Native-like β-structure in a Trifluoroethanol-induced Partially Folded State of the All-β-sheet Protein Tendamistat

Schönbrunner, Nancy; Wey, Josef; Engels, Joachim W.; Georg, Holger; Kiefhaber, Thomas

doi:10.1006/jmbi.1996.0412

Cited by 109 publications

(71 citation statements)

References 49 publications

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“…This study and our previous ones (Fontana et al, 1995;Polverino de Laureto et al, 1995a, 1995b show that proteolysis of proteins in their TFE-state by the TFE-resistant thermolysin is very selective and that it can be used as a procedure to produce nicked proteins or, in general, rather large protein fragments for protein chemistry and/or biophysical studies. Finally, the results reported here provide some insights into the partly folded TFE-state of proteins utilizing a proteolytic probe, and complement those obtained by other investigators using spectroscopic techniques (Buck et al, 1993(Buck et al, , 1995Alexandrescu et al, 1994;Shiraki et al, 1994;Schonbrunner et al, 1996). This study represents a continuation of our efforts to demonstrate that proteolytic enzymes can be used as reliable probes of protein structure and dynamics (Vita et al, 1985;Fontana et al, 1989Fontana et al, , 1993Signor et al, 1990;Polverino de Laureto et al, 1994a, 1994b, 1995a, 1995bVindigni et al, 1994).…”

Section: Discussionsupporting

confidence: 81%

“…The conformational and dynamic features of RNase in relatively high concentrations of TFE are not yet known in such detail as for other proteins, e.g., lysozyme (Buck et al, 1993(Buck et al, , 1995. a-lactalbumin (Alexandrescu et al, 1994), and tendamistat (Schonbrunner et al, 1996). Nevertheless, the results of CD and NMR measurements of RNase dissolved in aqueous TFE allow us to propose a possible model of the TFE state of RNase.…”

Section: Limited Proteolysismentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 81%

Section: Limited Proteolysismentioning

confidence: 99%

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

et al. 1997

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“…Interleukin-1b (Varley et al, 1993) and rat intestinal fatty acid binding protein (Ropson et al, 1990) also belong to this class of b-sheet proteins. In addition, the presence of only native long range interactions in folding intermediates of protein S-NTD resembles the TFE-induced intermediate state of the b-sheet protein tendamistat (Schonbrunner et al, 1997). Non-hierarchical folding pathways have been proposed for b-lactoglobulin (Shiraki et al, 1995) and cellular retinoic acid binding protein (Liu et al, 1994).…”

Section: Comparison With Studies On Other B B B-sheet Proteinsmentioning

confidence: 99%

Equilibrium folding intermediates of a greek key β-barrel protein

Bagby

Inouye

et al. 1998

Journal of Molecular Biology

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Protein S is a calcium-binding protein comprising two Greek key b-barrel domains. We have used NMR and optical spectroscopies to show that, in the absence of calcium, the N-terminal domain of protein S forms two equilibrium folding intermediates that are in slow exchange. The intermediates arise from differential calcium-dependent folding of subdomains which are not contiguous along the polypeptide chain. The structures of these intermediates are incompatible with several previously proposed folding mechanisms for Greek key b-barrel domains. We propose a different mechanism that involves multiple nucleation sites for folding and sequential acquisition of native long-range interactions.

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“…Destabilization of the tertiary structure of a protein by alcohol (Herskovits et al, 1970;Fink and Painter, 1987) and stabilization of secondary structure (Nelson and Kallenbach, 1989;Lehrman et al, 1990) has often led to the induction of partially folded intermediates (Bhattacharjya and Balaram, 1997;Cort and Anderson, 1997;Gast et al, 1999) in proteins, sometimes referred to as the O-state (Yang and Mayo, 1993) and is "molten-globule" like (Ptitsyn, 1987;Kuwajima, 1989). Thus, alcohol is now commonly used to induce partially folded states in proteins (Fan et al, 1993;Alexandrescu et al, 1994;Schonbrunner et al, 1996). However, the behaviors of plant cysteine proteases of the papain superfamily and their folding aspects in alcohol have not been reported extensively, and needs consideration.…”

Section: Introductionmentioning

confidence: 99%

Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

Kundu¹,

Sundd²,

Jagannadham³

2002

BMB Reports

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The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly α-helical to β-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the α-helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

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Native-like β-structure in a Trifluoroethanol-induced Partially Folded State of the All-β-sheet Protein Tendamistat

Cited by 109 publications

References 49 publications

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Equilibrium folding intermediates of a greek key β-barrel protein

Alcohol and Temperature Induced Conformational Transitions in Ervatamin B: Sequential Unfolding of Domains

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