Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Basic pancreatic trypsin inhibitor

The assignment of the ¹H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor with the use of two-dimensional ¹H nuclear magnetic resonance techniques at 500 MHz is described. The assignments are based entirely on the known amino acid sequence and the nuclear magnetic resonance data. Individual resonance assignments were obtained for all backbone and C^β protons, with the exception of those of Arg1, Pro2, Pro13 and the amide proton of Gly37. The side-chain resonance assignments are complete, with the exception of Pro2 and Pro13, the N^δ protons of Asn44 and the peripheral protons of the lysine residues and all but two of the arginine residues.     

Individual assignments of the methyl resonances in the 1H nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor.

In earlier work the resonances of the 20 methyl groups in the basic pancreatic trypsin inhibitor (BPTI) had been identified in the 360-MHz 1H nuclear magnetic resonance (NMR) spectra and most of the methyl lines had from spin-decoupling experiments been assigned to the different types of amino acid residues. The assignments to the different amino acid types were now completed by studies of the saturation transfer between the denatured and the globular forms of the inhibitor and by spin-decoupling experiments in nuclear Overhauser enhancement (NOE) difference spectra. These distinguished between the methyl resonances of Ala and Thr. Furthermore, for most of the methyl resonances, individual assignments to specific residues in the amino acid sequence were obtained from measurements of intramolecular proton-proton NOE's, use of lanthanide NMR shift and relaxation probes, and comparative studies of various chemically modified forms of BPTI. These data provide the basis for individual assignments of the methyl 13C NMR lines in BPTI and for detailed investigations of the relations between the spatial structure of the protein and the chemical shifts of the methyl groups. The methyl groups in BPTI are of particular interest since they are located almost exclusively on the surface of the protein and thus represent potential natural NMR probes for studies of the protein-protein interactions in the complexes formed between BPTI and a variety of proteases.     

(14-38, 30-51) double-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor: a two-dimensional 1H nuclear magnetic resonance study

An analogue of the BPTI folding intermediate that contains only the disulphide bonds between Cys14 and Cys38 and between Cys30 and Cys51 has been prepared in Escherichia coli by protein engineering methods. The other two Cys residues of native BPTI (at positions 5 and 55) have been replaced by Ser. Essentially complete proton resonance assignments of the analogue were obtained by employing two-dimensional 1H nuclear magnetic resonance techniques. The intermediate has a more extended conformation in the N-terminal (residues 1 to 7) region and there are other differences in the C-terminal (residues 55 to 58) region. The remainder of the protein is substantially identical to native BPTI. The conformational properties of the analogue can explain several aspects of the kinetic role that the normal (14-38, 30-51) intermediate plays in the folding of BPTI.     

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.     

Complete sequence-specific 1H nuclear magnetic resonance assignments for the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae

The 1H nuclear magnetic resonance (n.m.r.) spectrum of the alpha-amylase inhibitor Tendamistat was completely assigned with the use of phase-sensitive homonuclear two-dimensional n.m.r. The assignments include the non-labile protons of the 74 amino acid residues as well as the labile protons which exchange sufficiently slowly to be observed in H2O solution. The proton chemical shifts are listed at 50 degrees C and pH 3.2, which coincides with the conditions used for the determination of the three-dimensional structure of Tendamistat.     

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.     

A study of the lysyl residues in the basic pancreatic trypsin inhibitor using 1H nuclear magnetic resonance at 360 Mhz.

Fourier transform 1H nuclear magnetic resonance (NMR) experiments at 360 MHz using convolution difference techniques to improve the spectral resolution were employed to investigate the resonances of the lysyl residues in bovine pancreatic trypsin inhibitor. The observations in both native protein and in chemically modified protein containing Nepsilon-dimethyllsysine show that three of the four lysines extend predominantly freely into the solvent, whereas lysine-41 is involved in an intramolecular interaction with tyrosine-10. Since in the single crystal structure tyrosine-10 is involved in an intermolecular interaction with arginine-42 of the neighboring protein molecule, the NMR data thus reveal a local conformation difference for bovine pancreatic trypsin inhibitor in solution and in the crystalline form which appears to result primarily from intermolecular interaction in the crystal lattice.     

Complete sequence-specific 1H nuclear magnetic resonance assignments for mouse epidermal growth factor

The 1H NMR spectrum of the mouse epidermal growth factor (53 residues) was analyzed with the use of two-dimensional NMR techniques. All the observable 296 proton resonances were completely assigned in a sequential manner. For the spin system identification, two-dimensional homonuclear Hartmann-Hahn spectrum was useful, especially for arginine and proline residues. The easy spin system identification of these long-side-chain-bearing amino acid residues greatly facilitated the sequence-specific resonance assignment of the epidermal growth factor.     

Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance.

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 51 and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Protein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the beta-loop (residues 25-28) or C-terminal alpha-helix, respectively.     

Toward the complete assignment of the carbon nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor

A total of 54 of the 58 alpha-carbon resonances and numerous side-chain carbon signals were individually assigned in the basic pancreatic trypsin inhibitor by using two-dimensional heteronuclear correlated and relayed coherence transfer spectroscopy with proton detection. No isotope enrichment was used, and the spectra were recorded in 5-mm sample tubes. The pulse sequences were optimized to eliminate, prior to phase cycling, the signals of protons attached to 12C. We have concentrated on assignments of carbons bearing a single hydrogen in view of a relatively easy interpretation of carbon relaxation times, and most of these carbon resonances could be assigned. Furthermore, we demonstrate that two-dimensional heteronuclear correlated and relayed coherence transfer spectra can be used to elucidate connectivities between degenerate resonances within proton spin systems that often occur in threonines and aromatic side chains.     

Nitrotyrosine chelation of nuclear magnetic resonance shift probes in proteins: application to bovine pancreatic trypsin inhibitor

The interactions of Pr(III) and Eu(III) with specifically nitrated derivatives of the basic bovine pancreatic trypsin inhibitor have been studied using optical spectroscopy and nuclear magnetic resonance (NMR) at 250 and 270 MHz. Stability constants for proton and metal binding to nitrotyrosines 10 and 21 determined optically are in good agreement with those from NMR. Observations of the Eu(III)-induced NMR shifts of the ring protons of nitrotyrosine 21 allowed calibration of the magnetic interactions for this binding site. The Pr(III)-induced shifts for several resolved nonexchangeable backbone proton resonances were compared with calculated shifts using the known x-ray structure. With several simplifying assumptions, the Pr(III)-induced shifts were used to assign one alpha-CH and five NH protons to compatible sets of backbone positions which are consistent with the known pH dependence and resistance to exchange with solvent D2O. Some of the more general aspects of lanthanide-induced shifts are discussed with reference to their use in proteins. Due to the complexities of the analysis of the shift data, the most straightforward use of this technique is in conjunction with the relaxation probe Gd(III) for measurement of intramolecular distances.     

Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor

The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-Å resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.     

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.     

Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.     

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.