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Tryptophan Fluorescence Reveals the Presence of Long-Range Interactions in the Denatured State of Ribonuclease Sa

Roy W. Alston ^*, Mauricio Lasagna , Gerald R. Grimsley ^*, J. Martin Scholtz ^* , Gregory D. Reinhart  and C. Nick Pace ^* 

^* Department of Molecular and Cellular Medicine, Texas A&M University Health Science Center; and Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843

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

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, _max, does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at _max, I_F, differs significantly for the five Trp containing variants of RNase Sa; 3), the I_F differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I_F differences in the peptides are also present in the proteins; 4) the I_F values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.