Journal of Molecular Biology
Regular articleTyrosine hydrogen bonds make a large contribution to protein stability1
Introduction
Tyrosine is by far the least soluble amino acid: Tyr (0.05 g/100 g water), Trp (1.33), Leu (2.16), and Phe (2.82).1 (In contrast, the most soluble are: Pro (182), Arg (85), Asp (78), and Gly (25).) In the crystal, each Tyr forms eight out of nine possible hydrogen bonds to six neighboring molecules.2 The phenolic -OH group accepts a hydrogen bond from an amino group and donates a hydrogen bond to a carboxyl oxygen. If hydrogen bonds by the Tyr -OH group to water in solution were just as favorable as hydrogen bonds by the -OH group in the crystal, Tyr would probably not be the least soluble amino acid. Since a Tyr crystal bears some resemblance to the interior of a folded protein,3 these solubilities suggest that hydrogen bonds by tyrosine -OH groups will make a favorable contribution to protein stability. The results described here support this conclusion.
Experimental studies indicate that the intramolecular hydrogen bonds that proteins form on folding contribute significantly to their conformational stability,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 but theoretical studies suggest that they do not.18, 19, 20 It is not clear why. The experimental studies show that when the same mutation, e.g. Tyr⇒Phe, of a hydrogen bonded side-chain is made in different proteins or at different residues in the same protein, a wide range of effects on stability are observed.21 There is an excellent discussion of this subject by Barrick.22 It is important that we understand what factors contribute to this range of effects, and that is one goal of this study.
Ribonuclease Sa (RNase Sa) is a small enzyme (96 amino acids) that is synthesized by Streptomyces aureofaciens and then secreted into the growth medium.23 A crystal structure at 1.2 Å resolution is available,24 as well as a solution structure determined by NMR.25 We have published studies of the conformational stability and thermodynamics of folding of RNase Sa, and two close relatives, RNases Sa2 and Sa3.26 RNase Sa3 has three more amino acid residues at the amino terminus than RNase Sa, and the amino acid sequence is 69% identical. The crystal structures of the proteins are very similar (Figure 1). Both proteins have eight tyrosine residues with seven at equivalent structural positions. This is an exceptionally high content of tyrosine (8%) relative to an average protein (3%). Our first study of hydrogen bonding mutants of RNase Sa focused on the contribution of a conserved Asn residue in the microbial ribonuclease family (RNase Sa Asn39) to the stability.27 Here, we report studies of eight Tyr⇒Phe mutants in RNase Sa and eight Tyr⇒Phe mutants in RNase Sa3. Our goal is to reach a better understanding of the contribution of hydrogen bonding to protein stability.
Section snippets
Thermal denaturation
The pronounced positive circular dichroism (CD) band near 234 nm is lost when RNases Sa and Sa3 unfold, and this was the wavelength used to follow unfolding. In a previous paper, we showed CD spectra and a typical thermal denaturation curve for RNase Sa, and demonstrated that both proteins unfold reversibly by a two-state mechanism.26 Thermal denaturation curves were determined in the same way for each of the RNase Sa and Sa3 mutants. The data were analyzed as described below, and the results
Proteins
Proteins were expressed and purified as described.58 The mutants were prepared as described for RNase Sa.27 Protein concentrations were determined using a molar absorption coefficient at 278 nm of 12,300 M−1 cm−1 for wild-type RNase Sa and 17,550 for wild-type RNase Sa3.58 For the tyrosine to phenylalanine mutants, the molar absorption coefficients at 278 nm were reduced by 1570 M−1 cm−1 based on estimates of the contribution of tyrosine to the absorption coefficients of proteins.59 The protein
Acknowledgements
This research was supported by the National Institutes of Health (GM37039 and GM52483), the Robert A. Welch Foundation (BE-1060 and BE-1281), and the Texas Advanced Research Project Grant 010366-181. C.N.P. is supported by the Tom and Jean McMullin Professorship, and J.M.S. is an American Heart Association Established Investigator. Structural work was supported by Howard Hughes Medical Institute grant number 75195-547601 and the Slovak Academy of Sciences grant number 2/1070/96 (J.S. and L.U).
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Edited by C. R. Matthews