Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease.

Conditions for liposome adsorption and bilayer formation on BSA passivated solid supports.

Planar solid supported lipid membranes that include an intervening bovine serum albumen (BSA) cushion can greatly reduce undesirable interactions between reconstituted membrane proteins and the underlying substrate. These hetero-self-assemblies reduce frictional coupling by shielding reconstituted membrane proteins from the strong surface charge of the underlying substrate, thereby preventing them from strongly sticking to the substrate themselves. The motivation for this work is to describe the conditions necessary for liposome adsorption and bilayer formation on these hetero-self-assemblies. Described here are experiments that show that the state of BSA is critically important to whether a lipid bilayer is formed or intact liposomes are adsorbed to the BSA passivated surface. It is shown that a smooth layer of native BSA will readily promote lipid bilayer formation while BSA that has been denatured either chemically or by heat will not. Atomic force microscopy (AFM) and fluorescence microscopy was used to characterize the surfaces of native, heat denatured, and chemically reduced BSA. The mobility of several zwitterionic and negatively charged lipid combinations has been measured using fluorescence recovery after photobleaching (FRAP). From these measurements diffusion constants and percent recoveries have been determined and tabulated. The effect of high concentrations of beta-mercaptoethanol (ß-ME) on liposome formation as well as bilayer formation was also explored.

Near native binding of a fluorescent serotonin conjugate to serotonin type 3 receptors.

Described is the synthesis of 5-hydroxytryptamine-tetramethylrhodamine (5HT*); an indole nitrogen linked fluorescent conjugate of serotonin. Through a series fluorescence quenching experiments and experiments in the presence of a known competitive antagonist (Granisetron), it was shown that 5HT* specifically binds to purified homo-pentameric type-3 human serotonin receptors (5HT(3A)). The measured dissociation constant and Hill coefficient are K(d) = 83 ± 3 nM and n = 3.1 ± 0.3, respectively which is indicative of multi-ligand binding and cooperativity similar to that of unconjugated serotonin.