Characterization of inhibitory effects of NH 2 OH and its N-methyl derivatives on the O 2 -evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca 2+ and Cl - ) are essential for oxidation of H 2 O to O 2 by Photosystem II. The Mn reductants NH 2 OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H 2 O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O 2 evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl - , F - and SO 4 2- ) is able to slow NH 2 OH and CH 3 NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl - is capable of attenuating CH 3 NHOH and (CH 3 ) 2 NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl - fails to protect against NH 2 OH inhibition in salt-washed membranes. These results indicate that the attack by NH 2 OH and its N-methyl derivatives on Mn occurs at different sites in the O 2 -evolving complex. The small reductant NH 2 OH acts at a Cl - -insensitive site whereas the inhibitions by CH 3 NHOH and (CH 3 ) 2 NOH involve a site that is Cl - sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl - sensitivity of their binding to Mn in the O 2 -evolving complex.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43537/1/11120_2004_Article_BF00046773.pd

Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models

Phage display has demonstrated the utility of cyclic peptides as general protein ligands but cannot access proteins inside eukaryotic cells. Expanding a new chemical genetics tool, we describe the first expressed library of head-to-tail cyclic peptides in yeast (Saccharomyces cerevisiae). We applied the library to selections in a yeast model of alpha-synuclein toxicity that recapitulates much of the cellular pathology of Parkinson's disease. From a pool of 5 million transformants, we isolated two related cyclic peptide constructs that specifically reduced the toxicity of human alpha-synuclein. These expressed cyclic peptide constructs also prevented dopaminergic neuron loss in an established Caenorhabditis elegans Parkinson's model. This work highlights the speed and efficiency of using libraries of expressed cyclic peptides for forward chemical genetics in cellular models of human disease