Mapping peptide thiol accessibility in membranes using a quaternary ammonium isotope-coded mass tag (ICMT).

The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesized in both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing α-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the headgroup zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the headgroup abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral analysis to the presence of lipids will enable future facile investigation of membrane proteins in a cellular context.

Evaluation of organophosphorus chemicals-degrading enzymes: a comparison of Escherichia coli and human cytosolic aminopeptidase P.

An enzyme capable of hydrolyzing organophosphate compounds is of biological as well as environmental significance. We evaluated the possibility of human cytosolic aminopeptidase P (hcAMPP) as an attractive bioscavenger candidate by measuring the enzymatic rates of hydrolysis for a wide variety of organophosphorus compounds. The comparison of substrate specificity exhibited by hcAMPP and E. coli aminopeptidase P (E. coli AMPP) was studied. We cloned, expressed, and purified hcAMPP from HeLa cells and AMPP from Escherichia coli. The pH-rate profiles of hcAMPP were measured in the presence of organophosphate compound 3 or 5. All of the organophosphorus compounds, 1-19, were synthesized by using the approach of phosphorus chemistry described in a previous publication. The relative activity of hcAMPP and E. coli AMPP in hydrolyzing a series of organophosphorus analogues, 1-17, was evaluated in a spectrophotometric assay by monitoring the difference of accumulation of 4-nitrophenol at 400 nm. The overall substrate preference of hcAMPP is as follows: methylphosphonates>ethylphosphonates> or =organophosphates. Interestingly, the observed enhancement in the activity of hcAMPP with methyl phosphonates, 8, 10, 12, and 13, suggests that there is particularly special about the substructure of both methyl moiety and P=O ligand, since the values of specific activity with hcAMPP for the methylphosphonates 8, 10, 12, and 13 are 2- to 73-fold greater than those for the ethylphosphonates 14-17 and the organophosphates 1-7. Similarly, in E. coli AMPP toward ethylphosphonates 14-17, the results indicate that the regions of both MeO moiety and P=O ligand may be located in the vicinity of the substrate-binding site, which have not been altered within the active site of enzyme upon mutation of Trp88, Arg153, and Arg370. These studies demonstrate that E. coli AMPP and hcAMPP display different substrate preference toward organophosphorus compounds. Evidence here, therefore, represents the first example of hcAMPP that might serve as a valuable bioscavenger candidate as E. coli AMPP due to the promise from the hydrolysis of these toxic chemicals.