The real-time monitoring of the thermal unfolding of tetramethylrhodamine-labeled actin.
Modification of actin at Cys (374) with tetramethylrhodamine maleimide (TMR-actin) has been used for visualization of actin filaments and to produce high-resolution crystal structures of actin. We show that TMR-actin exhibits a 21% decrease in absorbance at 557 nm upon thermal unfolding, likely due to the movement of TMR to a more hydrophobic environment upon rapid unfolding and protein aggregation. We took advantage of this property to test models of actin protein unfolding. A transition temperature ( T m) of 60.2 +/- 0.2 degrees C for Ca (2+).ATP.TMR-actin was determined using A 557 and agreed with our own determinations employing different techniques and previous work with unlabeled actin. Our data show that the dependence of TMR-actin thermal stability on the bound nucleotide and cations follows a trend of Ca (2+).ATP > Mg (2+).ATP > Ca (2+).ADP > Mg (2+).ADP. The activation energies and frequency factors for the thermal unfolding of TMR-actin determined with two methods were in good agreement with those previously determined for unlabeled actin. We observed a biphasic trend in the T m of TMR-actin with increasing nucleotide concentrations, supporting a two-pathway model for actin protein unfolding where one pathway dominates at different concentrations of nucleotide. Additionally, TMR-actin bound by DNase I or gelsolin segment-1 exhibited elevated transition temperatures.
Perieteanu
Alexandru A
AA
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Sweeting
Braden
B
Dawson
John F
JF
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002645:Chickens; D006358:Hot Temperature; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000737:chemistry
D000199:Actins; D002413:Cations, Divalent; D012235:Rhodamines; D000244:Adenosine Diphosphate; C005358:tetramethylrhodamine; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium