Hydrophobic core manipulations in ribonuclease T1.


Abstract

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. Study holds ProTherm entries: 11637, 11638, 11639, 11640, 11641, 11642, 11643, 11644, 11645, 11646, 11647, 11648, 11649, 11650, 11651, 11652, 11653, 11654, 11655, 11656, 11657, 11658, 11659, 11660, 11661, 11662, 11663, 11664, 11665, 11666, 11667, 11668, 11669 Extra Details: cavity; hydrophobic core; sulfhydryl group; hydroxyl; methyl groups

Submission Details

ID: oTb25wZJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
De Vos S;Backmann J;Prévost M;Steyaert J;Loris R,Biochemistry (2001) Hydrophobic core manipulations in ribonuclease T1. PMID:11513591
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1B2M 1998-11-27T00:00:00+0000 2.0 THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS.
1BIR 1996-01-04T00:00:00+0000 1.8 RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP
1BU4 1998-09-11T00:00:00+0000 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
1BVI 1998-09-15T00:00:00+0000 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
1CH0 1999-03-30T00:00:00+0000 2.3 RNASE T1 VARIANT WITH ALTERED GUANINE BINDING SEGMENT
1DET 1996-02-20T00:00:00+0000 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
1FYS 2000-10-03T00:00:00+0000 2.0 Ribonuclease T1 V16C mutant
1FZU 2000-10-04T00:00:00+0000 1.8 RNAse T1 V78A mutant
1G02 2000-10-05T00:00:00+0000 1.86 Ribonuclease T1 V16S mutant
1GSP 1997-11-28T00:00:00+0000 2.2 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS, 1 DAY

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR