Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.
The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single-stranded pyrimidine in its RNA binding site has been examined. Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1-3 kcal/mol under standard binding conditions. Since the Phe, His and Cys 85 proteins formed UV photocrosslinks with iodouracil-containing RNA at the same rate as the wild-type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild-type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.
LeCuyer
K A
KA
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Behlen
L S
LS
Uhlenbeck
O C
OC
D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D016296:Mutagenesis; D010777:Photochemistry; D012313:RNA / Q000737:chemistry*; D015347:RNA Probes; D016601:RNA-Binding Proteins / Q000737:chemistry*; D013816:Thermodynamics
D036022:Capsid Proteins; D015347:RNA Probes; D016601:RNA-Binding Proteins; D012313:RNA