Structural Characterization of Conformational Substates in Cardiac Troponin C Using Nuclear Magnetic Resonance and Lanthanide Induced Pseudocontact Shifts

Every protein exists in numerous different conformational substates with similar free energies. For some proteins, e.g. myoglobin, it has been shown, that conformations higher in energy than the native state, so called low lying excited states, can determine protein function. Excited states are usually populated to only a few percent. This makes it impossible to obtain their 3D-structures with current methods. Nuelear Magnetic Resonance (NMR) is the only method available to detect motions associated with transitions between conformational substates in a residue specific manner and allows the determination of populations, rate constants and chemical shifts of the involved conformations as well, even if they are populated to less than one percent. Although these data can describe the excited state rather well, they are not sufficient to calculate a 3D- structure. The aim of the project is to develop a general method, that allows the structure determination of low lying excited states in proteins using NMR. As a model system cardiac troponin C, a calcium binding protein regulating muscle contraction, has been chosen, as there is good evidence that it exists in an inactive as well as an active, but low populated conformation. By binding lanthanide instead of calcium ions to troponin C, a distance dependent modulation of the measured chemical shifts will be introduced, which can be used to calculate the structure of the excited state of the protein. The structures of proteins in exited, active states obtained using this new method will be of great importance for the understanding of protein function.