Displacement affinity chromatography of protein phosphatase one (PP1) complexes

Background  

Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.     

Chemo- and regioselective cyclohydrocarbonylation of alpha-keto alkynes catalyzed by a zwitterionic rhodium complex and triphenyl phosphite

alpha-Keto alkynes react with CO and H(2) in the presence of catalytic quantities of the zwitterionic rhodium complex (eta(6)-C(6)H(5)BPh(3))(-)Rh(+)(1,5-COD) and triphenyl phosphite affording either the 2-, 2(3H)-, or 2(5H)-furanones in 61-93% yields. The cyclohydrocarbonylation is readily accomplished using substrates containing alkyl, aryl, vinyl, and alkoxy groups at the acetylenic terminal, as well as a variety of primary, secondary, and tertiary alkyl, aryl, and heteroaryl groups connected to the ketone functionality. Structural and electronic properties present in the starting materials mediate the chemo- and regioselectivity of the reaction.     

Hydrophobic interactions in a cyanobacterial plastocyanin-cytochrome f complex.

The complex of the photosynthetic redox partners plastocyanin and cytochrome f from the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and, to a lesser extent, the "eastern face" of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f. Rigid-body structure calculations using NMR-derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex are independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH results in limited reorganization of the complex interface, while the binding affinity is unaffected. Therefore, protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron-transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.     

Modulation of heme redox potential in the cytochrome c6 family

Cytochrome c6A is a unique dithio-cytochrome of green algae and plants. It has a very similar core structure to that of bacterial and algal cytochromes c6 but is unable to fulfill the same function of transferring electrons from cytochrome f to photosystem I. A key feature is that its heme midpoint potential is more than 200 mV below that of cytochrome c6 despite having His and Met as axial heme-iron ligands. To identify the molecular origins of the difference in potential, the structure of cytochrome c6 from the cyanobacterium Phormidium laminosum has been determined by X-ray crystallography and compared with the known structure of cytochrome c6A. One salient difference of the heme pockets is that a highly conserved Gln (Q51) in cytochrome c6 is replaced by Val (V52) in c6A. Using protein film voltammetry, we found that swapping these residues raised the c6A potential by +109 mV and decreased that of c6 by almost the same extent, -100 mV. X-ray crystallography of the V52Q protein showed that the Gln residue adopts the same configuration relative to the heme as in cytochrome c6 and we propose that this stereochemistry destabilizes the oxidized form of the heme. Consequently, replacement of Gln by Val was probably a key step in the evolution of cytochrome c6A from cytochrome c6, inhibiting reduction by the cytochrome b6f complex and facilitating establishment of a new function.