Characterization of folding dynamics and accelerated electron transfer in proteins

Abstract: The research presented in this thesis is divided in two: The first part is a study of the intracellular protein human copper zinc superoxide dismutase which has been associated with the fatale disease Amyotrophic lateral Selerosis (ALS). ALS is a progressive neurodegenerative disease of motor neurons, and in the cases where the disease is inheriled (fALS), roughly 15% is caused by mutations in human copper·zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 causes ALS is still unknown, but it is believed that mutant CuZnSOD proteins misfold followed by aggregation into high molecular species that ultimately lead to the death of motor neurons. In our study we have investigated the dynamical and structural differences between the wt of CuZnSOD and the ALS-associated variant G93A. 1H-15N·HSQC NMR spectroscopy was used to analyze hydrogen deuterium exchange at the backbone amide groups. The study showed that the mutation selectively destabilizes the remote metal binding region. This suggests that the metal binding region might be involved in intermolecular protein-protein interactions which may constitule the eariy stages in formation of aggregates. In another of our studies the monomer to dimer equilibrium as weil as the catalytic activity is investigated upon protein denaturation using CdmCI. The study showed persistent dimer interactions and high catalytic activity at GdmCllevels where the holo-protein according to CD measurements is fully unfolded.The second part of this thesis focuses on electron transfer (ET) in proteins. ET processes are fundamental in many biological processes such as respiration and photosynthesis. Biological ET reactions occur rapidly over large molecular distances (>20Å) and only minor structural changes around the active site arises during the ET event. Previous work on Rumodified P. aeruginosa azurin have demonstrated that optimized electron coupling through a θ-strand yield a distance decay constant of 1.1 Å-1. ET in biological systems often requires sub-millisecond charge transport over long molecular distances (>20A). This is not possible via direct tunneling through a θ-strand. It is believed that ET rates can be greatly enhanced by multistep tunneling ("hopping") in which redox-active amino acid side-chains act as intermediate donors or acceptors. In our work, rapid spectroscopic methods are used to investigate hopping through an intermediate tryptophan or tyrosine radical. Cu(l) to Re(II) electron tunneling in Re(H107) azurin occurs in the microsecond range, which is much faster than for previously studied Cu(l) to Ru(III) tunneling in Ru(H107). At first it was believed to be multistep tunneling, but further investigation disproved this. A more likely explanalion is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radieal), followed by electron tunneling from Cu(l) to the hole in the imidazole Iigand. In the other investigated system Cu(l) oxidation by a photoexcited Re(l) diimine at position 124 on a ß-strand (His124-Gly123-Trp122-Met121) takes place in nanoseconds, which is remarkable and more than two orders of magnitude faster than for single-step ET at a 19 Adonoracceptor distance. This system is the first model system to show that an intervening tryptophan residue between donor and acceptor can accelerate the ET rate. Therefore this work was published in Science.