Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2.

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

A New Suite of Plasmid Vectors for Fluorescence-Based Imaging of Root Colonizing Pseudomonads.

In the terrestrial ecosystem, plant-microbe symbiotic associations are ecologically and economically important processes. To better understand these associations at structural and functional levels, different molecular and biochemical tools are applied. In this study, we have constructed a suite of vectors that incorporates several new elements into the rhizosphere stable, broad-host vector pME6031. The new vectors are useful for studies requiring multi-color tagging and visualization of plant-associated, Gram-negative bacterial strains such as Pseudomonas plant growth promotion and biocontrol strains. A number of genetic elements, including constitutive promoters and signal peptides that target secretion to the periplasm, have been evaluated. Several next generation fluorescent proteins, namely mTurquoise2, mNeonGreen, mRuby2, DsRed-Express2 and E2-Crimson have been incorporated into the vectors for whole cell labeling or protein tagging. Secretion of mTurquoise2 and mNeonGreen into the periplasm of Pseudomonas fluorescens SBW25 has also been demonstrated, providing a vehicle for tagging proteins in the periplasmic compartment. A higher copy number version of select plasmids has been produced by introduction of a previously described repA mutation, affording an increase in protein expression levels. The utility of these plasmids for fluorescence-based imaging is demonstrated by root colonization of Solanum lycopersicum seedlings by P. fluorescens SBW25 in a hydroponic growth system. The plasmids are stably maintained during root colonization in the absence of selective pressure for more than 2 weeks.

pH dependence of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL.

Equilibrium and kinetic properties of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL (SmFixLH and BjFixLH) have been investigated between pH5 and 11. KD determinations were made at integral pH values, with the strongest binding at pH9 for both ligands. KD for the cyanide complexes of BjFixLH and SmFixLH is 0.15±0.09 and 0.50±0.20µM, respectively, and 0.70±0.01mM for imido-BjFixLH. The association rate constants are pH dependent with maximum values of 443±8 and 252±61M(-1)s(-1) for cyano complexes of BjFixLH and SmFixLH and (5.0±0.3)×10(4) and (7.0±1.4)×10(4)M(-1)s(-1) for the imidazole complexes. The dissociation rate constants are essentially independent of pH above pH5; (1.2±0.3)×10(-4) and (1.7±0.3)×10(-4)s(-1) for the cyano complexes of BjFixLH and SmFixLH, and (73±19) and (77±14) s(-1) for the imidazole complexes. Two ionizable groups in FixLH affect the rate of ligand binding. The more acidic group, identified as the heme 6 propionic acid, has a pKa of 7.6±0.2 in BjFixLH and 6.8±0.2 in SmFixLH. The second ionization is due to formation of hydroxy-FixLH with pKa values of 9.64±0.05 for BjFixLH and 9.61±0.05 for SmFixLH. Imidazole binding is limited by the rate of heme pocket opening with maximum observed values of 680 and 1270s(-1) for BjFixLH and SmFixLH, respectively.

Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor.

Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.

Large-scale muLC-MS/MS for silver- and Coomassie blue-stained polyacrylamide gels.

2-DE combined with LC-MS/MS has become a routine, reliable protein separation and identification technology for proteome analysis. The demand for large-scale protein identifications after 2-DE separation requires a sensitive and high-throughput LC-MS/MS method. In this report, a simple, splitless, fully automated capillary LC-MS/MS system was described for the large-scale identification of proteins from gels stained with either silver or CBB. The gel samples were digested and peptides were extracted using an in-gel digestion workstation. The peptides were automatically introduced into a capillary column by an autosampler connected to an HPLC pump. A nanoLC pump was then used to deliver the gradient and elute the peptides from the capillary column directly into an LCQ IT mass spectrometer. Neither a peptide trapping setting nor a flow split is needed in this simple setup. The collected MS/MS spectra were then automatically searched by SEQUEST, and filtered and organized by DTASelect. Hundreds of silver-stained or CBB-stained Shewanella oneidensis, Geobacter sulfurreducens, and Geobacter metallireducens proteins separated by denaturing or nondenaturing 2-DE were digested and routinely analyzed using this fully automated muLC-MS/MS system. High peptide hits and sequence coverage were achieved for most CBB-stained gel spots. About 75% of the spots were found to contain multiple proteins. Although silver staining is not commonly thought to be optimal for MS analysis, protein identifications were successfully obtained from silver-stained 2-DE spots detected using methods with and without formaldehyde for protein fixation.