Quantitative Determination of Helical Propensities from Trifluoroethanol Titration Curves

Jasanoff, Alan; Fersht, Alan R.

doi:10.1021/bi00174a020

Cited by 362 publications

(431 citation statements)

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“…Low concentrations of TFE can induce an a-helical conformation in short peptides which were characterized by CD [21]. T284-294 and T284-295 reveal a rather low tendency to adopt an a-helical structure even at high concentrations of TFE.…”

Section: Discussionmentioning

confidence: 99%

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

Jung¹,

Windhaber²,

Palm³

et al. 1995

FEBS Letters

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Section: Discussionmentioning

confidence: 99%

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

Jung¹,

Windhaber²,

Palm³

et al. 1995

FEBS Letters

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“…These include synthetic peptides of de novo design (some recent reviews: Chakrabartty & Baldwin, 1995; Scholtz & Baldwin, 1995;Kallenbach et al, 1996), and protein fragments (e.g., Goodman & Kim, 1989;Waltho et al, 1993;Myers et al, 1996). Structured peptides can serve as models for investigating the contribution of local interactions to protein folding and stability (Muiioz & Serrano, 1996;Myers et al, 1996).2,2,2-TrifluoroethanoI (TFE) is a co-solvent that has been shown to stabilize helical structure in peptides (Goodman et al, 1963;Nelson & Kallenbach, 1986;Jasanoff & Fersht, 1994; CammersGoodwin et al, 1996; Luo & Baldwin, 1997 and references therein). Peptide fragments of protein helices often show little helix formation in water, and TFE is frequently used to induce helix formation (Sonnischen et al, 1992;Hamada et al, 1995;Kemmink & Creighton, 1995;Yang et al, 1995;Bolin et al, 1996).…”

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confidence: 99%

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T₁

1998

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Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the a-helix of ribonuclease T I . There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T I helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine-based peptides and the ribonuclease T I peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context. Keywords: a-helix; helical propensity; peptide; ribonuclease TI ; TFE; trifluoroethanol The tendency of some short peptides to form helical structure in solution has led to their use as models for protein folding and stability. These include synthetic peptides of de novo design (some recent reviews: Chakrabartty & Baldwin, 1995; Scholtz & Baldwin, 1995;Kallenbach et al., 1996), and protein fragments (e.g., Goodman & Kim, 1989;Waltho et al., 1993;Myers et al., 1996). Structured peptides can serve as models for investigating the contribution of local interactions to protein folding and stability (Muiioz & Serrano, 1996;Myers et al., 1996).2,2,2-TrifluoroethanoI (TFE) is a co-solvent that has been shown to stabilize helical structure in peptides (Goodman et al., 1963;Nelson & Kallenbach, 1986;Jasanoff & Fersht, 1994; CammersGoodwin et al., 1996; Luo & Baldwin, 1997 and references therein). Peptide fragments of protein helices often show little helix formation in water, and TFE is frequently used to induce helix formation (Sonnischen et al., 1992;Hamada et al., 1995;Kemmink & Creighton, 1995;Yang et al., 1995;Bolin et al., 1996). Although its use is widespread, the mechanism of TFE stabilization of secondary structure is not clear. One important question is whether helix propensities of the amino acids measured in water still apply in aqueous TFE solutions. This question has been addressed recently in alanine-based peptides by Baldwin and co-workers (Rohl et al., 1996;Luo & Baldwin, 1997). For reasons that are not completely clear, in water the helix propensities are twice as large in alaninebased peptides than in peptides based on natural protein sequences. Because TFE is used so widely, it is important to compare its effect on helix formation in natural peptide sequences.Recently, we have developed a variant of the small protein ribonuclease TI (RNase T I ) and a peptide corresponding to the single a-helix of RNase T I as a model system. We have used the T I peptide/protein system previously to compare directly helix propensities in peptides and proteins (Myers et al., 1997a(Myers et al., , 1...

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“…The precise physical mechanism of a-helix induction by TFE is not fully understood and is the subject of intense and recent investigations (Thomas & Dill, 1993;Jasanoff & Fersht, 1994;Shiraki et al, 1994;Cammers-Goodwin et al, 1996). Current views are that TFE favors the formation of the intramolecular hydrogen bonds of the a-helical conformation rather than hydrogen bonds to solvent.…”

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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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Quantitative Determination of Helical Propensities from Trifluoroethanol Titration Curves

Cited by 362 publications

References 32 publications

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T₁

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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Quantitative Determination of Helical Propensities from Trifluoroethanol Titration Curves

Cited by 362 publications

References 32 publications

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

NMR and circular dichroism studies of synthetic peptides derived from the third intracellular loop of the β‐adrenoceptor

Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

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Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T₁