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Alan R Fersht - One of the best experts on this subject based on the ideXlab platform.
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protein folding from a highly disordered denatured state the folding pathway of Chymotrypsin Inhibitor 2 at atomic resolution
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Steven L Kazmirski, Stefan M V Freund, Alan R Fersht, Kambo Wong, Valerie DaggettAbstract:Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of Chymotrypsin Inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.
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upper limit of the time scale for diffusion and chain collapse in Chymotrypsin Inhibitor 2
Nature Structural & Molecular Biology, 1999Co-Authors: Andreas G Ladurner, Alan R FershtAbstract:The rates of folding of wild–type Chymotrypsin Inhibitor 2 (CI2) (t 1/2 = 12 ms) and of faster (t 1/2 = 2 ms) and slower (t 1/2 = 350 ms) folding mutants are accelerated in parallel by increasing concentrations of sucrose, despite the increases in viscosity. At a viscosity 26 times that of water, the folding rate constant of wild–type CI2 is accelerated four–fold (t 1/2 = 2.7 ms). From this, we can estimate that the diffusional chain collapse in CI2 occurs in less than 100 μs in water, and is not rate–determining in folding.
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folding of circular and permuted Chymotrypsin Inhibitor 2 retention of the folding nucleus
Biochemistry, 1998Co-Authors: Daniel E Otzen, Alan R FershtAbstract:The 64-residue Chymotrypsin Inhibitor 2 (CI2) folds by a two-state nucleation−condensation mechanism, whereby secondary and tertiary structure coalesce concomitantly in the transition state around Ala 16 in the helical N-cap. Permutation of the SH3-domain of α-spectrin apparently shifts its folding nucleus to another region of the protein, suggesting that a protein's transition state may be altered by altering the protein's connectivity. We have characterized the structure of the transition state of a circular and a permuted version of CI2 by a protein engineering study encompassing 11 mutations. Circular CI2 was obtained by the introduction of cysteines at residues 3 and 63 and linking them by disulfide bond formation. Subsequent cyanogen−bromide cleavage of the scissile bond, Met 40−Glu 41, yielded permuted CI2. Circular and permuted CI2 also fold according to a two-state mechanism. Permutation does not affect the folding rate constant, but circularization increases it 7-fold. The transition states of c...
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real time nmr studies on folding of mutants of barnase and Chymotrypsin Inhibitor 2
FEBS Letters, 1998Co-Authors: Thomas R Killick, Stefan M V Freund, Alan R FershtAbstract:The folding and unfolding of proteins is generally assumed to be so co-operative that the overall process may be followed by a single probe, such as tryptophan fluorescence. Folding kinetics of three mutants of barnase and Chymotrypsin Inhibitor 2 (CI2) were studied by real-time NMR. Rate constants for changes in individual residues during the unfolding or refolding of the mutants studied by real-time NMR are all within experimental error of the overall process of folding/unfolding measured by stopped-flow measurements of tryptophan fluorescence. Folding of these mutants is thus highly co-operative. Changes in the tryptophan fluorescence give accurate measurements of the protein folding process.
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strain in the folding nucleus of Chymotrypsin Inhibitor 2
Folding and Design, 1997Co-Authors: Andreas G Ladurner, Laura S Itzhaki, Alan R FershtAbstract:Background: Chymotrypsin Inhibitor 2 (CI2) is a member of the class of fast-folding small proteins, which is very suitable for testing theories of folding. CI2 folds around a diffuse extended nucleus consisting of the single α helix and a set of hydrophobic residues. In particular, Ala16 has been predicted and independently found to interact with Leu49 and Ile57, hydrophobic residues that are highly conserved among homologues. We have characterised in detail the interactions between these residues in the folding nucleus of the protein by using double-mutant cycles. Results: Surprisingly, we find that there is some destabilising strain in the transition state for folding of the wild-type protein between Ala16 and Ile57. Further, we find that the strain is larger in the native state of the protein. This is shown directly in the unfolding kinetics, which clearly show a release of strain. The net result of this is that the presence of both residues speeds up folding. Ala16 and Leu49 interact favourably in the transition state, but have no net interaction energy in the native state. Conclusions: Part of the folding nucleus of the protein fits together more snugly in the transition state than it does in the native state. Interactions between some of the closely packed residues in the folding nucleus of CI2 may perhaps be optimised for the rate of folding and not for stability.
T B Ng - One of the best experts on this subject based on the ideXlab platform.
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a dimeric high molecular weight Chymotrypsin Inhibitor with antitumor and hiv 1 reverse transcriptase Inhibitory activities from seeds of acacia confusa
Phytomedicine, 2010Co-Authors: T B NgAbstract:Abstract A dimeric 70-kDa Chymotrypsin Inhibitor with substantial N-terminal sequence homology to serine protease Inhibitors was isolated from Acacia confusa seeds. The Chymotrypsin Inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The Chymotrypsin Inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its Chymotrypsin Inhibitory activity was stable from pH 3 to 10 and from 0 to 50 °C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC 50 of 10.7±4.2 μM. It inhibited HIV-1 reverse transcriptase with an IC 50 of 8±1.5 μM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the Chymotrypsin Inhibitor included dimeric nature, a high molecular mass, lack of trypsin Inhibitory activity, highly potent HIV-1 reverse transcriptase Inhibitory activity, specific antitumor activity and relatively high pH-stability.
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A dimeric high-molecular-weight Chymotrypsin Inhibitor with antitumor and HIV-1 reverse transcriptase Inhibitory activities from seeds of Acacia confusa.
Phytomedicine : international journal of phytotherapy and phytopharmacology, 2009Co-Authors: T B NgAbstract:A dimeric 70-kDa Chymotrypsin Inhibitor with substantial N-terminal sequence homology to serine protease Inhibitors was isolated from Acacia confusa seeds. The Chymotrypsin Inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The Chymotrypsin Inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its Chymotrypsin Inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the Chymotrypsin Inhibitor included dimeric nature, a high molecular mass, lack of trypsin Inhibitory activity, highly potent HIV-1 reverse transcriptase Inhibitory activity, specific antitumor activity and relatively high pH-stability.
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a bowman birk type trypsin Chymotrypsin Inhibitor from broad beans
Biochemical and Biophysical Research Communications, 2001Co-Authors: X Y Ye, T B NgAbstract:An isolation procedure comprising affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and fast protein liquid chromatography on Mono S was used to purify a peptide from broad beans which manifested antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea. The peptide demonstrated a molecular mass of 7.5 kDa. N-terminal sequence analysis disclosed the identity of the antifungal peptide to be a trypsin-Chymotrypsin Inhibitor. The trypsin-Chymotrypsin Inhibitor also exerted an Inhibitory action on Chymotrypsin activity and HIV-1 reverse transcriptase activity. Proliferation of murine splenocytes was stimulated in the presence of the trypsin-Chymotrypsin Inhibitor. This report constitutes the first observation of antifungal activity of a leguminous peptidic protease Inhibitor.
Longsen Chang - One of the best experts on this subject based on the ideXlab platform.
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nmr solution structure of a Chymotrypsin Inhibitor from the taiwan cobra naja naja atra
Molecules, 2013Co-Authors: Teppei Ikeya, Peter Guntert, Longsen ChangAbstract:The Taiwan cobra (Naja naja atra) Chymotrypsin Inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type Inhibitors such as bovine pancreatic trypsin Inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another Chymotrypsin Inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 A for the backbone atoms and 0.73 A for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 A between NACI and BPTI and 1.27 A between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14–Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14–Cys38 disulfide bond.
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Mutagenesis studies on the N-terminus and Thr54 of Naja naja atra (Taiwan cobra) Chymotrypsin Inhibitor
Protein Journal, 2006Co-Authors: Ching-ping Chen, Yunching Cheng, Longsen ChangAbstract:Ala-screening mutagenesis studies on Arg1, Pro2, Arg3, Phe4 and Thr54 of Naja naja atra (Taiwan cobra) Chymotrypsin Inhibitor showed that Inhibitory potency and gross conformation of the mutants were not significantly different from those of wild-type Inhibitor. Nevertheless, the R1A mutant had an appreciable decrease in the structural stability underlying thermal unfolding and urea-induced denaturation. Alternatively, deleting the first three residues at the N-terminus caused a reduction in structural stability as well as Inhibitory potency. In sharp contrast to wild-type and other mutated Inhibitors, R1A mutant and truncated mutant completely lost their Inhibitory activity when the Inhibitors were incubated with Chymotrypsin for periods of up to 3 h. The loss of activity correlated with chymotryptic cleavage of Inhibitors as evidenced by SDA-PAGE. Taken together, these results reflect that the globally structural rigidity of N. naja atra Chymotrypsin Inhibitor functionally affects the sustainable period in inhibiting Chymotrypsin activity, and that the intact N-terminus might contribute to this event.
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taiwan cobra Chymotrypsin Inhibitor cloning functional expression and gene organization
Biochimica et Biophysica Acta, 2005Co-Authors: Yunching Cheng, Longsen ChangAbstract:Abstract A cDNA encoding Chymotrypsin Inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the Inhibitory constant (Ki) against Chymotrypsin. The genomic DNA encoding the Chymotrypsin Inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the β-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra Chymotrypsin Inhibitor and β-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease Inhibitors and neurotoxic homologs may have originated from a common ancestor.
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purification and characterization of a Chymotrypsin Inhibitor from the venom of ophiophagus hannah king cobra
Biochemical and Biophysical Research Communications, 2001Co-Authors: Longsen Chang, Charling Chung, Hsienbin HuangAbstract:Abstract A Chymotrypsin Inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease Inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other Chymotrypsin Inhibitors, a P1 Asn in the novel Inhibitor may cause a weak binding (Ki = 3.52 μM) with Chymotrypsin. Phylogenetic analysis suggests that the functional variations of the Chymotrypsin Inhibitor and other Kunitz-type Inhibitors probably distinguish from dendrotoxins by accelerated evolution.
Valerie Daggett - One of the best experts on this subject based on the ideXlab platform.
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sensitivity of the folding unfolding transition state ensemble of Chymotrypsin Inhibitor 2 to changes in temperature and solvent
Protein Science, 2005Co-Authors: Valerie DaggettAbstract:To better characterize the transition state for folding/unfolding and its sensitivity to environmental changes, we have run multiple molecular dynamics simulations of Chymotrypsin Inhibitor 2 (CI2) under varying solvent conditions and temperature. The transition state structures agree well with experiment, and are similar under all of the conditions investigated here. Increasing the temperature leads to some movement in the position of the transition state along several reaction coordinates, as measured by changes in properties of the transition state structures. These structural changes are in the direction of a more native-like transition state as denaturation conditions become more severe, as expected for a Hammond effect. These structural changes are not, however, reflected in the global structure as measured by the total number of contacts or the average S-values. These results suggest that the small changes in average Φ-values with temperature seen by experiment may be due to an increase in the sensitivity of the transition state to mutation rather than a change in the average structure of the transition state. A simple analysis of the rates of unfolding indicates that the free energy barrier to unfolding decreases with increasing temperature, but even in our very high temperature simulations there is a small free energy barrier.
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protein folding from a highly disordered denatured state the folding pathway of Chymotrypsin Inhibitor 2 at atomic resolution
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Steven L Kazmirski, Stefan M V Freund, Alan R Fersht, Kambo Wong, Valerie DaggettAbstract:Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of Chymotrypsin Inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.
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identification and characterization of the unfolding transition state of Chymotrypsin Inhibitor 2 by molecular dynamics simulations
Journal of Molecular Biology, 1996Co-Authors: Aijun Li, Valerie DaggettAbstract:Abstract 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≥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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characterization of the transition state of protein unfolding by use of molecular dynamics Chymotrypsin Inhibitor 2
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Aijun Li, Valerie DaggettAbstract:Abstract 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.
Sonia Maria De Freitas - One of the best experts on this subject based on the ideXlab platform.
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purification and ph stability characterization of a Chymotrypsin Inhibitor from schizolobium parahyba seeds
Phytochemistry, 2004Co-Authors: Rozeni C.l. Teles, Elizabeth Maria Talá De Souza, Leonardo A Calderon, Sonia Maria De FreitasAbstract:Abstract Schizolobium parahyba Chymotrypsin Inhibitor (SPCI) was completely purified as a single polypeptide chain with two disulfide bonds, by TCA precipitation and ion exchange chromatography. This purification method is faster and more efficient than that previously reported: SPCI is stable from pH 2 to 12 at 25 °C, and is highly specific for Chymotrypsin at pH 7–12. It weakly inhibits elastase and has no significant Inhibitory effect against trypsin and α-amylase. SPCI is a thermostable protein and resists thermolysin digestion up to 70 °C.
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Topographical Analysis of Schizolobium Parahyba Chymotrypsin Inhibitor (Spci) by Atomic Force Microscopy
Protein and Peptide Letters, 2002Co-Authors: José Roberto S. A. Leite, Luciano P. Silva, Clarice Cunha Taveira, Rozeni C.l. Teles, Sonia Maria De Freitas, Ricardo B. AzevedoAbstract:Atomic Force Microscopy (AFM) has been a useful tool for molecular surface analysis and to estimate topographical properties of proteins. Here we report a topographical study of a Chymotrypsin Inhibitor from Schizolobium parahyba seeds (SPCI) by AFM. The underlying structure of SPCI oligomers has been resolved in nanometer order resolution. SPCI oligomerize in hexagonal, ellipsoid, comet, pyramidal, and “Z“ shaped. The hexagonal was the most observed oligomer shape.
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analysis of the black eyed pea trypsin and Chymotrypsin Inhibitor α Chymotrypsin complex
FEBS Letters, 1997Co-Authors: Sonia Maria De Freitas, Luciane V Mello, Maria Cristina Mattar Da Silva, G Vriend, Goran Neshich, Manuel Mateus VenturaAbstract:The black-eyed pea trypsin and Chymotrypsin Inhibitor (BTCI) is a member of the Bowman-Birk protease Inhibitor (BBI) family. The three-dimensional model of the BTCI-Chymotrypsin complex was built based on the homology to Bowman-Birk Inhibitors with known structures. An extensive theoretical and experimental study of these known structures has been performed. The model confirms the ideas about Bowman-Birk Inhibitor structure-function relations and agrees well with our experimental data (circular dichroism, IR and light scattering). The electrostatic potentials at the enzyme-Inhibitor contact surface reveal a pattern of complementary electrostatic potentials from which mutations can be inferred that could give these Inhibitors an altered specificity.