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

Abstract

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.

Identification and Characterization of an 8-kDa Light Chain Associated with Dictyostelium discoideum MyoB, a Class I Myosin

“…The dispersion of backbone H N resonances increased, particularly in the 8.0 -8.5 ppm region, and additional resonances appeared at greater than 8.5 ppm in the 1 H dimension. The extent of the Ca 2ϩ -induced spectral change is comparable with that observed for EF-hand proteins, such as CaM, troponin C, and S100 family members (28,29), and is consistent with a Ca 2ϩ -mediated stabilization or reorientation of the MlcB secondary structure.…”

Solution Structure and Main Chain Dynamics of the Regulatory Domain (Residues 1–91) of Human Cardiac Troponin C

“…In the N-terminal domain the low affinity sites of TnC bind calcium ions when they are released from the sarcoplasmic reticulum of skeletal or cardiac muscle myocytes. The conformational changes brought about by the calcium binding to the N-terminal domain of skeletal troponin C (sTnC) have been followed by NMR (1)(2)(3)(4). The data confirm the early hypothesis of an open and a closed conformation (5)(6)(7).…”

“…In the CPMG spin echo pulse sequence the delay (2⌬) was 900 s (23). The 15 N spin lock strength in T 1 measurements (4,8,15,30,46,61,76,106, and 122 ms) was limited to 1.7 kHz due to experimental restrictions. The crosscorrelation rate () was determined by the relaxation interference measurement (24).…”

Calcium-Induced Structural Transition in the Regulatory Domain of Human Cardiac Troponin C,

“…The secondary structure of apo-and Ca 2+ -cNTnC is similar to that found in the apo-and Ca 2+ -sNTnC solution structures (Gagné et al, 1994) and the N domain of the intact sTnC solution structure (Slupsky et al, 1995b). The N and A-D helices and the antiparallel -sheet span similar lengths over analogous residues in the cardiac protein.…”