title: Solution properties of tetramethylrhodamine-modified G-actin.
abstract: In the recently solved structure of TMR-modified ADP-G-actin, the nucleotide cleft is in a closed state conformation, and the D-loop contains an alpha-helix (L. R. Otterbein, P. Graceffa, and R. Dominguez, 2001, Science, 293:708-711). Subsequently, questions were raised regarding the possible role of the TMR label on Cys(374) in determining these aspects of G-actin structure. We show here that the susceptibility of D-loop on G-actin to subtilisin cleavage, and ATP/ADP-dependent changes in this cleavage, are not affected by TMR-labeling of actin. The TMR modification inhibits nucleotide exchange, but has no effect on DNase I binding and the fast phase of tryptic digestion of actin. These results show an absence of allosteric effects of TMR on subdomain 2, while confirming ATP/ADP-dependent changes in D-loop structure. In conjunction with similar results obtained on actin-gelsolin segment 1 complex, this works reveals the limitations of solution methods in probing the putative open and closed nucleotide cleft states of G-actin.
mesh_terms: D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D016477:Artifacts; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D018260:Gelsolin / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008422:Materials Testing / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012235:Rhodamines / Q000737:chemistry*; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D020860:Subtilisin / Q000737:chemistry*
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D018260:Gelsolin; D012235:Rhodamines; D012996:Solutions; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I; D020860:Subtilisin; D008274:Magnesium; D002118:Calcium