Ca2+S1-Induced Movement of Troponin T on Reconstituted Skeletal Muscle Thin Filaments Observed by Fluorescence Energy Transfer Spectroscopy

Abstract

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).

The Rates of Switching Movement of Troponin T between Three States of Skeletal Muscle Thin Filaments Determined by Fluorescence Resonance Energy Transfer

“…From steady-state fluorescence measurements, we reported previously that a large difference in the transfer efficiency between these donor and acceptor probes was observed between the presence and absence of Ca 2ϩ (15). The change in the transfer efficiency upon Ca 2ϩ binding corresponds to the increase in the distance by 3.6 Å for AEDANS-T60-Tn and 7 Å for AEDANS-T250-Tn (15). This Ca 2ϩ -dependent conformational change was measured by changing the free Ca 2ϩ concentrations.…”

“…␣␣Tm was extracted from rabbit hearts as reported previously (10). Single cysteine TnT25k mutants (E60C and S250C) were prepared as reported previously (15). Actin was labeled at Cys-374 with DABMI as mentioned previously (14).…”

“…The Tn complex labeled at Cys-133 of TnI with AEDANS (AEDANS-I133-Tn) was prepared as previously reported (14). TnT25k mutants were labeled with IAEDANS, and reconstitution of ternary Tn complexes with AEDANSlabeled TnT25k mutants (AEDANS-T60-Tn and AEDANS-T250-Tn) was carried out as reported previously (15). Cys-60 is located at the second residue from the N terminus, and Cys-250 is located at the seventeenth residue from the C terminus of the 25-kDa fragment of TnT.…”

“…Cys-60 is located at the second residue from the N terminus, and Cys-250 is located at the seventeenth residue from the C terminus of the 25-kDa fragment of TnT. ATPase measurements indicated that Tn complexes reconstituted with the AEDANS-labeled TnT25k mutants retained the regulatory activity as well as the wild-type Tn (15). For stopped-flow measurements, equimolar phalloidin to actin was added to stabilize the actin filament.…”

“…FRET between probes attached to TnI and actin showed a significant extent of Ca 2ϩ -induced and also S1-induced movement of TnI on the reconstituted thin filament (10 -14). Not only TnI but also TnT changes positions on the actin filament corresponding to three states of the thin filament (15). The movements of TnI and TnT between three states are impaired on the thin filament reconstituted with a functionally deficient mutant Tm (D234Tm) (14,16).…”