The environment of the high-affinity cation binding site on actin and the separation between cation and ATP sites as revealed by proton NMR and fluorescence spectroscopy.
The lanthanide ions Lu3+ (diamagnetic) and Gd3+ (paramagnetic broadening probe) were used to displace Ca2+ from the high-affinity cation binding site on G-actin. The effects of these higher-affinity ions on the proton nuclear magnetic resonance spectrum of actin were recorded. The aliphatic proton envelope in the Gd-actin sample exhibited a complex array of changes due to the proximity of Gd to several aliphatic residues. No such changes were observed in the diamagnetic Lu-actin control spectrum. By contrast, the aromatic proton envelope remained largely unaffected in both Gd-actin and Lu-actin samples. However, the adenosine moiety on the actin-bound ATP became increasingly mobilized without the triphosphate chain being released from the ATP binding site. Maximum adenosine mobilization occurred with approximately 1 mol of lanthanide ion bound per mol of actin. The absence of changes in the aromatic proton envelope suggests that the high-affinity cation binding site is in a region well removed from the adenosine moiety of bound ATP as well as any aromatic side-chains. The separation of the ATP and cation sites was further explored using the fluorescent ATP analogues FTP and epsilon-ATP. Tb3+ bound to the high-affinity cation site was found to be separated by 16 A from the FTP chromophore bound to the nucleotide binding site on actin. Since this distance is greater than can be accommodated on a model of the Tb-ATP complex, we conclude that the sites are physically separate. This conclusion was further reinforced by experiments involving the quenching of epsilon-ATP fluorescence by Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002412:Cations; D004735:Energy Transfer; D005682:Gadolinium / Q000494:pharmacology; D007700:Kinetics; D008187:Lutetium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods
D000199:Actins; D002412:Cations; D008187:Lutetium; D000255:Adenosine Triphosphate; D005682:Gadolinium