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)

2,2,2-Trifluoroethanol (TFE)-induced nonnative alpha-helical structure in peptides and proteins has been extensively studied with circular dichroism (CD) spectroscopy. However, to date, complementary information from infrared (IR) spectroscopy has not been reported. Using both IR and CD spectroscopy, we demonstrate here that the TFE-induced nonnative alpha-helical structure in two beta-sheet-predominant proteins, beta-lactoglobulin and alpha-chymotrypsin, is unstable in comparison with those found in the alpha-helix-predominant proteins myoglobin and cytochrome c under identical conditions. IR spectra showed that, immediately after dissolution of the beta-sheet proteins in 50% (v/v) TFE, a strong amide I band component appears at 1654 cm-1 in H2O and at 1650 cm-1 in D2O, which is ascribed to alpha-helical structure. However, the intensities of the alpha-helical bands decrease as a function of time, concomitant with the appearance of two new band components near 1620 and 1695 cm-1 in H2O and 1612 and 1684 cm-1 in D2O, a typical IR spectral pattern for an intermolecular beta-sheet aggregate. Clear gels begin to develop within 30 min. No similar spectral changes were observed for the alpha-helical proteins. CD spectra suggested initially that the TFE-induced alpha-helix was retained in the gelled state. However, further analysis of the spectra, and Gaussian function modeling with basic spectra, indicated that the apparent alpha-helix signal was actually due to a combination of signals from intermolecular beta-sheet and residual alpha-helix. These results indicate that the TFE-induced nonnative alpha-helix structure in predominantly beta-sheet proteins is unstable and readily converts to an intermolecular beta-sheet aggregate.
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PMID:Intermolecular beta-sheet results from trifluoroethanol-induced nonnative alpha-helical structure in beta-sheet predominant proteins: infrared and circular dichroism spectroscopic study. 967 38

A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed.
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PMID:Identification of kinetically hot residues in proteins. 986 46

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.
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PMID:Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes. 1044 60

Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.
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PMID:Effects of molecular crowding by saccharides on alpha-chymotrypsin dimerization. 1196 57

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.
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PMID:Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects. 1197 78

The effects of protein entrapment on the structure and phase behavior of periodically curved lipid mesostructures have been examined by synchrotron small-angle X-ray diffraction and FT-IR spectroscopy. The study was directed towards a better understanding of the effect of confinement in a lipid environment on the stability and unfolding behavior of alpha-chymotrypsin, and, vice versa, the effect of the entrapped protein on the lipid's mesophase structure and temperature- and pressure-dependent phase behavior. We compare the interaction of protein molecules of two different sizes (cytochrome c, 12.4 kDa, and alpha-chymotrypsin, 25.8 kDa) with the cubic Ia3d phase of monoolein (MO), which forms spontaneously in water. The cubic structure changes significantly when cyt c is incorporated: above a protein concentration of 0.2 wt.%, the interaction between the positively charged protein and the lipid headgroups leads to an increase in interfacial curvature which promotes the formation of a new micellar cubic phase, presumably of crystallographic space group P4(3)32, which the lipid system does not form on its own. The larger alpha-chymotrypsin leads to a different scenario. On the basis of an examination of the calculated geometric parameters and water volume fractions, it is concluded that the alpha-chymotrypsin molecules cannot be located exclusively in the water channels of the cubic Ia3d or P4(3)32 phases, but rather form new, less ordered (presumably cubic Pn3m) structures. The new structure disappears above the unfolding temperature of chymotrypsin and exhibits a pressure stability, which-- in contrast to cyt c in MO-- decreases with increasing chymotrypsin concentration in the system. While the secondary structure of cyt c remains unaffected in the confining lipid environment, the structure of alpha-chymotrypsin gets destabilized slightly, and the protein tends to aggregate even at relatively low concentrations.
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PMID:Incorporation of alpha-chymotrypsin into the 3D channels of bicontinuous cubic lipid mesophases. 1633 Feb 64

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.
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PMID:Modification by homocysteine thiolactone affects redox status of cytochrome C. 1747 17

Gold nanoparticles (NPs) functionalized with L-amino acid-terminated monolayers provide an effective platform for the recognition of protein surfaces. Isothermal titration calorimetry (ITC) was used to quantify the binding thermodynamics of these functional NPs with alpha-chymotrypsin (ChT), histone, and cytochrome c (CytC). The enthalpy and entropy changes for the complex formation depend upon the nanoparticle structure and the surface characteristics of the proteins, e.g., distributions of charged and hydrophobic residues on the surface. Enthalpy-entropy compensation studies on these NP-protein systems indicate an excellent linear correlation between DeltaH and TDeltaS with a slope (alpha) of 1.07 and an intercept (TDeltaS0) of 35.2 kJ mol(-1). This behavior is closer to those of native protein-protein systems (alpha = 0.92 and TDeltaS0 = 41.1 kJ mol(-1)) than other protein-ligand and synthetic host-guest systems.
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PMID:Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study. 1767 56

Amino acid and dipeptide-functionalized gold nanoparticles (NPs) possessing L/D-leucine and/or L/D-phenylalanine residues have been constructed in order to target the surfaces of alpha-chymotrypsin (ChT) and cytochrome c (CytC). Isothermal titration calorimetry (ITC) was conducted to evaluate the binding thermodynamics and selectivity of these NP-protein interactions. The chirality of the NP end-groups substantially affects the resultant complex stability, with up to 20-fold differences seen between particles of identical hydrophobicity, demonstrating that structural information from the ligands can be used to control protein recognition.
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PMID:Isomeric control of protein recognition with amino acid- and dipeptide-functionalized gold nanoparticles. 1797 62

The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.
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PMID:Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts. 1804 9


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