This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.104.050377v1 87/6/4036    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alston, R. W. Articles by Pace, C. N. Search for Related Content PubMed PubMed Citation Articles by Alston, R. W. Articles by Pace, C. N.

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

Roy W. Alston ^* , Lubica Urbanikova , Jozef Sevcik , Mauricio Lasagna , Gregory D. Reinhart  , J. Martin Scholtz ^*   and C. Nick Pace ^*  

^* Department of Medical Biochemistry and Genetics, Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas; Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic; and Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas

Correspondence: Address reprint requests to C. Nick Pace, Tel.: 979-845-1788; Fax: 979-847-9481; E-mail: nickpace{at}tamu.edu.

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 Å 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 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.

This article has been cited by other articles:

				R. W. Alston, M. Lasagna, G. R. Grimsley, J. M. Scholtz, G. D. Reinhart, and C. N. Pace Peptide Sequence and Conformation Strongly Influence Tryptophan Fluorescence Biophys. J., March 15, 2008; 94(6): 2280 - 2287. [Abstract] [Full Text] [PDF]

				R. W. Alston, M. Lasagna, G. R. Grimsley, J. M. Scholtz, G. D. Reinhart, and C. N. Pace Tryptophan Fluorescence Reveals the Presence of Long-Range Interactions in the Denatured State of Ribonuclease Sa Biophys. J., March 15, 2008; 94(6): 2288 - 2296. [Abstract] [Full Text] [PDF]

				J. M. Trefethen, C. N. Pace, J. M. Scholtz, and D. N. Brems Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa Protein Sci., July 1, 2005; 14(7): 1934 - 1938. [Abstract] [Full Text] [PDF]