title: Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments.
abstract: In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-¿[(dimethylamino)phenyl]azo¿phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.
authors.affiliation: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore 21201, USA.
authors.forename: T
authors.initials: T
authors.lastname: Kobayashi
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Z
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
authors.forename: J H
authors.initials: JH
authors.lastname: Collins
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates / Q000737:chemistry; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism*; D019210:Troponin I / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014335:Tropomyosin; D019210:Troponin I; D020106:Acrylamide; C048876:1,5-AEDANS; D003545:Cysteine; D002118:Calcium