Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin.
To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.
Chen
Weizu
W
Department of Biochemistry, University of Iowa, Carver College of Medicine, Iowa City, Iowa 52242, USA.
Wen
Kuo-Kuang
KK
Sens
Ashley E
AE
Rubenstein
Peter A
PA
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism; D011485:Protein Binding; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
D000199:Actins; D011721:Pyrenes; D014335:Tropomyosin; D014336:Troponin; C030984:pyrene; D002118:Calcium