Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.
We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.
Nadler
S G
SG
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Kapouch
J L
JL
Elliott
J I
JI
Williams
K R
KR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009696:Nucleic Acids / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D011063:Poly A-U / Q000378:metabolism; D039102:Poly(A)-Binding Proteins; D012331:RNA, Fungal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002352:Carrier Proteins; D005656:Fungal Proteins; D009696:Nucleic Acids; D010446:Peptide Fragments; D039102:Poly(A)-Binding Proteins; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D011063:Poly A-U; D011061:Poly A