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)

Although the rates of chemical reactions become faster with increasing temperature, the converse may be observed with protein-folding reactions. The rate constant for folding initially increases with temperature, goes through a maximum, and then decreases. The activation enthalpy is thus highly temperature dependent because of a large change in specific heat (delta Cp). Such a delta Cp term is usually presumed to be a consequence of a large decrease in exposure of hydrophobic surfaces to water as the reaction proceeds from the denatured state to the transition state for folding: the hydrophobic side chains are surrounded by "icebergs" of water that melt with increasing temperature, thus making a large contribution to the Cp of the denatured state and a smaller one to the more compact transition state. The rate could also be affected by temperature-induced changes in the conformational population of the ground state: the heat required for the progressive melting of residual structure in the denatured state will contribute to delta Cp. By examining two proteins with different refolding mechanisms, we are able to find both of these two processes; barley chymotrypsin inhibitor 2, which refolds from a highly unfolded state, fits well to a hydrophobic interaction model with a constant delta Cp of activation, whereas barnase, which refolds from a more structured denatured state, deviates from this ideal behavior.
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PMID:Negative activation enthalpies in the kinetics of protein folding. 756 45

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state kinetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a function of concentration of denaturant. The transition state was found to move progressively closer to the folded state on destabilization of the protein by mutation. Different regions of CI2 all show a similar sensitivity to changes in the energy of the transition state. This is in contrast to the behavior of barnase on mutation for which the position of the transition state for its unfolding is sensitive to mutation in some regions, especially in its major alpha-helix, but not in others. The transition state for the folding and unfolding of CI2 resembles an expanded version of the folded state and is formed in a concerted manner, in contrast to that for barnase, in which some regions of structure are fully formed and others fully unfolded. The reason for the general sensitivity of the position of the transition state of CI2 to mutation is presumably the relatively uniform degree of structure formation in the transition state and the concerted nature of its formation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Movement of the position of the transition state in protein folding. 757 56

The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL. The chaperonin retards the rate of refolding of wild type and 32 representative point mutants. The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on the mutation. It is seen qualitatively that truncation of large hydrophobic side chains to smaller side chains weakens binding. Analysis of the magnitude of the rates of retardation shows further that hydrophobic and positively charged side chains tend to interact favorably with GroEL whereas negatively charged side chains tend to repel. There is an inverse correlation between the strength of hydrophobic interactions and the rate constant for refolding of the GroEL-complexed protein: the better the binding, the slower the folding. This shows directly that hydrophobic (and other favorable) interactions between the chaperonin and substrate are weakened during the refolding process and implies that unfolding can be catalyzed by the gain of such interactions.
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PMID:Nature and consequences of GroEL-protein interactions. 757 64

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.
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PMID:Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases. 760 23

The solution structure of Cucurbita maxima trypsin inhibitor-V (CMTI-V), which is also a specific inhibitor of the blood coagulation protein, factor XIIa, was determined by 1H NMR spectroscopy in combination with a distance-geometry and simulated annealing algorithm. Sequence-specific resonance assignments were made for all the main-chain and most of the side-chain hydrogens. Stereospecific assignments were also made for some of the beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens. The ring conformations of all six prolines in the inhibitor were determined on the basis of 1H-1H vicinal coupling constant patterns; most of the proline ring hydrogens were stereospecifically assigned on the basis of vicinal coupling constant and intraresidue nuclear Overhauser effect (NOE) patterns. Distance constraints were determined on the basis of NOEs between pairs of hydrogens. Dihedral angle constraints were determined from estimates of scalar coupling constants and intraresidue NOEs. On the basis of 727 interproton distance and 111 torsion angle constraints, which included backbone phi angles and side-chain chi 1, chi 2, chi 3, and chi 4 angles, 22 structures were calculated by a distance geometry algorithm and refined by energy minimization and simulated annealing methods. Both main-chain and side-chain atoms are well-defined, except for a loop region, two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 22 individual structures and the mean structure obtained by averaging their coordinates is 0.58 +/- 0.06 A for the main-chain atoms and 1.01 +/- 0.07 A for all the non-hydrogen atoms of residues 3-40 and 49-67. These structures were compared to the X-ray crystallographic structure of another protein of the same inhibitor family-chymotrypsin inhibitor-2 from barley seeds [CI-2; McPhalen, C. A., & James, M. N. G. (1987) Biochemistry 26, 261-269]. The main-chain folding patterns are highly similar for the two proteins, which possess 62% sequence differences. However, major differences are noted in the N- and C-terminal segments, which may be due to the presence of a disulfide bridge in CMTI-V, but not in CI-2.
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PMID:Three-dimensional solution structure of Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy. 771 Oct 40

beta-Sheet propensities of different amino acids depend on the context of both secondary and tertiary structure. In an attempt to establish general empirical relationships that determine this context dependence, we have determined the free energy of unfolding of a series of mutants at six positions in the beta-sheet of chymotrypsin inhibitor 2 (CI2). We have generated the series Val-->Ala-->Gly and Val<==>Thr at five positions, as well as the side-chain deletion Ile-->Val at residue 49 and Ala-->Gly at residue 77. In the series Val-->Ala-->Gly, the ranking order in terms of stability is Val > Ala > Gly at all positions. However, the change in free energy on deletion of methylene groups varies greatly. When Val and Thr are interchanged, the wild-type residue is always the more stable, but by a different amount at each position. We have attempted to rationalize the data by relating it to changes in solvent-accessible surface area, packing density, and statistically derived pseudo-energy functions that depend on phi, psi angles. There is no significant correlation of the energies with any of the variables except with the pseudo-energy function, but the deviations from these values are large. We conclude that thermodynamic scales for beta-sheet propensity are currently of insufficient precision for general design purposes, although they may be useful in special cases.
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PMID:Side-chain determinants of beta-sheet stability. 772 32

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.
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PMID:Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. 773 65

The structures of all the intermediates and transition states, from the unfolded state to the native structure, are being determined at the level of individual residues in the folding pathways of barnase and chymotrypsin inhibitor 2 (CI2), using a combination of protein engineering and nuclear magnetic resonance methods. Barnase appears to refold according to a classical framework model in which elements of secondary structure are flickeringly present in the denatured state, consolidate as the reaction proceeds and, when nearly fully formed, dock in the rate-determining step. Unlike barnase, CI2 folds without a kinetically significant folding intermediate. The transition state for its formation has no fully formed elements of secondary structure, and the transition state is like an expanded form of the native structure. CI2 probably represents the folding of an individual domain in a larger protein, whereas barnase represents the folding of a multi-domain protein. The protein engineering methods are being extended to map the pathway in the presence of molecular chaperones. There are parallels between the folding of barnase when bound to GroEL and in solution.
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PMID:Mapping the structures of transition states and intermediates in folding: delineation of pathways at high resolution. 777 Apr 80

Two fragments of chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), derived from cyanogen bromide cleavage at Met-59, associate to give a native-like structure. We analyze the kinetics and equilibria of association of mutant fragments derived from cleaving mutant proteins at the same methionine residue. The changes in free energy of association have been measured both from isothermal studies of the binding of fragments and from thermal denaturation of the complexes. In general, there is a good correlation between the changes on mutation of the free energy of association of fragments and the changes in free energy of folding of the uncleaved parent protein. The notable exceptions are for residues in regions of the fragments that form nonnative hydrophobic clusters in the isolated fragments; mutation of the hydrophobic residues involved in these clusters decreases the equilibrium constant for formation of the noncovalent complex less than it does the equilibrium constant for folding of intact protein. The dissociated fragments must be destabilized by mutation of those hydrophobic residues, but to a lesser extent than is the complex itself. These clusters are thus less important energetically in the denatured state of the intact protein. The second-order rate constants for the major phase of association change with mutation, similar results being obtained from fluorescence measurements of the regain of tertiary structure and from circular dichroism measurements of the regain of secondary structure. The rate constants for association correlate well, in general, with the rate constants of refolding of the respective uncleaved proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein fragments as models for events in protein folding pathways: protein engineering analysis of the association of two complementary fragments of the barley chymotrypsin inhibitor 2 (CI-2). 784 29

The backbone dynamics of uniformly 15N-labeled chymotrypsin inhibitor 2 (CI2) and of the complex formed by the association of two fragments consisting of residues 20-59 and 60-83 have been studied. A data set consisting of 15N longitudinal (T1) and transverse (T1 rho) relaxation times and (1H)-15N NOE enhancements has been measured for all backbone NH groups in both proteins. Information on internal motions has been extracted from these data using the model-free approach to determine order parameters (S2) and effective internal correlation times (tau e). The data indicate that most of the backbone of CI2 is highly constrained (S2 approximately 0.9) with the exception of residues in the binding loop (residues 54-64), which have slightly lower order parameters. Most of the residues in the CI2(20-59).(60-83) complex are also highly constrained (S2 approximately 0.9). However, the loss of the covalent bond between Met59 and Glu60 leads to a large increase in the mobility of residues in the loop region. The residues in the first half of the loop region have significantly lower order parameters than those in the second half of the loop. This observation suggests that the NH2 group that is released on cleavage of the scissile bond remains anchored in its original position, inhibiting the attack of water on the acyl-enzyme that is formed between the protease and the cleaved inhibitor. More importantly, the NH2 group is optimally placed for reversing the formation of the acyl-enzyme so that the equilibrium between the cleaved and uncleaved inhibitor, bound to the protease, greatly favors the uncleaved complex.
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PMID:Backbone dynamics of chymotrypsin inhibitor 2: effect of breaking the active site bond and its implications for the mechanism of inhibition of serine proteases. 785 34


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