Introduction of a donor-acceptor pair by a single protein modification. Förster energy transfer distance measurements from trapped 1,N6-ethenoadenosine diphosphate to chromophoric cross-linking reagents on the critical thiols of myosin subfragment.

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.

Section: Discussionsupporting

confidence: 54%

Calcium dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements

Tao¹,

Gowell²,

Strasburg³

et al. 1989

“…Secondly, the fact that the randomly and the specifically labeled samples gave the same results suggests that the value of κ 2 is the same regardless of the location of the donor and the acceptor, most likely the isotropically averaged value of 2 / 3 . Finally, these values are considerably smaller than the theoretical maximum of 0.4, indicating substantial rapid reorientation of the donor's transition moment via either mixing of electronic states (Haas et al, 1978;Perkins et al, 1984) or rapid probe rotation or both, further justifying our assumption that κ 2 is 2 / 3 under all conditions.…”

Section: Discussionmentioning

confidence: 59%

Troponin T and Ca²⁺ Dependence of the Distance between Cys48 and Cys133 of Troponin I in the Ternary Troponin Complex and Reconstituted Thin Filaments

Luo

Gergely

et al. 1997

Contraction of vertebrate striated muscle is regulated by the interaction of Ca2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibitory subunit, responds to the binding of Ca2+ to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N- and C-terminal domains, respectively, of TnI. A mutant rabbit skeletal TnI, TnI48/133 (C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of approximately 41 A for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 A in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca2+ to TnC. In the reconstituted thin filament, this distance remained to be 50 A in the presence of saturating Ca2+, but increased to approximately 66 A on removing Ca2+ with EGTA in the presence of Mg2+. Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca2+-dependent change in the global conformation of TnI in the Tn complex free in solution, the removal of Ca2+ caused a substantial separation of the N- and C-terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the relaxed state.

“…Fluorescence resonance energy transfer measurements place SH, (and SH2) some 30-40 Á from the active site (Tao & Lamkin, 1981;Perkins et al, 1984), too far for direct interaction with ATP. DNA and protein sequence studies of myosin heavy chains, while indicating general conservation of the sequence between SH, and SH2 (see Table III), show SH, is replaced by threonine in slime mold myosin (Warrick et al, 1986) and by alanine in myosin II from Acanthamoeba (Hammer et al, 1987), demonstrating the nonessential nature of this residue.…”

Section: Discussionmentioning

confidence: 99%

Flexibility of the myosin heavy chain: direct evidence that the region containing SH1 and SH2 can move 10 .ANG. under the influence of nucleotide binding

Huston¹,

Grammer²,

Yount³

1988

Previous experiments demonstrated that two thiols of skeletal myosin subfragment 1 (SF1) could be oxidized to a disulfide bond by treatment with a 2-fold excess of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of MgADP [Wells, J. A., & Yount, R. G. (1980) Biochemistry 19, 1711-1717]. The resulting characteristic changes in the ATPase activities of SF1 and the fact that MgADP was stably trapped at the active site [Wells, J. A., & Yount, R. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970] suggested that the two thiols cross-linked were SH1 (Cys-707) and SH2 (Cys-697) from the myosin heavy chain. To verify this suggestion, SF1, after DTNB treatment as above, was treated with an excess of N-ethylmaleimide to block all accessible thiols. The single protein disulfide produced by DTNB oxidation was reduced with dithioerythritol and the modified SF1 internally cross-linked with equimolar [14C]p-phenylenedimaleimide (pPDM) in the presence of MgADP. After extensive trypsinization, the major 14C-labeled peptide was isolated, characterized, and shown to be Cys-Asn-Gly-Val-Leu-Gly-Ile-Arg-Ile-Cys-Arg, in which the two cysteines were cross-linked by pPDM. This peptide is known to contain SH2 and SH1 in this order and to come from residues 697-708 in the rabbit skeletal myosin heavy chain [Elzinga, M., & Collins, J. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4281-4284; M. Elzinga, personal communication].(ABSTRACT TRUNCATED AT 250 WORDS)