Structure and stability of the P93G variant of ribonuclease A

Abstract

Abstract:The peptide bonds preceding Pro 93 and Pro I14 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 w.This structure is essentially identical to that of the wild-type protein, except for the 9 1-94 p-turn containing the substitution. In the wild-type protein, the p-turn is of type Vla. In the P93G variant, this turn is of type I1 with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of AAGm, which reports on the stability lost in the variants, is 1 S-fold greater for the PI 14G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type I1 turn, which has a preference for a glycine residue in its i + 2 position.

Back to the future: Ribonuclease A

“…This is in sharp contrast to the previous study where the introduction of the two nipecotic acid six-membered rings at position 113 and 114 only increased the DT m by (1.2 6 0.3)8C. RNase has been a focus of protein folding studies by the Scheraga group which have shown multiple folding pathways depending on the redox reagent (DTT versus glutathione) 119,120 or the location of the four disulfide bonds (RNase versus onconase). 119,121 It would be intriguing to analyze the pathways associated with folding of the Pro114dmP mutant.…”

“…19 The discussion centered on generating a construct similar to RNase S, but which explored the role of the C-terminal segment of RNase contributing His119 to the catalytic site. Ultimately, the semisynthetic ribonuclease, RNase-(1-118)- (111)(112)(113)(114)(115)(116)(117)(118)(119)(120)(121)(122)(123)(124), was evolved to study the role of the C-terminal segment in recognition and catalysis. [45][46][47][48] A three-component system, combining equimolar amounts of RNase fragments 1-20, 21-118, and 111-124, retained 30% of the enzymatic activity of intact RNase.…”

“…The midpoint of the thermal transition for dmP114 RNase was increased, DT m ¼ (2.8 6 0.3)8C or DDG m ¼ 4.6 6 0.4 kJ mol À1 . For comparison, replacing Pro114 with glycine or L-alanine causes a very significant decrease in conformational stability (DT m ¼ À108C, DDG m ¼ 3.6 kJ mol À1 ), 119 suggesting an important role of this reverse turn and its associated b-hairpin in stabilizing RNase.…”

“…RNase has been a focus of protein folding studies by the Scheraga group which have shown multiple folding pathways depending on the redox reagent (DTT versus glutathione) 119,120 or the location of the four disulfide bonds (RNase versus onconase). 119,121 It would be intriguing to analyze the pathways associated with folding of the Pro114dmP mutant. Other proline analogs impact cis-trans amide isomerization and, therefore, type-VI reverse-turn formation.…”

Protein prosthesis: β‐peptides as reverse‐turn surrogates

“…This finding is not rote, as replacing Asn113 and Pro114 with R-Nip-RNip (a diastereomer of R-Nip-S-Nip) eliminates enzymatic activity. Similarly, the transition temperatures for the P114A and P114G variants are decreased by $10 C. [23][24][25] Thus, b 2 -hAla-b 3 -hAla and R-Nip-S-Nip are more than passive linkers 26 -both support native protein structure. This conclusion is supported by the indistinguishable CD spectra of both RNase A and b 2 b 3 hAla RNase A (Supporting Information Fig.…”