Binding of calcium ions to an avian flight muscle troponin T.
Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its N-terminal hypervariable region, but the functions of these isoforms are not completely understood. Here for the first time, we discovered that a chicken fast TnT isoform with a unique Tx motif (HEEAH)(n) binds calcium. The metal binding behavior of this TnT isoform was first investigated using terbium as a calcium analogue due to its more readily detectable fluorescence variation upon TnT binding. Both intact TnT and TnT N-terminal fragment (TnT N47) bound terbium with high affinity indicating that the N-terminal sequence was the site of binding. Since terbium often substitutes at calcium-binding sites, radioactive calcium was tested and found to bind both intact TnT and TnT N47. Fluorescence measurements using the calcium-sensitive fluorescent dye, calcium green 5N, confirmed that calcium bound to the tertiary complex of TnT and the tropomyosin dimer with a fast on-rate (10(6)-10(7) M(-1) s(-1)) as detected in stopped-flow analysis. Consistent with these observations, computational predictions suggest that TnT N47 might fold into an elongated structure with at least one high-affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. These results suggest that TnT may play a role in modulating the calcium-mediated regulatory process of striated muscle contraction.
Zhang
Zhiling
Z
Department of Biological Sciences, Division of Biochemistry, University of North Texas, Denton, Texas 76203-5220, USA.
Jin
Jian-Ping
JP
Root
Douglas D
DD
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent; D002645:Chickens; D003198:Computer Simulation; D005426:Flight, Animal / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D013725:Terbium / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D020033:Protein Isoforms; D020107:Troponin T; D013725:Terbium; D002118:Calcium