BACKBONE AND METHYL DYNAMICS OF THE REGULATORY DOMAIN OF TROPONIN-C -ANISOTROPIC ROTATIONAL DIFFUSION AND CONTRIBUTION OF CONFORMATIONAL ENTROPY TO CALCIUM AFFINITY

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium io n to each of its two calcium binding loops. In order to characterize t he backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed N-15 NMR relaxation parameters T-1, T-2 and NOE at H-1 NMR frequencies of 500 and 600 MHz. The overall rotational cor relation time of NTnC-apo at 29.6-degrees-C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion anistropy D-2 /D-1 of 1.10. Additionally, the dynamic properties of side-chains havi ng a methyl group were derived from H-2 relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristic s of TnC, two different levels of ''fine tuning'' of the calcium affin ity are presented. Significantly lower backbone order parameters (S-2) , were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational en tropy of sites I and II. This is consistent with different dissociatio n constants previously measured for sites I and II of 16 muM and 1.7 m uM, respectively. In addition to the direct role of binding loop dynam ics, the side-chain methyl group dynamics play an indirect role throug h the energetics of the calcium-induced structural change form aclosed to an open state. Our results show thatn the side-chains which will b e exposed upon calcium binding have reduced motion in the apo state, s uggesting that conformational entropic contributions can be used to of fset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characte ristics is necessary in order to fully understand how TnC and other pr oteins are fine tuned to appropriately carry out their function.