenase (IDH) in the mosquito Aedes (Stegomyia) albopictus. Only one locus coding for -GPDH was revealed on agar gels by applying adult homogenates. Two loci for IDH were observed using either fourth-instar larvae, pupae, or adults. This study was restricted to the more anodal Idh-2 of the two loci, and -Gpdh. Both -Gpdh and Idh-2 encode dimeric enzymes. Thirteen backcrosses indicated that the -Gpdh and Idh-2 loci are arranged in linkage group 2 in the following order: p (pigmented pupa)—(ca. 2 map units)—Wb (white-body)—(7.5–17.8)—Idh-2—(13.1)—-Gpdh. Females exhibited more recombination than males.This work was supported by a Grant-in-Aid for Co-operative Research from the Ministry of Education, Japan.     

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50%H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H^N–H^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The -proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H₂ –H^NEth is indicative of proximity between C- and N-termini.     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

13C NMR chemical shifts can predict disulfide bond formation

The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography, and otherwise must be inferred by chemical methods. In this study we demonstrate that ¹³C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine ¹³C C and C chemical shifts was constructed from the BMRB and Sheffield databases, and published journals. Statistical analysis indicated that the C shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C/C chemical shift map. On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.     

The 13C chemical shifts of amino acids in aqueous solution containing organic solvents: Application to the secondary structure characterization of peptides in aqueous trifluoroethanol solution

Summary The ¹³C chemical shifts for all of the protonated carbons of the 20 common amino acid residues in the protected linear pentapeptide Gly-Gly-X-Gly-Gly have been obtained in water at low pH as well as in aqueous solution containing 10, 20 and 30% acetonitrile or trifluoroethanol. Dioxane was used as an internal reference and its carbon chemical shift value was found to be 66.6 ppm relative to external TMS in water. Comparison of the different referencing methods for ¹³C chemical shifts in organic cosolvent mixtures showed that an external standard (either TMS or TSP capillary) was the most appropriate. In the present study, external TSP was chosen to define the 0 ppm of the ¹³C chemical shift scale. When the difference in referencing the dioxane carbon resonance is taken into account, the carbon chemical shift values of the amino acids in aqueous solution are similar to those previously reported (Richarz and Wüthrich (1978) Biopolymers, 17, 2133–2141; Howarth and Lilley (1979) Prog. NMR Spectrosc., 12, 1–40). The pentapeptides studied were assumed to be in a random coil conformation and the measured ¹³C chemical shifts were used as reference values to correlate carbon chemical shifts with the secondary structure of two well-characterized peptides, bombesin and the 1–29 amino acid fragment of Nle²⁷ human growth hormone-releasing factor. In both cases, the C chemical shifts exhibited a characteristic positive deviation from the random coil values, which indicates the presence of -helices.     

Precise vicinal coupling constants3JHNα in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments

Summary Improved experimental schemes for the recently introduced J-modulated [¹⁵N,¹H]-correlation experiment for measurements of the homonuclear amide proton-C proton vicinal coupling constants.³J_HN, in uniformly¹⁵N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of³J_HN. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M=7.3 kDa), where at 13 C precise values of³J_HN in the range 2.0–9.5 Hz were obtained for all residues with resolved¹⁵N-¹H cross peaks. It was then applied to theAntennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex 18 kDa). The³J_HN values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.Abbreviations and symbols NOE nuclear Overhauser effect - COSY two-dimensional correlated spectroscopy - ³J_HN or J homonuclear vicinal amide proton-C proton coupling constant - 434 repressor(1–69) N-terminal DNA-binding domain of the 434 repressor comprising 69 residues     

Tolerance to Diazepam-Induced Motor Impairment: A Study with GABAA Receptor α6 Subunit Knockout Mice

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0–7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell–restricted GABA_A receptor 6 subunit and their wild-type controls. The 6–/– mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4–7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the 6–/– brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the 6–/– and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive 6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.     

Quantitative measurement of small through-hydrogen-bond and ‘through-space’1H-113Cd and1H-199Hg J couplings in metal-substituted rubredoxin fromPyrococcus furiosus

Summary A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring¹H-¹¹³Cd and¹H-¹⁹⁹Hg J couplings in metal-substituted rubredoxin (M_r 5.4 kDa) fromPyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a through-space coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to¹⁹⁹Hg are significantly larger and fall in the 0.4–4 Hz range.     

Solution Structures of Casein Peptides: NMR, FTIR, CD, and Molecular Modeling Studies of αs1-Casein, 1–23

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine _s1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional -helical or -sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30–40% turns, 25–30% extended) that were highly stable from 5°C to 25°C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of _s1-casein (1–23) in facilitating casein-casein interactions.     

Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy

Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR₁C (1–77) and TR₂C (78–148). Experiments were done in H₂O, D₂O and D₂O/trifluoroethanol (TFE) mixtures. Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure. Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca²⁺-saturated state. Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR₁C domain. The maximum wavenumber for the amide I band of the two domains of CaM in D20 was markedly different (1642 cm^–1 for TR₁C versus 1646/1648 cm^–1 for Ca ²⁺ and apo-TR₂C). This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of -helices with slightly different hydrogen bonding patterns.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Molecular and crystal structure of Nα-(9-fluorenyl)- methoxycarbonyl-L-ornithine hydrochloride diethyl ether solvate

The solid-state conformation of the first N-protected ornithine derivative has been established by X-ray analysis. The hydrochloride of N-(9-fluorenyl)methoxycarbonyl-L-ornithine crystallises as diethyl ether solvate. The backbone (₀, , , ¹) torsion angles are (174.9°, –84.0°, 145.9°, –171.0°). The conformation of the urethane amide bond is trans. The ornithine aliphatic side chain adopts preferred fully extended conformation which is stabilised by the hydrogen bonding of the –NH₃ ⁺ group to the diethyl ether molecule, carboxyl group and Cl^- anions.     

A method for the calculation of protein α-CH chemical shifts

Summary The chemical shifts of CH protons have been calculated for 9 proteins, based on coordinates taken from high-resolution crystal structures. Chemical shifts were calculated using ring-current shifts, shifts arising from magnetic anisotropies of bonds, and shifts arising from the polarizing effect of polar atoms on the C-H bond. The parameters used were refined iteratively to give the best fit to (experimental — random coil) shifts over the set of 9 proteins. A further small correction was made to the averaged Gly CH shift. The calculated shifts match observed shifts with correlation coefficients varying between 0.45 and 0.86, with a standard deviation of about 0.3 ppm. The differences between calculated and observed shifts have been studied in detail, including an analysis of different crystal structures of the same protein, and indicate that most of the differences can be accounted for by small differences between the structure in solution and in the crystal. Calculations using NMR-derived structures give a poor fit. The calculations reproduce the experimentally observed differences between chemical shifts for CH in -helix and -sheet. Most of the differentiation in secondary structure-dependent shifts arises from electric field effects, although magnetic anisotropy also makes a large contribution to the net shift. Applications of the calculations to assignment (including stereospecific assignment) and structure determination are discussed.     

α-Globin gene deletions associated with αAand αG Philadelphiagenes in an Algerian family that includes two Hb G homozygotes

Summary An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed -globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of 3.7 kb deletion resulting from the rightward type of recombination event between ₂ and ₁ genes on both the ^A/and the ^G chromosomes. Such data indicate that the –^A/ and –^G/ haplotypes are involved and suggest that the –^G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (–^A/–^G/) or (–^G/–^G/), the output of the remaining genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (–^A/–^A/) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the –^A/ haplotypes are heterogeneous in Algerians.