Mobility and orientation of spin probes attached to nucleotides incorporated into actin.
Each actin molecule contains a nucleotide, tightly bound in a deep cleft that divides the molecule. To probe conformational changes within this region of the molecule, we have incorporated two spin label analogues of ATP into actin. In both analogs the spin label was attached to the 6 position on the adenine ring, either directly (6nSLATP) or via a longer thioacetamido linker (6sSLATP). Electron paramagnetic resonance spectra of randomly oriented actin filaments showed that both the probes possessed considerable rotational mobility relative to the protein surface. The 6nSLADP has two degrees of rotational mobility that can be approximately modeled by rapid diffusion within cones with half angles of 30 +/- 1 degrees and 42 +/- 1.5 degrees. The 6sSLADP displayed one degree of rotational mobility approximated by rapid motion within a cone with a half-angle of 38 +/- 1 degrees. The rotational mobility of the probes is determined by the protein structure surrounding them, and changes in this structure should alter the mobility. The mobility of the probes was unchanged by addition of 20 mM Pi, which forms an ADP-Pi complex. However, binding of myosin heads (S1) shifted the population of 6nSLADP toward the more highly restricted cone, while binding of DNase-I shifted it toward the less restricted cone. We conclude that this region of actin is unchanged by binding of phosphate, while the binding of S1 or DNase-I produces only a modest shift in conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Naber
N
N
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0524.
Cooke
R
R
D000199:Actins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D003850:Deoxyribonuclease I / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005782:Gels; D066298:In Vitro Techniques; D009038:Motion; D015879:Myosin Subfragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013113:Spin Labels
D000199:Actins; D005782:Gels; D015879:Myosin Subfragments; D010710:Phosphates; D013113:Spin Labels; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I