Comparison of the calcium- and magnesium-induced structural changes of troponin-C

Levine, Barry A.; Thornton, Janet M.; Fernandes, Rodney A.; Kelly, Camille M.; Mercola, Dan

doi:10.1016/0005-2795(78)90028-4

Cited by 53 publications

(29 citation statements)

References 16 publications

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“…Conditions of slowintermediate exchange are observed for the backbone CaH signals which are notably broadened during the titration, indicative of faster kinetics for the readjustment at the binding loops. The global alteration in structure in the C-terminal half of TnC upon replacement of Mg2+ is similar to that observed in previous spectral studies of the intact protein [22].…”

Section: A P -Trzc and Cation Bindingsupporting

confidence: 85%

Solution conformation of the C‐terminal domain of skeletal troponin C

Drabikowski

Levine

Gergely

et al. 1985

European Journal of Biochemistry

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Proton magnetic resonance spectroscopy has been used to study the cation(Mg2+, Ca2 +)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.Contractile activity of skeletal and cardiac muscle is triggerd by calcium binding to the troponin C (TnC) subunit of the troponin (Tn) complex which is located on the thin filaments [l, 21. The calcium-activated switch is held to involve the transmission of the effects of Ca2+ binding via the other troponin subunits, TnI and TnT, and thus the relay of information through the contractile apparatus. In order to investigate both the overall structural events and the kinetics of the transitions associated with the binding of calcium to the four coordination regions of skeletal TnC (numbered I -IV from the N-terminus), we have undertaken studies by highresolution proton magnetic resonance (PMR) spectroscopy. Our approach to the study of the structural factors that determine the protein's cation affinity and the functional binding response has utilized specific peptide fragments of TnC. PMR studies on peptides comprising different single PMR spectral studies reported here deal with the C-terininal domain of TnC using the tryptic peptide TR2C (residues 89 -159) and the smaller tryptic peptide TR2E (residues 101 -159 [6]) in a variety of cation-bound and free states, at different pH and temperatures. The spectral assignment of PMR signals that derive from different portions of the molecule provides reporter groups for the environment at the binding loops and the flanking helical segments of the C-terminal half of the TnC molecule. MATERIALS AND METHODSTroponin C was isolated from rabbit back and leg muscles by published procedures [6, 91. Troponin C was labeled at Cys-98 using 3-iodoacetamido-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl obtained from Syva (Palo Alto, CA). Tryptic digestion was carried out in the presence of CaC12 or EDTA and the resulting peptides were separated by preparative electrophoresis following the procedure previously described [6,10, 111. The purity of the fragments was checked by electrophoresis on gels containing 6 M urea in the presence and absence of Ca2+ [ll]. Although the TR2E peptide (residues 101 -159) exhibited a single band upon gel electrophoresis, it was further purified on a TnI-Sepharose column using methods described in [ll] in order to remove trace quantities of a smaller peptide, TR2'E (residues 101 -153). The peptides were lyophilized from a 10mM...

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Section: A P -Trzc and Cation Bindingsupporting

confidence: 85%

Solution conformation of the C‐terminal domain of skeletal troponin C

Drabikowski

Levine

Gergely

et al. 1985

European Journal of Biochemistry

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“…They deduced from NMR studies at 270 MHz, that calcium binding to the low affinity sites causes changes in the environment of largely hydrophobic residues. The results were later verified by Levine et al (1978), Evans et al (1980), and Gagne et al (1994).…”

Section: V R Q M K E D a K G K S E E E L A N C I~~a~~d I E E L G E mentioning

confidence: 66%

“…This model suggests that, when calcium binds to sites I and 11, the major conformational transition is a movement of the B/C helix pair away from the A/D helix pair, exposing a patch of hydrophobic residues, providing a binding site for other muscle proteins. This model has strong support from several lines of evidence, including NMR (Levine et al, 1977(Levine et al, , 1978Evans et al, 1980;Gagne et al, 1994) and site-directed mutagenesis (Fujimori et al, 1990;Grabarek et al, 1990;Gusev et al, 1991;Pearlstone et al, 1992aPearlstone et al, , 1992b. There have been several studies of TnC that have focused on the function of the central helix (Reinach & Karlsson, 1988;Xu & Hitchcock-DeGregori, 1988;Gulati et al, 1990Gulati et al, , 1993Dobrowolski et al, 1991;Babu et al, 1993;Wang et al, 1993).…”

Section: ~mentioning

confidence: 83%

Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy

et al. 1995

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The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The ' H , I5N, and I3C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the 414 angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular 4/$ angles in the crystal structure, producing a kink in the B helix, whereas in calciumsaturated TnC, Glu 41 has helical +/$ angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.

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“…For example, calcium binding to troponin occurs simultaneously at calcium-magnesium sites and calcium-specific sites at physiological levels of magnesium (Levine, Thornton, Fernandes, Kelly & Mercola, 1978), but a relatively slow conformational rearrangement following displacement of magnesium by calcium may contribute to the myofibril priming delay if the resulting state of the molecule were an essential forerunner of activation and contraction. Alternatively, if latency relaxation were involved in structural alterations, the myofibril priming delay may result from transitory interference with normal crossbridge action.…”

Section: Discussionmentioning

confidence: 99%

Activation delays in frog twitch muscle fibres.

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1981

The Journal of Physiology

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SUMMARY1. The length dependence of the mechanical latent period and early tension development, uncomplicated by latency relaxation, was determined from responses of single muscle fibres to restimulation at the peak of the isometric twitch at 15 'C.2. The onset of the reactivation response showed little (< 1x X 1O-7 N) or no latency relaxation.3. Reactivation latency was minimal (2-8 msec) and constant at 1P9-21 ,tm sarcomere length and it increased by about 3 msec with sarcomere extension to 3-2,sm.4. Reactivation responses showed two stages of early tension development, an initial phase in which tension acceleration increased, and a phase of maximum responsiveness in which tension acceleration was constant; the transition between the two phases occurred about 4-5 msec after the start of the stimulus at 2-2 jum sarcomere length and was delayed about 4 msec with increase in sarcomere length to 3-2 ,um.5. The square root of the maximum tension acceleration was directly proportional to the degree of overlap of thick and thin filaments in the sarcomere length range 2-3-3-2 ,um.6. It is proposed that the onset of the phase of constant tension acceleration marks the end of the period during which the activator, calcium, is distributed throughout the sarcomere.7. Analysis of early tension transients in relation to myofibril structure showed that length-dependent changes in reactivation latency and time of onset of constant tension acceleration were probably brought about mainly by alteration of the kinetics of distribution of activator within the myofibril and by changes in the diffusion distance between activator-release sites near the end of the sarcomere and the tension-generating sites.8. There was a 2 msec myofibril priming delay in the rise of tension in twitch responses that was not seen in reactivation responses; the possible origin of that delay is discussed in relation to structural changes accompanying activation and to competition between calcium-binding structures.9. The onset of twitch latency relaxation occurred within about 250 ,gsec after the time corresponding to latency of the earliest reactivation responses and appeared to signal the start of a process that took place after the arrival of calcium among the myofilaments. The origin of latency relaxation is discussed.

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Comparison of the calcium- and magnesium-induced structural changes of troponin-C

Cited by 53 publications

References 16 publications

Solution conformation of the C‐terminal domain of skeletal troponin C

Solution conformation of the C‐terminal domain of skeletal troponin C

Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy

Activation delays in frog twitch muscle fibres.

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