Molecular dynamics studied by NMR relaxation experiments. Characterization of functional dynamics in the FK506 binding protein FKBP12

Sammanfattning: The presented thesis work is concerned with the study of molecular dynamics using liquid nuclear magnetic resonance (NMR) spectroscopy. Spin relaxation dispersion experiments in the rotating frame (R1ρ) are employed to provide a detailed, atomic-resolution view of protein dynamics. Comprehensive investigations of 15N backbone and 13C methyl side-chain conformational exchange in the FK506 binding protein, FKBP12, were carried out. The human enzyme FKBP12 catalyzes the cis-trans isomerization of peptidyl-prolyl linkages in proteins, thereby increasing the rate of protein folding. Recombinant protein production with 13C/2H-enrichment yielded 13CHD2 isotopomers and allowed for development of novel R1ρ experiments aimed to characterize the methyl-bearing side-chains. The joint analysis of backbone and side-chain dynamics revealed a collective conformational exchange process with a correlation time of 121 ± 26 microseconds in the free enzyme. The locations of the exchanging nuclei showed close correspondence with residues previously shown to be important for the catalytic activity, which emphasize the connection between protein function and dynamics. The FKBP12 isomerase activity is strongly inhibited by the small organic molecule FK506. In addition, the bound conformation of FK506 exhibit distinct structural transition state analog characteristics. Conformational exchange dynamics studies of the FKBP12 – FK506 complex demonstrated a striking rigidity of the backbone atoms, in line with the established theory that the transition state is more constrained than the free state. Conformational exchange dynamics found for peripheral methyl groups is suggested to play a role in the subsequent binding of the FKBP12 – FK506 complex to other target molecules. A specific isotope labeling strategy using [1-13C]-glucose was shown to produce isolated 13C nuclei in the side-chains of Phe/Tyr/Trp/His. Proteins with this characteristic labeling provide nuclei amenable to R1ρ experiments, which were previously excluded from analysis due to complications arising from uniform carbon labeling. The method thus extended the collection of dynamic probes to include the frequently occurring and often functionally important aromatic side-chains. R1ρ studies of the C-terminal part of a mutant Calmodulin protein revealed microsecond time scale dynamics contributions to Tyr80Cδ, confirming previous reports of conformational exchange present for the protein backbone nuclei. Detection of conformational exchange in isotropic media requires a chemical shift difference between the exchanging states. However, such chemical shift difference can be replaced by differential residual dipolar couplings (RDC) in the exchanging states for molecules dissolved in partially aligning media. The RDC-induced contribution to the exchange-free transverse relaxation rate can be suppressed using relaxation dispersion experiments, thus revealing details of the conformational exchange characteristics. The theory was described, and the method was demonstrated using the model molecule N,N-dimethyltrichloroacetamide.