Conformational Studies of Equilibrium Structures in Fragments of Horse Heart Cytochrome <i>c</i>

Toniolo, Claudio; Fontana, Angelo; Scoffone, Ernesto

doi:10.1111/j.1432-1033.1975.tb09812.x

Cited by 54 publications

(81 citation statements)

References 42 publications

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“…In the presence of TFE, cytochrome c is digested to relatively few and rather large protein fragments. After 24-48 h in 50% (v/v) TFE, the protein is completely digested to two major fragments, which show electrophoretic mobilities similar to those of the BrCN-fragments 1-65 and 66-104 of cytochrome c [21] (Fig. 1B).…”

Section: Proteolysis Of Cytochrome Cmentioning

confidence: 94%

Limited proteolysis of cytochrome c in trifluoroethanol

et al. 1995

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Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25°C, 24 h) at the Gly56-Ile 57 peptide bond of the 104-residue chain of the protein.Additional, but anyway minor, fragmentation at the Gly4S-Phe 46 and MetS°-Ile s~ peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE-resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low a-helix propensity. globular proteins are characterized by enhanced chain flexibility, since a correlation exists between sites of proteolysis and sites of high segmental mobility, these last determined crystallographically [13].Here we show that horse heart cytochrome c in the presence of aqueous TFE is cleaved by the TFE-resistant proteolytic enzyme thermolysin [18] to few fragments only, whereas in aqueous solution and in the absence of alcohol many small peptides are formed. The preferential proteolysis was interpreted on the basis of the conformational properties of cytochrome c dissolved in aqueous TFE, as deduced from circular dichroism (CD) and fluorescence emission measurements. Materials and methods

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Section: Proteolysis Of Cytochrome Cmentioning

confidence: 94%

Limited proteolysis of cytochrome c in trifluoroethanol

et al. 1995

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“…The last type of hydrogen bond forms a five-membered ring, but only limited experimental evidence for such structures exist so far. [25][26][27] In the XRAYLJLM simulation, using the reduced mass Lennard-Jones liquid, one observes a breaking up of the P-pleated sheet, and the occupancies of 2-Abu-5-Val and 7-Ala-11-MeVal are reduced by almost 50%. The y-turn formed by 2-Ala and 11-MeVal is still present; however, the 7-Ala-5-Val hydrogen bond is not present any- more.…”

Section: Comparison Of the Simulations Starting From The (Mdc) Structmentioning

confidence: 99%

On the dependence of molecular conformation on the type of solvent environment: A molecular dynamics study of cyclosporin A

et al. 1990

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The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.

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“…Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al, 1975; …”

Section: Introductionmentioning

confidence: 99%

“…Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al, 1975; …”

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Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42"C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Thl) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

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Conformational Studies of Equilibrium Structures in Fragments of Horse Heart Cytochrome c

Cited by 54 publications

References 42 publications

Limited proteolysis of cytochrome c in trifluoroethanol

Limited proteolysis of cytochrome c in trifluoroethanol

On the dependence of molecular conformation on the type of solvent environment: A molecular dynamics study of cyclosporin A

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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