Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa

Biophysical Journal
Roy W AlstonC Nick Pace

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 Tr...Continue Reading

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Citations

Jan 3, 2014·Journal of Pharmaceutical Sciences·Harshit KhasaC Russell Middaugh
Nov 26, 2015·Computers in Biology and Medicine·Abhigyan Nath, Karthikeyan Subbiah
Jan 5, 2016·Journal of Biomolecular Structure & Dynamics·Lumbini R YadavAshok K Varma
Sep 8, 2007·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Sudha D KamathK K Mahato
Jun 7, 2005·Protein Science : a Publication of the Protein Society·Jared M TrefethenDavid N Brems
Mar 4, 2010·Protein Science : a Publication of the Protein Society·C Nick PaceGerald R Grimsley
Dec 11, 2007·Biophysical Journal·Roy W AlstonC Nick Pace
Feb 17, 2005·Journal of Molecular Biology·Sudharsan SridharanJames C Sacchettini
Jun 14, 2014·Biochemical and Biophysical Research Communications·Lumbini R YadavAshok K Varma
Feb 7, 2014·The Journal of Biological Chemistry·Jiri VlachJamil S Saad
Aug 26, 2019·Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy·Preeti GuptaMd Imtaiyaz Hassan
Oct 21, 2009·Biochemistry·Lidija KovacicIgor Krizaj
Apr 24, 2020·Analytical Chemistry·Dillen AugustijnÅsmund Rinnan

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