Hydrophobic core manipulations in ribonuclease T1.
Differential scanning calorimetry, urea denaturation, and X-ray crystallography were combined to study the structural and energetic consequences of refilling an engineered cavity in the hydrophobic core of RNase T1 with CH(3), SH, and OH groups. Three valines that cluster together in the major hydrophobic core of T1 were each replaced with Ala, Ser, Thr, and Cys. Compared to the wild-type protein, all these mutants reduce the thermodynamic stability of the enzyme considerably. The relative order of stability at all three positions is as follows: Val > Ala approximately equal to Thr > Ser. The effect of introducing a sulfhydryl group is more variable. Surprisingly, a Val --> Cys mutation in a hydrophobic environment can be as or even more destabilizing than a Val --> Ser mutation. Furthermore, our results reveal that the penalty for introducing an OH group into a hydrophobic cavity is roughly the same as the gain obtained from filling the cavity with a CH(3) group. The inverse equivalence of the behavior of hydroxyl and methyl groups seems to be crucial for the unique three-dimensional structure of the proteins. The importance of negative design elements in this context is highlighted.
De Vos
S
S
Laboratorium voor Ultrastructuur, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium. stedevos@vub.ac.be
Backmann
J
J
Prévost
M
M
Steyaert
J
J
Loris
R
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014508:Urea
D011994:Recombinant Proteins; D014508:Urea; D006163:Ribonuclease T1