NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin.
The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000577:Amides; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism; D012995:Solubility
D000199:Actins; D000577:Amides; D009842:Oligopeptides; D011122:Polyphosphates; D000255:Adenosine Triphosphate