Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The loop region of chymotrypsin inhibitor 2 from barley has been employed as a scaffold for testing the intrinsic propensity of a peptide fragment to form a secondary structure. The helix formation of the nine amino acid residue segment Lys-Gln-Ala-Val-Asp-Asn-Ala-Tyr-Ala of helix E from subtilisin Carlsberg has been studied by the construction of a hybrid consisting of chymotrypsin inhibitor 2 (CI2) where part of the active loop has been replaced by the nonapeptide. An expression system for a truncated form of CI2 where the 19 structureless residues of the N-terminus have been removed and Leu20 replaced by methionyl was constructed from the entire 83-residue wild-type CI2 gene by polymerase chain reaction methodology. The gene encoding the hybrid was constructed from the truncated inhibitor gene. The stability of the truncated inhibitor and of the hybrid toward guanidinium chloride denaturation was examined. From these measurements, the energy of unfolding in pure water was extrapolated to 30.5 +/- 1.0 kJ/mol for the truncated inhibitor and 10.9 +/- 0.3 kJ/mol for the hybrid. These energies show that the stability of CI2 is unaffected by the N-terminal truncation but severely decreased by the loop mutations. The three-dimensional structure of the hybrid protein has been determined in solution by nuclear magnetic resonance spectroscopy using 893 distance restraints and 84 torsional angle restraints. The average root-mean-square deviation (rmsd) of 15 structures compared to their geometrical average was 0.8 +/- 0.2 A for heavy backbone atoms and 1.3 +/- 0.2 A for all heavy atoms.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Context dependence of protein secondary structure formation: the three-dimensional structure and stability of a hybrid between chymotrypsin inhibitor 2 and helix E from subtilisin Carlsberg. 821 65

Hydrophobic residues in the core of a truncated form of chymotrypsin inhibitor 2 (CI2) have been mutated in order to measure their contribution to the stability of the protein. The free energy of unfolding of wild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry. The two methods give results for the changes in free energy on mutation that agree to within 1% or 2%. The average change in the free energy of unfolding (+/- standard deviation) for an Ile-->Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val-->Ala mutation 3.4 +/- 1.5 kcal mol-1, and for either an Ile-->Ala or a Leu-->Ala mutation 3.6 +/- 0.6 kcal mol-1. This gives an average change in the free energy of unfolding for deleting one methylene group of 1.3 +/- 0.5 kcal mol-1. Two significant correlations were found between the change in the free energy of unfolding between wild-type and mutant, delta delta GU-F, and the environment of the mutated residue in the protein. The first is between delta delta GU-F and the difference in side-chain solvent-accessible area buried between wild-type and mutant (correlation coefficient = 0.81, 10 points). The second and slightly better correlation was found between delta delta GU-F and N, the number of methyl/methylene groups within a 6-A radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points). The latter correlation is very similar to that found previously for barnase, suggesting that this relationship is general and applies to the hydrophobic cores of other globular proteins. The combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the change in the solvent-accessible surface area (correlation coefficient = 0.82, 30 points). This indicates that the packing density around a particular residue is important in determining the contribution the residue makes to protein stability. In one case, Ile-->Val76, a mutation which deletes the C delta 1 methyl group of a buried side chain, a surprising result was obtained. This mutant was found to be more stable than wild-type by 0.2 +/- 0.1 kcal mol-1. We have solved and analyzed the crystal structure of this mutant and find that there are small movements of side chains in the core, the largest of which, 0.7 A, is a movement of the side chain that has been mutated.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2. 821 91

Variants of subtilisin BPN' that possess improved specificity towards isoleucine compared with alanine at the P4 position of small peptide substrates, were analysed for their ability to bind chymotrypsin inhibitor 2. The binding of the inhibitor with isoleucine (wild-type) and with alanine as the P4 residue parallels the hydrolysis of tetrapeptide substrates. There is a linear relationship between the free energy of binding of the transition state of the substrate and the free energy of binding of the inhibitor with a slope of 2.0. The data suggest that the inhibitor uses predominantly ground state rather than transition state binding energy.
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PMID:Hydrolysis of small peptide substrates parallels binding of chymotrypsin inhibitor 2 for mutants of subtilisin BPN'. 826 82

The structural basis for the stability of N termini of helices has been analyzed by thermodynamic and crystallographic studies of three suitably engineered mutants of the barley chymotrypsin inhibitor 2 with Ser, Gly, or Ala at the N-cap position (residue 31). Each mutant has a well-organized shell of hydration of the terminal NH groups of the helix. The three structures are virtually superimposable (rms separations for all atoms, including the common water molecules, are 0.15-0.17 A) and show neither changes in conformation at the site of substitution nor changes in the crystal packing. The only changes on going from Ser-31 to Ala-31 to Gly-31 are in the position of a water molecule (Wat-116). This is bound to the Ser-O gamma atom in the Ser-31 structure but is in a weak hydrogen bonding position with the NH of residue 34 (O ... N = 3.28 A) in the Ala-31 mutant, partly replacing the strong Ser-31-O gamma ... N34 hydrogen bond (O ... N = 2.65 A). The corresponding water molecule completely replaces the Ser hydroxyl hydrogen bond to N34 on mutation to Gly (2.74 A). The only other change between the three structures is an additional water molecule in the Ala-31 structure (Wat-150) that partly compensates for the weak Wat-116 ... N34 hydrogen bond. Perturbation of solvation by the side chain of Ala is consistent with earlier hypotheses on the importance of exposure of the termini of helices to the aqueous solvent.
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PMID:Direct observation of better hydration at the N terminus of an alpha-helix with glycine rather than alanine as the N-cap residue. 827 84

Recombinant chymotrypsin inhibitor 2 (CI-2) and the three mutants Ile39-->Val, Ile39-->Leu, and Arg67-->Ala were successfully enriched with [2-13C]tryptophan at position 24 within the hydrophobic core of the protein. Carbon-13 NMR relaxation measurements were then used to investigate the effect of these mutations on the dynamics of the tryptophan residue. In addition, the stability of wild-type and mutant CI-2s was measured by their susceptibility to unfolding by guanidine hydrochloride. The mutant proteins were all found to be less stable, giving delta delta GU values relative to wild-type of 1.17, 1.96, and 1.21 kcal mol-1, respectively. The indole moiety of the tryptophan residue was found to be more mobile in all the mutants studied than in wild-type CI-2. Order parameters of 0.69, 0.60, 0.56, and 0.44 were derived for wild-type, Ile39-->Val, Ile39-->Leu, and Arg67-->Ala CI-2, respectively. It is concluded that there is a correlation between the protein stability and the picosecond dynamics within the hydrophobic core and that mutations can influence the dynamic behavior of the residues that are relatively distant in the three-dimensional structure.
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PMID:13C NMR study of the effects of mutation on the tryptophan dynamics in chymotrypsin inhibitor 2: correlations with structure and stability. 842 72

Experimental and simulation studies show that small monomeric proteins fold in one kinetic step, which entails overcoming the free-energy barrier between the unfolded and the native protein through a transition state. Two models of transition state formation have been proposed: a 'nonspecific' one in which it depends on the formation of a sufficient number of native-like contacts regardless of what amino acids are involved, and a 'specific' one, in which it depends on formation of a specific subset of the native structure (a folding nucleus). The latter requires that some amino acids form most of their contacts in the transition state, whereas others only do so on reaching the native conformation. If so, mutations affecting the stability of the transition state nucleus should have a greater effect on the folding kinetics than mutations elsewhere, and the residues involved should be evolutionarily conserved. Lattice-model simulations and experiments suggest that such mutations exist. Here we present a method for determining the folding nucleus of a protein with known structure with two-state folding kinetics. This method is based on the alignment of many sequences designed to fold into the native conformation of a protein to identify the positions where amino acids are most conserved in designed sequences. The method is applied to chymotrypsin inhibitor 2 (CI2), a protein whose transition state has been previously studied by protein engineering. The involvement of residues in folding nucleus of CI2 is clearly correlated with their conservation in design, and the residues forming the nucleus are highly conserved in 23 natural sequences homologous to CI2.
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PMID:Conserved residues and the mechanism of protein folding. 853 50

Temperature-induced unfolding of chymotrypsin inhibitor 2 (CI2) in water has been investigated using molecular dynamics simulations. One simulation (2.2 ns) has been analyzed in detail and three additional simulations (each > or = 1 ns) were performed to check the generality of the results. Concurrent loss of secondary and tertiary structure during unfolding was observed in all the simulations. For each simulation, the major transition state of unfolding was identified based on conformational analysis of protein structures along the unfolding trajectory. The transition state has a considerably weakened hydrophobic core and disrupted secondary structure. Nevertheless, the overall structure of the transition state is closer to the native state than to the unfolded state. The disruption of the hydrophobic core appears to be rate limiting. However, other energy barriers have to be overcome before reaching the major transition state. A method is described to quantitatively compare the structure of the simulated transition state with that characterized by protein engineering experiments. Good agreement with the experimental data is obtained for all four transition state models (the correlation coefficient R = 0.80 to 0.93) and the average over all four models gives the best correlation (R = 0.94). These simulations provide the first comprehensive atomic-level view of what the unfolding transition state of C12 may look like.
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PMID:Identification and characterization of the unfolding transition state of chymotrypsin inhibitor 2 by molecular dynamics simulations. 860 33

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the alpha-helix is weakened but partially intact and the beta-sheet is quite disrupted. As inferred from the phi f values and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a "folding core" comprising a patch on the alpha-helix and a portion of the beta-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived phi values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting phi F values and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.
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PMID:Structure of the transition state for folding of a protein derived from experiment and simulation. 860 34

The stability changes in peptides and proteins caused by the substitution of a single amino acid, which can be measured experimentally by the change in folding free energy, are evaluated here using effective potentials derived from known protein structures. The analysis is focused on mutations of residues that are accessible to the solvent. These represent in total 106 mutations, introduced at different sites in barnase, bacteriophage T4 lysozyme and chymotrypsin inhibitor 2, and in a synthetic helical peptide. Assuming that the mutations do not modify the backbone structure, the changes in folding free energies are computed using various types of database-derived potentials and are compared with the measured ones. Distance-dependent residue-residue potentials are found to be inadequate for estimating the stability changes caused by these mutations, as they are dominated by hydrophobic interactions, which do not play an essential role at the protein surface. On the contrary, the potentials based on backbone torsion angle propensities yield quite good results. Indeed, for a subset of 96 out of the 106 mutations, the computed and measured changes in folding free energy correlate with a linear correlation coefficient of 0.87. Moreover, the ten mutations that are excluded from the correlation either seem to cause modifications of the backbone structure or to involve strong hydrophobic interactions, which are atypical for solvent-accessible residues. We find furthermore that raising the ionic strength of the solvent used for measuring the changes in folding free energies improves the correlation, as it tends to mask the electrostatic interactions. When adding to these 106 mutations 44 mutations performed in staphylococcal nuclease and chemotactic protein, which were first discarded because some of them were suspected to affect the backbone conformation or the denatured state, the correlation between measured and computed folding free energy changes remains quite good: the correlation coefficient is 0.86 for 135 out of the 150 mutations. The success of the backbone torsion potentials in predicting stability changes indicates that the approximations made for deriving these potentials are adequate. It suggests moreover that the local interactions along the chain dominate at the protein surface.
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PMID:Stability changes upon mutation of solvent-accessible residues in proteins evaluated by database-derived potentials. 863 71

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.
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PMID:Solution structure and backbone dynamics of recombinant Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy. 863 82


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