title: Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins.
abstract: The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.
authors.affiliation: Muscle Research Group, Boston Biomedical Research Institute, Boston, MA, 02114, USA. yinluo@bbfru,irg
authors.forename: Y
authors.initials: Y
authors.lastname: Luo
authors.forename: J L
authors.initials: JL
authors.lastname: Wu
authors.forename: B
authors.initials: B
authors.lastname: Li
authors.forename: K
authors.initials: K
authors.lastname: Langsetmo
authors.forename: J
authors.initials: J
authors.lastname: Gergely
authors.forename: T
authors.initials: T
authors.lastname: Tao
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001577:Benzophenones / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism; D008301:Maleimides / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D018482:Muscle, Skeletal; D009154:Mutation / Q000235:genetics; D017319:Photosensitizing Agents / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020107:Troponin T / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D000199:Actins; D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D008840:Microfilament Proteins; D017319:Photosensitizing Agents; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; C046109:benzophenone-4-maleimide; D003545:Cysteine; D002118:Calcium