Toward Protein Structure In Situ: Comparison of Two Bifunctional Rhodamine Adducts of Troponin C

Abstract

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.

Conformation of the Troponin Core Complex in the Thin Filaments of Skeletal Muscle during Relaxation and Active Contraction

“…The distribution of BR dipole orientations in the muscle fiber was also expressed using a Gaussian orientation model with mean angle θ g between the probe dipole and the thin filament axis and angular dispersion σ g . 20 In relaxed muscle, θ g was 56.9 ± 0.3°(mean ± SD, n = 5 fibers) and σ g was 19.4 ± 2.0°( Table 1). The corresponding values for actively contracting muscle were 48.1 ± 1.0°and 27.2 ± 0.9°, respectively.…”

Orientation of the N-Terminal Lobe of the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

“…The polarization of the excitation and emitted beams were set either parallel or perpendicular to the fiber axis, allowing determination of the three-order parameters, hP 2d i, hP 2 i, and hP 4 i that describe the dipole orientation in the fiber (24). The orientation distribution of individual BR-RLCs with respect to the fiber axis was determined by one-dimensional maximum entropy (ME) analysis (25). The orientation of the N-lobe of the RLC was calculated by combining the data from the A, B, C, and D helix smRLC probes using two-dimensional ME analysis (15,26).…”

“…The angular changes represented by these differences were estimated by calculating the smoothest distribution of angles between the probe dipole and the fiber axis consistent with each set of hP 2 i and hP 4 i values. This one-dimensional maximum entropy (ME) distribution (25) was then represented by its mean (q ME ) and standard deviation (s ME ). q ME and s ME were similar, within a few degrees, for the BR-skRLC-D and BR-smRLC-D data in all conditions studied ( Table 2).…”

Single molecule optical measurements of orientation and rotations of biological macromolecules

“…Probes with two separate reactive groups that bind to the macromolecule can achieve markedly reduced local motions [48], and fixed local orientation relative to the structure. Bifunctional rhodamine (BR) contains two iodoacetamide groups that flank the chromophore and can crosslink two cysteines which are 7 or 8 residues (~1.1 nm) apart on an α -helix.…”