Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Native-like folded conformations of bovine pancreatic trypsin inhibitor protein are calculated by searching for conformations with the lowest possible potential energy. Twenty-five random starting structures are subjected to soft-atom restrained energy minimization with respect to both the torsion angles and the atomic Cartesian co-ordinates. The restraints used to limit the search include the three disulphide bridges and the 16 main-chain hydrogen bonds that define the native secondary structure. The potential energy functions used are detailed and include terms that allow bond stretching, bond angle bending, bond twisting, van der Waals' forces and hydrogen bonds. Novel features of the methods used include soft-atoms to make restrained energy minimization work, writhing numbers to classify chain threadings, and molecular dynamics followed by energy minimization to anneal the conformations and reduce their energies further. Conformations are analysed using writhing numbers, torsion angle distributions, hydrogen bonds and accessible surface areas. The resulting conformations are very diverse in their chain threadings, energies and root-mean-square deviations from the X-ray structure. There is a relationship between the root-mean-square deviation and the energy, in that the lowest energy conformations are also closest to the X-ray structure. The best conformation calculated here has a root-mean-square deviation of only 3 A and shows the same special threading found in the X-ray structure. The methods introduced here have wide ranging applications; they can be used to build models of protein conformations that have low energy values and obey a wide variety of restraints.
J Mol Biol 1983 Nov 05
PMID:Protein folding by restrained energy minimization and molecular dynamics. 619 46

The stability parameters delta Gst, delta Hst and delta Sst of native basic pancreatic trypsin inhibitor (BPTI) have been characterized by microcalorimetric unfolding studies in various buffer solutions, at different pH values and in the presence of guanidine hydrochloride. The unfolding enthalpy of BPTI, in contrast ot other globular proteins, exhibits a very small dependence on temperature, which results in a characteristic different temperature dependence of the Gibbs energy of stabilization. BPTI has a very high specific Gibbs energy of stabilization, which renders the slow exchange rates of amide protons understandable. Comparison of the unfolding entropy of BPTI at 110 degrees C with corresponding values of other proteins, revealed that the delta S values of BPTI are lower by 2.9 J/(K X residue). This lower value of the unfolding entropy is in good agreement with predictions of a theoretical study by Poland & Scheraga (1965) where the influence of crosslinks on the configurational entropy has been studied. Additionally, we were able to calculate an interaction enthalpy per site of -5.6 kJ/mol based on the measurements of unfolding of BPTI in 6 M-guanidine hydrochloride.
J Mol Biol 1983 Nov 05
PMID:Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters. 619 47

A powerful method of conformational energy minimization which uses both first and second derivatives of the energy function is applied both to a small globular protein, bovine pancreatic trypsin inhibitor (BPTI), consisting of 58 amino acid residues and to its chemical derivative obtained by carboxamidomethylation of cysteinyl residues of the 14-38 disulphide bond. Conformational fluctuations are also calculated from the second derivative matrix obtained at the respective minimum energy conformations. Appreciable conformational changes upon chemical modification are observed only in the vicinity of the site of the modification. The nuclear magnetic resonance data on both BPTI and the modified BPTI are analyzed to compare with the calculated conformational changes upon chemical modification. Good correlations are found between the theoretically predicted and experimentally deduced conformational changes. The theoretical method employed here has a general application for the calculation of small conformational changes of globular proteins upon their chemical modification or an amino acid substitution.
J Mol Biol 1983 Nov 15
PMID:Conformational change of a globular protein elucidated at atomic resolution. Theoretical and nuclear magnetic resonance study. 619 87

The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.
J Mol Biol 1984 Apr 05
PMID:A new two-disulphide intermediate in the refolding of reduced bovine pancreatic trypsin inhibitor. 620 19

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.
J Mol Biol 1984 Jul 05
PMID:Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human urinary kallikrein and to porcine pancreatic beta-kallikreins A and B. 620 56

Knowledge about the architecture of macromolecules has been derived primarily from crystallography. Therefore, it has been a matter of concern whether the conformation of a macromolecule in solution, namely in vivo, might be different from that in the crystalline state. To determine the difference between the conformations, a protein (trypsin inhibitor) dissolved in water has been simulated using the method of molecular dynamics and the results are compared with those obtained from a simulation of the full crystalline unit cell. We report here that no significant difference was found for backbone atoms, except for two more or less flexible loops extending from the core of the protein and the very flexible carboxyterminal residues. The side-chains in which the conformation in solution differs considerably from that in the crystal all belong to polar residues.
J Mol Biol 1984 Jul 15
PMID:Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state. 620 58

Large orthorhombic crystals of the complex formed by bovine trypsinogen and a semisynthetic homologous bovine pancreatic trypsin inhibitor with the reactive-site lysine residue replaced by an arginine residue [( Arg15]PTI) have been obtained which are isomorphous with the crystals of PTI-trypsinogen [Bode, W., Schwager, P. and Huber, R. (1978) J. Mol. Biol. 118, 99-112]. The X-ray crystal structure of the ternary complex of trypsinogen-[Arg15]PTI with the dipeptide Val-Val has been determined by X-ray data to 2.2-A (0.22-nm) resolution by means of difference Fourier methods and has been crystallographically refined to a final R-value of 0.17. Replacement of the reactive-site Lys15 by an arginine residue is accompanied in the complex by small movements of polar side groups of trypsin and enclosed solvent molecules within the specificity pocket. Only solvent molecule 414 OH which mediates the hydrogen bond interactions between Lys15 NZ and Asp189 carboxylate is expelled, thus allowing the bulkier guanidyl group to approach this carboxylate. The dipeptide Val-Val binds in the pocket accepting the Ile-Val N-terminus in trypsin. The cavity left by the CD-methyl group of Ile16 upon replacement by a valine residue is only partially filled by slight rearrangements of neighbouring peptide side chains. Part of the positive free energy change observed upon replacement of Ile-Val may allow for the maintenance of this cavity.
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PMID:The refined 2.2-A (0.22-nm) X-ray crystal structure of the ternary complex formed by bovine trypsinogen, valine-valine and the Arg15 analogue of bovine pancreatic trypsin inhibitor. 620 21

The properties have been determined of a recently identified form of bovine pancreatic trypsin inhibitor, with only two of the three disulphide bonds of the native protein, but possessing a native-like conformation. The kinetics of refolding of the reduced inhibitor were re-measured to elucidate the kinetic role in folding of this two-disulphide species; it is formed both directly from a minor one-disulphide intermediate and by rearrangement of the other two-disulphide intermediates. It is not a productive intermediate because the Cys30 and Cys51 thiols are buried and unreactive. The previous kinetic analysis was extended by using both intra- and intermolecular disulphide reagents. Entirely consistent kinetic parameters for the rates of all the intramolecular steps of the pathway were obtained, and use of both types of reagents permits a detailed dissection of the kinetic pathway. In the process, the energetics of the folding transition were measured more thoroughly. The unique information available about the formation and stabilities of the disulphides during refolding of reduced bovine pancreatic trypsin inhibitor provides a useful description of the way in which numerous weak interactions within a protein co-operate to produce a stable folded conformation.
J Mol Biol 1984 Nov 05
PMID:Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. 621 Mar 70

The pathway of unfolding and refolding of a circular form of bovine pancreatic trypsin inhibitor, in which the termini were linked together in a peptide bond, has been examined by trapping and identifying the disulphide-containing intermediates, as was done previously for the unmodified protein. The folding pathway of the circular protein was essentially the same as that of the unmodified inhibitor, although there were differences in the distribution of intermediates that accumulated and in the rates of some steps. The effects of the cross-link between the termini on the stabilities of the folding intermediates and the native state were determined by measuring the rates of the interconversions making up the folding transition, and comparing them with those measured for the unmodified protein. The major effect of the cross-link was to stabilize an intermediate containing two native disulphides, (30-51, 14-38), but lacking the disulphide nearest the termini, 5-55. The native conformation was not measurably stabilized by the cross-link, in spite of the expected reduction of entropy of the unfolded state, indicating that the native state of the circular protein had a slightly strained conformation. The stabilities of the major one-disulphide intermediates were not significantly affected by the cross-link, suggesting that the termini of bovine pancreatic trypsin inhibitor do not tend to interact during the early stage of folding.
J Mol Biol 1984 Nov 05
PMID:Folding pathway of a circular form of bovine pancreatic trypsin inhibitor. 621 Mar 71

The structure of form II crystals of bovine pancreatic trypsin inhibitor has been investigated by joint refinement of X-ray and neutron data. Crystallographic R factors for the final model were 0.200 for the X-ray data extending to 1 A resolution and 0.197 for the 1.8 A neutron data. This model was strongly restrained, with 0.020 A root-mean-square (r.m.s.) departure of bond lengths from their ideal values and 0.019 A r.m.s. departure of planar groups from planarity. The resulting structure was very similar to that of crystal form I (r.m.s. deviation for main chain atoms was 0.40 A); nevertheless larger deviations were observed in particular regions of the chain. Twenty out of 63 ordered water molecules occupy similar positions (deviation less than 1 A) in both models. Eleven amide hydrogens were found to be protected from exchange after three months of soaking the crystals in deuterated mother liquor at pH 8.2. Their locations were in excellent agreement with the results obtained by two-dimensional nuclear magnetic resonance, but the rates of exchange are much lower in the crystalline state.
J Mol Biol 1984 Dec 05
PMID:Structure of bovine pancreatic trypsin inhibitor. Results of joint neutron and X-ray refinement of crystal form II. 621 Mar 73


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