Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry

A determination of the solution conformation of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA) is described. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain a list of 202 distance constraints between individually assigned hydrogen atoms of the polypeptide chain, to identify the positions of the three disulfide bridges, and to locate the single cis peptide bond. Supplementary geometric constraints were derived from the vicinal spin-spin couplings and the locations of certain hydrogen bonds, as determined by nuclear magnetic resonance (n.m.r.). Using a new distance geometry program (DISGEO) which is capable of computing all-atom structures for proteins the size of BUSI IIA, five conformers were computed from the NOE distance constraints alone, and another five were computed with the supplementary constraints included. Comparison of the different structures computed from the n.m.r. data among themselves and with the crystal structures of two homologous proteins shows that the global features of the conformation of BUSI IIA (i.e. the overall dimensions of the molecule and the threading of the polypeptide chain) were well-defined by the available n.m.r. data. In the Appendix, we describe a preliminary energy refinement of the structure, which showed that the constraints derived from the n.m.r. data are compatible with a low energy spatial structure.     

Secondary structure in the solution conformation of the proteinase inhibitor IIA from bull seminal plasma by nuclear magnetic resonance

Nuclear magnetic resonance data on the protease inhibitor IIA from bull seminal plasma were used to determine the secondary structure elements in the solution conformation of the protein. The experimental data were obtained from analyses of two-dimensional 1H nuclear magnetic resonance spectra at 500 and 360 MHz and include details of inter-residue nuclear Overhauser enhancements, vicinal spin-spin coupling constants and the sequence location of slowly exchanging amide protons. Accurate measurement of coupling constants and reliable assignments of nuclear Overhauser enhancements were facilitated by the use of absorption mode two-dimensional spectroscopy and large data matrices. It is shown that the peptide backbone is extended from residues 4 to 7, followed by a poorly defined helical region from residues 8 to 13 with a marked change of direction at residue Phe10. Residues 15 to 19 are extended and there is a kink at residue Glu20. Residues 22 to 27 form the central strand of a triple-stranded antiparallel beta-sheet, of which the other two strands are residues 29 to 33 and 49 to 53. Residues 34 to 46 form a helix. The tight turn in the beta-sheet is of type I geometry, and there is a beta-bulge at residue His53.     

Method of modeling protein structure by the two-dimensional nuclear magnetic resonance spectroscopy data; application to the proteinase inhibitor BUSI IIA from bull seminal plasma

A new approach is suggested to model the spatial structure of protein molecules in solution based on combined use of the methods of theoretical conformational analysis and NMR spectroscopy data. At the first stage, special means are used to convert d connectivity information into the most probable values of dihedral angles. This allows search for possible spatial structures in the limited regions of the conformational space at further stages using the methods of the theoretical conformational analysis. The suggested approach was verified in reconstructing the spatial backbone structure of the fragment 17-57 of the proteinase inhibitor BUSI IIA from the bull seminal plasma. The structural model parameters are compared with the corresponding characteristics obtained from the X-ray analysis data for the homologic proteinase inhibitor from the Japanese quail ovomucoid. The suggested approach is shown to correctly reproduce both the general molecule topology and the conformations of individual amino acid residues.     

Characterization of the proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance. Stability, amide proton exchange and mobility of aromatic residues

The isoinhibitor IIA from bull seminal plasma was investigated in aqueous solution by 1H nuclear magnetic resonance (n.m.r.). The analysis of the 1H n.m.r. data was based on individual resonance assignments, which are described in the following paper. Large conformation-dependent chemical shifts for aliphatic amino acid side-chains, numerous slowly exchanging amide protons and unusual pH titrations of two aromatic residues show that this protein forms a compact, globular conformation. This form of the protein is stable between pH 4 and 12 at 25 degrees C, and between 5 and 50 degrees C at pH 4.9. At temperatures above 50 degrees C there is evidence for an equilibrium between several different conformations, with the rate of exchange between the different species being in the intermediate range on the n.m.r. time-scale. Preliminary data are presented for the individual exchange rates of 18 backbone amide protons. Among the four aromatic rings, Phe10, Phe38 and Tyr16 undergo rapid 180 flips over the entire temperature range, whereas for Tyr32 a temperature-dependent transition from low-frequency to high-frequency flipping motions was observed.     

Assignment of the 1H nuclear magnetic resonance spectrum of the trypsin inhibitor homologue K from Dendroaspis polylepis polylepis. Two-dimensional nuclear magnetic resonance at 360 and 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the trypsin inhibitor homologue K from the venom of Dendroaspis polylepis polylepis is described and documented. The assignments are based entirely on the amino acid sequence and on 2-dimensional n.m.r. experiments at 360 and 500 M Hz. Individual assignments were obtained for the backbone and C beta protons of all 57 residues of the inhibitor homologue K, with the exceptions of the N-terminal amino group, the amide protons of Arg16, Gly37 and Gly40 and the C beta protons of Arg16 and Pro19. The assignments for the non-labile protons of the amino acid side-chains are complete, with the exception of Gln29, Glu49 and all the proline, lysine and arginine residues. For Asn and Trp the labile side-chain protons have also been assigned. The chemical shifts for the assigned resonances are listed for an aqueous solution at 50 degrees C and pH 3.4.     

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man₈GlcNAc₂Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Assignment of resonances in the 1H nuclear magnetic resonance spectrum of the carbon monoxide complex of sperm whale myoglobin by phase-sensitive two-dimensional techniques

Phase-sensitive two-dimensional nuclear magnetic resonance (n.m.r.) experiments have been used to obtain extensive proton resonance assignments for the carbon monoxide complex of sperm whale myoglobin. Multiple quantum experiments were particularly important in the assignment procedure. The assignments are the most complete yet reported for a protein of such high molecular weight (approximately 18,000) and make possible new and comprehensive studies of the structure and dynamics of carbonmonoxymyoglobin in solution. Assignments for seven of the histidine residues are reported, including the critical proximal and distal histidines. Most of these are at variance with the assignments already in the literature. The present n.m.r. data indicate that histidines 24 (B5) and 119 (GH1) are hydrogen bonded to each other and, in contrast to neutron diffraction data, show that His24 does not protonate at pH greater than 5. The aromatic rings of all the phenylalanine and tyrosine residues undergo rapid flips about the ring axis. The side-chains of Leu89 (F4) and Phe138 (H15), which border a large hydrophobic cavity, are particularly mobile.     

Assignment of aromatic spin systems in the 1H nuclear magnetic resonance spectrum of adenylate kinase

A combination of selective spin decoupling, two-dimensional double quantum spectroscopy, correlated spectroscopy (COSY), and pH titration experiments brought about the assignment of all tyrosyl spin systems and completed the assignment of the histidyl spin systems in porcine adenylate kinase. In the detection of the tyrosyl spin systems it proved to be advantageous to resort to the COSY method rather than to two-dimensional double quantum spectroscopy. In the titration experiments, His189 revealed a second apparent pK value at pH 8.3, which is explained by deprotonation of the adjacent residue Cys187. None of the seven tyrosyl side-chains shows any evidence for deprotonation up to the point of denaturation of the protein, which took place around pH 10.     

Amide proton exchange in the alpha-amylase polypeptide inhibitor Tendamistat studied by two-dimensional 1H nuclear magnetic resonance

The individual amide proton exchange rates in Tendamistat at pH 3.0 and 50 degrees C were measured by using two-dimensional 1H nuclear magnetic resonance. Overall, it was found that the distribution of exchange rates along the sequence is dominated by the interstrand hydrogen bonds of the beta-sheet structures. The slowly exchanging protons in the core of the two beta-sheets were shown to exchange via an EX2 mechanism. Further analysis of the data indicates that different large-scale structure fluctuations are responsible for the exchange from the two beta-sheets, even though the three-dimensional structure of Tendamistat appears to consist of a single structural domain.     

Epidermal growth factor from the mouse. Structural characterization by proton nuclear magnetic resonance and nuclear overhauser experiments at 500 MHz

Mouse epidermal growth factor (mEGF), a protein hormone effector molecule that regulates cellular development and division, has been investigated by using proton nuclear magnetic resonance techniques at 500 MHz. Well-resolved downfield aromatic and alpha-CH proton resonances (5.0-8.0 ppm) and an upfield ring current shifted isoleucine delta-methyl resonance (approximately 0.5 ppm) have been examined by using principally nuclear Overhauser methods. The data are analyzed in terms of model building based on the predictive Chou-Fasman secondary structure algorithm applied to mEGF [Holladay, L. A., Savage, C. R., Cohen, S., & Puett, D. (1976) Biochemistry 15, 2624-2633], which suggests the existence of some beta-structure and little or no alpha-helicity. Proximity relationships derived from nuclear Overhauser data among Tyr-3, -10, and -13, His-22, and Ile-23 allow refinement of some aspects of the predicted secondary structure and render additional information on how the protein backbone in mEGF is folded (i.e., tertiary structure). Nuclear Overhauser effects (NOEs) from irradiation of several alpha-CH proton resonances give evidence for tiered beta-sheet structure in mEGF. Such proximity relationships derived from NOE data place stringent limitations on possible models for the molecule. pH titration data demonstrate a His-22 pKa of 7.1, indicating either a salt bridge or hydrogen-bond formation between His-22 and another residue. The His-22 pKa is also reflected in the chemical shift changes of several other resonances as a function of pH. Nuclear Overhauser methods, used to differentiate direct (protonation) and indirect (conformation) effects on the chemical shift changes in the spectra of mEGF by varying the pH, yield evidence for a pH-induced conformational transition in the protein hormone associated with the breaking of the His-22 salt bridge or hydrogen bond.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Residue-specific assignments of resonances in the 1H nuclear magnetic resonance spectrum of ribosomal protein E-L30 by systematic application of two-dimensional Fourier transform nuclear magnetic resonance methods

A two-dimensional Fourier transform nuclear magnetic resonance study of the ribosomal protein E-L30 is reported. Five two-dimensional techniques, namely: nuclear magnetic resonance J-resolved spectroscopy, correlated spectroscopy, double quantum spectroscopy, relayed coherence transfer and nuclear Overhauser enhancement spectroscopy were used. Qualitative inspection of the spectra obtained by these techniques provided evidence that the E-L30 molecule has a well-defined structure in solution. This analysis indicated that, despite the fact that the protein is stable only at moderate temperatures and neutral pH, a structural analysis of the molecule would be feasible. A detailed analysis of the spectra permitted unambiguous discrimination between the spin systems of different amino acids, resulting in residue-specific resonance assignments. We were able to assign all resonances of all six threonine, four valine, five alanine, two histidine, two serine, one phenylalanine, one asparagine and one aspartic acid residue of E-L30. Complete resonance assignment was obtained for two glycine residues. Partial assignments became available for all six isoleucine, three glycine and one glutamine residue. These results form a sound basis for the structure determination of the protein described in the accompanying paper.     

Assignment of the 13C nuclear magnetic resonance spectrum of a short DNA-duplex with 1H-detected two-dimensional heteronuclear correlation spectroscopy.

Proton-detected 1H-13C heteronuclear correlated spectroscopy [( 1H,13C]-COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC)2. Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [1H,13C]-COSY.     

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).     

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 aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.