pH-dependent structural transition in rabbit skeletal troponin C.

Fluorescence energy transfer measurements were carried out between landmarks on wheat germ calmodulin to measure the interdomain distance. Tb3+ ions bound at the four Ca2+-binding sites were used as energy donors, and an organic chromophore, [4-(dimethylamino)-phenyl-4'-azophenyl]maleimide, attached to the single cysteine residue at position 27, was used as the acceptor. At pH's near neutrality all bound Tb3+ ions emit luminescence with shortened lifetimes as a result of energy transfer to the acceptor; at pH 5, however, part of the metal emission becomes unquenched. When the protein is subjected to limited digestion with trypsin in the presence of Ca2+, resulting in the formation of two fragments, each corresponding to half of the molecule, the decay of Tb3+ emission is no longer pH sensitive. These results suggest that, like rabbit skeletal troponin C [Wang, C.-L. A., Zhan, Q., Tao, T., & Gergely, J. (1987) J. Biol. Chem. 257, 8372-8375], wheat germ calmodulin exists in a relatively compact conformation at neutral pH's, but becomes more elongated at pH 5.

Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

pH-dependent conformational changes of wheat germ calmodulin

Wang¹

1989

“…Both of these observations are consistent with the fact that troponin C is larger than calmodulin, having an additional 14 residues. Uncertainties concerning aggregation effects have, to date, prevented a complete analysis of troponin C data collected at pH 7.4 in the presence of Ca2+, or at pH 5.5 where fluorescent energy transfer measurements indicate a large conformational rearrangement (Wang et al, 1986;Wang & Cheung, 1985). It would be extremely valuable to be able to identify solution conditions for which either calmodulin or troponin C (or both) was stabilized in the crystal conformation in a monodisperse solution, but efforts to identify such conditions by use of buffers related to those used for crystallization have failed due to aggregation problems.…”

Section: Discussionmentioning

confidence: 99%

“…In view of the potential flexibility in the interconnecting helix, it is important to examine the effects of a solution environment as well as changes in pH on the overall structure of these proteins. Fluorescence energy transfer measurements (Wang et al, 1986; Wang & Cheung, 1985) on skeletal troponin C suggest that it undergoes a large conformational rearrangement involving a change in the distance between the two calcium 0006-2960/88/0427-0909501.50/0 © 1988 American Chemical Society binding domains between pH 5.0 and pH 6.7. Small-angle scattering can be useful in exploring changes in the overall shape of molecules in solution under a variety of conditions.…”

mentioning

confidence: 99%

Comparison of the crystal and solution structures of calmodulin and troponin C

Heidorn

Trewhella²

1988

338

284

X-ray solution scattering data from skeletal muscle troponin C and from calmodulin have been measured. Modeling studies based on the crystal structure coordinates for these proteins show discrepancies between the solution data and the crystal structure that indicate that if the size and shape of the globular domains are the same in solution as in the crystal, the distances between them must be smaller by several angstroms. Bringing the globular domains closer together requires structural changes in the interconnecting helix that joins them.

“…The second label, DABMI, was conjugated to DANZ-TnC as described by Wang et al (1987), except that a larger excess of DABMI was used. The degree of labeling was measured by using concentrations of protein determined by a comparison of the ellipticities at 220 nm of the conjugate and native TnC for identical conditions and by absorbance: DAB, = 24 800…”

Section: Methodsmentioning

confidence: 99%

Interactions of troponin I and its inhibitory fragment (residues 104-115) with troponin C and calmodulin

Lan¹,

Albaugh²,

Steiner³

1989

Fluorescent probes have been used to study the interaction of troponin I and its inhibitory peptide TnIp with troponin C, calmodulin, and the proteolytic fragments of calmodulin. The probes used included the noncovalently bound ligand TNS and the covalently attached labels dansyl and AEDANS. The fluorescence intensity of TNS bound to troponin C, calmodulin, or the calmodulin fragments was greatly enhanced by the presence of TnIp. This effect was used to estimate the corresponding binding constants. It was found that TnIp is bound by the C-terminal half-molecule of calmodulin, TR2C, with an affinity comparable to that of intact calmodulin or troponin C, while the binding affinity of the N-terminal half-molecule, TR1C, was an order of magnitude less, suggesting that the TnIp-containing portion of troponin I combines with the C-terminal half of calmodulin or troponin C. The fluorescence properties of an AEDANS group linked to Cys-98 of troponin C were modified by interaction with troponin I or TnIp. The fluorescence properties of the same group linked to Cys-27 of wheat germ calmodulin were affected by TnI, but not TnIp. TnI had a small effect upon the fluorescence of a dansyl group linked to Met-25 of troponin C. TnIp also inhibited the tryptic hydrolysis of the midpoint of the central connecting strand of calmodulin and troponin C.(ABSTRACT TRUNCATED AT 250 WORDS)