Contribution of Single Tryptophan Residues to the Fluorescence and Stability of Ribonuclease Sa

Abstract

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.

Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins

“…The unusual positive CD band at 235 nm observed for folded RNase Sa has been discussed previously. 54 The CD spectrum of thermally denatured RNase Sa differs substantially from that of the urea denatured protein, as has been observed in studies of other proteins. 55 The CD spectrum of the protein in 8.5M urea is consistent with the presence of substantial amounts of polyproline II (PPII) conformation.…”

Charge–charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

“…To monitor the folding and unfolding of WT RNase Sa and the charge‐reversal variant, D17R, by fluorescence spectroscopy, a Trp residue was added in place of Tyr 81. The crystal structure of Y81W showed that the Trp is 87% buried, and the variant is 0.4 kcal/mol less stable than WT (Alston et al 2004). For Y81W RNase Sa, there is a 2.5‐fold increase in fluorescence intensity at 319 nm when the protein folds (Alston et al 2004).…”

Solution Structure of Calmodulin Bound to the Binding Domain of the HIV-1 Matrix Protein

“…Typically, changes observed in tryptophan fluorescence signal are induced when the indole ring is involved in binding or upon occurrence of conformational changes in the protein, which involve a change in the environment of the tryptophan residue (e.g. protein unfolding) (83). Indeed, CaM-induced MA unfolding leads to exposure of both Trp 16 and Trp 36 rings and is likely to contribute to the observed changes in the fluorescence signal.…”