Sequence analysis of an artificial family of RNA-binding peptides.
Diverse peptide sequences recognizing the lambda boxB RNA hairpin were previously isolated from a library encoding the 22-residue lambda N peptide with random amino acids at positions 13-22 using mRNA display. We have statistically analyzed amino acid distributions in 65 unique sequences from rounds 11 and 12 of this selection and evaluated the resulting structural and functional predictions by alanine-scanning mutagenesis and circular dichroism spectrometry. This artificial sequence family has a consensus structure that continues the bent alpha helix of lambda N up to position 17 when bound to lambda boxB. A charge pair (E(14)R(15)) and hydrophobic patch (A(21)L(22) or V(21)L(22)) have important functional roles in this context. Notably, amino acid covariance reveals six specific pairs of random region positions with >95% significant linkage and strong overall helical (i+1, i+3, and i+4) couplings. The covariance analysis suggests that (1) the sequence context of every residue in each insert has been optimized, (2) selected sequences are local optima on a rugged fitness landscape, and (3) it is possible to detect more subtle structural features with artificial protein sequence families than natural homologs. Our results provide a framework for investigating the structures of in vitro selected proteins by functional minimization, reselection, and covariance analysis.
Barrick
Jeffrey E
JE
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Roberts
Richard W
RW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D016384:Consensus Sequence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D020539:Sequence Analysis, Protein
D000596:Amino Acids; D010455:Peptides; D016601:RNA-Binding Proteins; D012313:RNA