Design of an adenosine analogue that selectively improves the affinity of a mutant U1A protein for RNA.
The RNA recognition motif (RRM), one of the most common RNA binding domains, contains three highly conserved aromatic amino acids that participate in stacking interactions with RNA bases. We have investigated the contribution of these highly conserved aromatic amino acids to the affinity of the complex formed between the N-terminal RRM of the U1A protein and stem loop 2 of U1 snRNA. Previously, we found that substitution of one of these conserved aromatic amino acids, Phe56, with Ala resulted in a large destabilization of the complex. Here, we have modified A6, the base in stem loop 2 RNA that stacks with Phe56, to compensate for a portion of the destabilization caused by the Phe56Ala mutation. We have designed two modified adenosines, A-3CPh and A-4CPh, in which a phenyl group is linked to the adenosine such that it may replace the phenyl group that is eliminated by the Phe56Ala mutation in the complex. We have found that incorporation of A-3CPh into stem loop 2 RNA stabilizes the complex formed with Phe56Ala by 0.6 kcal/mol, while incorporation of A-4CPh into stem loop 2 RNA stabilizes this complex by 1.8 kcal/mol. Either base modification destabilizes the wild-type complex by 0.8-0.9 kcal/mol. Experiments with other U1A mutant proteins suggest that the stabilization of the complex between the Phe56Ala U1A protein and stem loop 2 RNA is due to a specific interaction between the Phe56Ala U1A protein and A6-4CPh stem loop 2 RNA.
Zhao
Ying
Y
Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
Baranger
Anne M
AM
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D019943:Amino Acid Substitution; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D012313:RNA; D000241:Adenosine