Intramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studies.
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ABSTRACT:
SUBMITTER: Genov NC
PROVIDER: S-EPMC1152410 | biostudies-other | 1983 Nov
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the D-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524-35530). To address the implications of a D-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (DeltaT(m), +9 degrees C) which corresponded to the formation of S-OVA, whereas the increment in T(m) for both S164A and S320A was only 4.5 degrees C. Furthermore, the T(m) value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T(m) of mutant R142A after alkaline treatment was 5.8 degrees C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T(m) value was compared before and after alkaline treatment. As expected, the increase in T(m) for the double mutant was only 1.2 degrees C. Taken together, the data suggest that D-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA.
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