Stereospecific reaction of muscle fiber proteins with the 5' or 6' isomer of (iodoacetamido)tetramethylrhodamine

Abstract

The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5'-IATR there are two probes per myosin sulfhydryl 1 (SH1). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SH1 when S1 is labeled with 5'-IATR (5'-IATR-S1) and that there are one covalent and two noncovalent probes per S1 when S1 is labeled with 6'-IATR (6'-IATR-S1). The 5'- and 6'-IATR probes have similar fluorescent lifetimes when bound to S1, but quenching studies with potassium iodide show that 5'-IATR-S1 has a single class of strongly bound chromophores while 6'-IATR-S1 has two or more classes of chromophores. It is possible that 5'-IATR labels SH1 as a dimer. The polarization anisotropies of 5'- and 6'-IATR-S1 indicate that 5'-IATR is immobilized, while 6'-IATR is moving independently, on the surface of S1. The emission spectrum from 5'-IATR-S1 is unaffected by the addition of MgATP, while 6'-IATR-S1 shows a spectral shift and total intensity change. When labeling muscle fibers, 5'-IATR labels myosin SH1 and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515]. 6'-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SH1 indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5'-IATR with its binding pocket on the surface of S1.

Probes bound to myosin Cys-707 rotate during length transients in contraction

“…The distribution of probes in Fig. 1 agrees with earlier work indicating that 77-86% of the total probe intensity is from the myosin heavy chain, 8-14% from actin, 4-7% from myosin light chain 1, and 2-5% from ␣-actinin (14,27,29). Fig.…”

“…Rabbit psoas muscle fibers were obtained as described (21) and kept in a relaxing solution containing 50% (vol͞vol) glycerol at Ϫ15°C for up to several weeks. We prepared 5ЈIATR-labeled glycerinated muscle fibers also as described (27). We reinvestigated the location of 5ЈIATR within the components of modified fibers by extracting the proteins from modified fiber bundles and separating them by molecular weight by using SDS͞PAGE (28).…”

“…The SH1 labeling specificity was evaluated by comparison of the Ca 2ϩ -and K ϩ -EDTA ATPase activities of myosin extracted from labeled fibers (27,28,30,31). All of the 5ЈIATR on the heavy chain resides on SH1 (28).…”

Structural Determination of Lipid-bound ApoA-I Using Fluorescence Resonance Energy Transfer

“…The choice of the fluorescein/rhodamine probe pair was made because their distance-dependent transfer efficiency falls intermediate between the two different models. Thus for this acceptor/donor probe pair, one would predict approximately 50% transfer efficiency (R 0 ) between 45 and 50Å (35)(36)(37)(38)(39). Therefore, when the donor and acceptor probes are configured in the belt orientation, nearly 100% transfer efficiency would be observed, whereas, if the donor and acceptor probes are configured in the picket fence, nearly 0% transfer efficiency would be observed.…”

“…rHDL did not change these results. This indicates that rhodamine dimer formation, although not of particularly high affinity (39), drives the preferential pairing of acceptor apoA-I monomers during the formation of rHDL. The conclusions that can be drawn from analysis of rhodamine self-quenching in DMPC rHDL are first, that the residues at position 132 in the two monomers of rHDL are sufficiently close together to allow formation of non-covalent rhodamine dimers.…”