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 contributions of the components of a type I reverse turn to the stability of chymotrypsin inhibitor-2 (Lys43-Pro44-Gly45) have been determined by protein engineering methods. A double-mutant cycle was used to determine the interaction between Lys43 and Glu45 by replacing them with alanine. We also mutated Pro44, which gives the geometry of the turn, to alanine and analysed the stability of the resulting mutants compared with wild-type chymotrypsin inhibitor-2, using equilibrium denaturation induced by guanidinium chloride. There are decreases in stability (in kcal/mol) of 0.64 +/- 0.06 for Lys43-->Ala, 0.57 +/- 0.15 for Glu45-->Ala, 0.95 +/- 0.06 for Lys43-->Ala/Glu45-->Ala and 1.93 +/- 0.09 for Pro44-->Ala. The free energy of interaction between Lys43 and Glu45 is calculated to be only 0.25 +/- 0.09 kcal/mol. From the changes in denaturation midpoint, Tm measured by circular dichroism, we estimate the energy of interaction between Lys43 and Glu45 to be 0.36 +/- 0.07 kcal/mol whereas the contribution of Pro44 is approximately 2.0 kcal/mol. The contribution of the salt bridge to the stability of the protein is very small and the residue Pro44 plays the key role in stabilizing the turn.
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PMID:Contribution of a proline residue and a salt bridge to the stability of a type I reverse turn in chymotrypsin inhibitor-2. 790 35

The N-terminus of the helix of the chymotrypsin inhibitor 2 from barley (CI2) has an N-capping box (Ser at the first position in the helix and Glu at position 4) as well as a frequently found Glu at position 3. The energetic importance of this motif has been studied by determining the free energy of unfolding of the wild-type and protein mutants derived from those residues using guanidinium chloride-induced denaturation and differential scanning microcalorimetry. Mutating N-cap residue Ser31 to either Ala or Gly destabilizes CI2 by 0.8-1 kcal mol-1. Truncation of the box in the mutants SA31EA33EA34 or SG31EA33EA34 destablizes the protein by 1.5-2 kcal mol-1. The N-capping box is an important motif in stabilizing proteins and delineating the beginning of alpha-helices in the pathway of protein folding.
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PMID:Mutational analysis of the N-capping box of the alpha-helix of chymotrypsin inhibitor 2. 793 8

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure. The structure of the transition state appears to be an expanded form of the native state in which secondary and tertiary elements have been partly formed concurrently. This is consistent with a "global collapse" model of folding rather than a framework model in which folding is initiated from fully preformed local secondary structural elements. This may be a general feature for the folding of proteins lacking a folding intermediate and is perhaps representative of the early stages of folding for multidomain or multimodule proteins. The major transition state for the folding of barnase, for example, has some fully formed secondary and tertiary structural elements in the major transition state, and barnase appears to form by a framework process. However, the fully formed framework may be preceded by a global collapse, and a unified folding scheme is presented.
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PMID:Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. 793 67

Protein engineering and kinetic experiments indicate that some regions of proteins have partially formed structure in the transition state for protein folding. A crucial question is whether there is a genuine single transition state that has interactions that are weakened in those regions or there are parallel pathways involving many transition states, some with the interactions fully formed and others with the structural elements fully unfolded. We describe a kinetic test to distinguish between these possibilities. The kinetics rule out those mechanisms that involve a mixture of fully formed or fully unfolded structures for regions of the barley chymotrypsin inhibitor 2 and barnase, and so those regions are genuinely only partially folded in the transition state. The implications for modeling of protein folding pathways are discussed.
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PMID:Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. 793 68

Temperature-induced unfolding of chymotrypsin inhibitor 2 in water was investigated by molecular dynamics simulations. The major transition state of unfolding was identified on the basis of structural and conformational changes in the protein during the unfolding reaction. The native tertiary contacts in the hydrophobic core were considerably disrupted in the transition state, whereas the secondary structure was partially intact. The extent of structural change of the protein around a particular residue was represented quantitatively by the ratio of the number of contacts the residue makes in the transition state relative to the native state, phi MD, which allows quantitative comparison with the experimentally determined phi F values. For the region of the unfolding trajectory that is identified as the transition state, the phi MD and phi F values are in good agreement, suggesting that the transition state identified in the unfolding simulation corresponds to that probed with protein engineering methods. Although speculative, the transition state identified in the simulation is consistent with available experimental data and provides an in-depth view of what the transition state of unfolding may look like.
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PMID:Characterization of the transition state of protein unfolding by use of molecular dynamics: chymotrypsin inhibitor 2. 793 69

Residues in the active site loop of the serine protease inhibitor, chymotrypsin inhibitor 2, thought to play an important role in loop stability and inhibitory activity, have been investigated by site-directed mutagenesis. Substitutions at residues 58 (threonine in wild type) and 60 (glutamic acid in wild type), which flank the scissile bond (Met-59-Glu-60) and are conserved among the potato inhibitor I family of serine protease inhibitors, are found to be of some importance in the global stability of the protein, as measured by guanidinium chloride-induced denaturation, but are essential for its inhibitory activity. Mutation of either Thr-58 or Glu-60 to alanine results in a decrease in stability of 0.7 +/- 0.1 kcal mol-1. These values reflect the loss of hydrogen bonds between the hydroxyl group of Thr-58 with Glu-60 and Arg-67 and hydrogen bonds and a salt bridge between Glu-60 and Arg-62 and Arg-65. In addition, these mutants were found to be much weaker inhibitors of the serine protease subtilisin BPN'. The dissociation constants for inhibition, Ki, were found to be (7.0 +/- 0.4, 540 +/- 30, and 980 +/- 50) x 10(-13) M, for wild type, T58A, and E60A, respectively. Further, we find that these mutants are only temporary inhibitors of subtilisin BPN', unlike wild type. Over long time scales, we observe a reversal of inhibition because of hydrolysis of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of residues in the reactive site loop of chymotrypsin inhibitor 2 to protein stability and activity. 794 96

Possible early events in protein folding may be studied by dissecting proteins into complementary fragments. Two fragments of chymotrypsin inhibitor 2 [CI2-(20-59) and CI2-(60-83)] associate to form a native-like structure in a second-order reaction that combines collision and rearrangement. The transition state of the reaction, analyzed by the protein engineering method on 17 mutants, is remarkably similar to that for the folding of the intact protein--a structure that resembles an expanded version of the folded structure with most interactions significantly weakened. The exception is that the N-terminal region of the single alpha-helix (the N-capping box) is completely formed in the transition state for association of the fragments, whereas it is reasonably well formed for the intact protein. Preliminary evidence on the structures of the individual fragments indicates that both are mainly nonnative, lacking native secondary structure and having regions of nonnative buried hydrophobic clusters. The association reaction does not result from the collision of a subpopulation of two fully native-like fragments but involves a considerable rearrangement of structure.
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PMID:The structure of the transition state for the association of two fragments of the barley chymotrypsin inhibitor 2 to generate native-like protein: implications for mechanisms of protein folding. 797 88

The suitability of the barley chymotrypsin inhibitor-2 for study by fragmentation and complementation has been analyzed. The primary residue for binding to proteases, Met-59 (the unique methionine in the sequence), lies in a broad, solvent-exposed loop. The bond between Met-59 and Glu-60 was cleaved by cyanogen bromide. The two fragments thus obtained, i.e., CI-2(20-59) and CI-2(60-83), associate (KD = 42 nM) to yield a complex that has fluorescence and circular dichroism spectra identical to those of uncleaved chymotrypsin inhibitor-2. Recovery of native-like structure is further indicated by the ability of the complex to inhibit chymotrypsin, although the [I]50% is 140-fold higher than for the uncleaved inhibitor. CI-2(60-83) appears to be highly disordered in water, but fragment CI(20-59) forms significative structure, as judged by its circular dichroism spectra and evidence from one-dimensional NMR. The circular dichroism spectra of CI-2(20-59) approach the baseline in 4 M guanidinium chloride but display characteristics of an alpha-helix in the presence of trifluoroethanol. Analytical ultracentrifugation shows no concentration-dependent change in the molecular weight of the monomer of CI-2(20-59). Both one- and two-dimensional NMR of the complex [CI-2(20-59).(60-83)] show unequivocally the presence of a folded structure, which appears to be slightly different from the uncleaved native protein.
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PMID:Generation of a family of protein fragments for structure-folding studies. 1. Folding complementation of two fragments of chymotrypsin inhibitor-2 formed by cleavage at its unique methionine residue. 801 58

The kinetics of association of the fragments of the barely chymotrypsin inhibitor-2, CI-2(20-59) and CI-2(60-83), to form a native-like structure follows two phases. There is a major second-order component with rate constant (3.7 +/- 0.3) x 10(3) M-1 s-1 and a slow first-order phase of rate constant 0.011 +/- 0.001 s-1. The major phase contains a cooperative folding process as judged by the secondary structure recovery in parallel with the fluorescence change. The time course for structure formation has uniform changes at all of the wavelengths of the circular dichroism spectra, suggesting that all elements of secondary structure are formed simultaneously. A series of kinetic experiments suggest that the association and folding occur in the second-order step and that the first-order step probably results from a cis-trans peptidylprolyl isomerization in the fragment CI-2(20-59). This was confirmed by experiments on fragments derived from two mutants whose parent proteins fold more slowly than wild-type CI-2. Those fragments display lower second-order rate constants, but the rate constants of the first-order phase are the same as for wild type. The experiments suggest that the mechanism of the association/folding of mutant fragments may be studied by a protein-engineering analysis.
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PMID:Generation of a family of protein fragments for structure-folding studies. 2. Kinetics of association of the two chymotrypsin inhibitor-2 fragments. 801 59

The solvation of polar groups at the N-terminal end of alpha-helices was studied by comparing the crystal structures of T4 lysozyme, barley chymotrypsin inhibitor 2 (CI2), barnase and their respective N-cap mutants. Whether or not the N3 residue is solvated on mutating the N-cap Thr/Ser to Ala or Gly appears to be related to the identities and the side-chain conformations of the N2 and N3 residues. When these two residues are alanines, as is in the pseudo-wild-type CI2 (E33A/E34A), the main-chain NH at the N3 position is exposed to the solvent and can be solvated. If the N2 residue is an Asp or a Glu, it is more likely that the side-chain of these residues will form a surrogate N-cap with the amide NH at N3 to compensate for the lost -OH group. In this case, no additional solvation will be observed. In general, Gly can be more stable than Ala at the N-cap because its small side-chain allows nearby polar groups to form hydrogen bonds with optimal geometry with solvent molecules or other polar groups.
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PMID:Stability and solvation of Thr/Ser to Ala and Gly mutations at the N-cap of alpha-helices. 803 23


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