2664-Pos Board B434 Molecular Determinants of the Human Zinc Transporter, Hzip4 Robert Dempski, Sagar Antala, Tuong-Vi Nguyen. Worcester Polytechnic Institute, Worcester, MA, USA. Zinc is an essential micronutrient which is required for the function of hun- dreds of cellular enzymes. In addition, zinc is the second most abundant transition metal found in biological systems (iron is most abundant). How- ever, the concentration of free zinc is nano to picomolar since most zinc is bound to proteins. This makes investigating the mechanism of zinc trans- port across the plasma membrane a challenge. Our interest has been to elu- cidate the mechanism of zinc transport mediated by one member of the ZIP family of proteins. To this end, we have developed a radiometric uptake assay to study the mechanism of zinc transport. In addition, we have ex- pressed and purified specific domains of the protein to examine the role of these domains in zinc coordination and transport. Thus, using a mixture of biochemical and biophysical techniques, we have begun to describe the mechanism of zinc transport to gain insight into the function of this protein. 2665-Pos Board B435 Molecular Dynamics Approach to Determine the Importance of an Aspar- agine Residue on Chloride-Dependence in the Human Serotonin Trans- porter Igor Zdravkovic 1 , Keith L. Henry 2 , Eric S. Dawson 3 , Sergei Y. Noskov 1 . 1 University of Calgary, Calgary, AB, Canada, 2 University of North Dakota, Grand Forks, ND, USA, 3 Vanderbilt University School of Medicine, Nashville, TN, USA. The brain helps regulate all of our body’s activities by maintaining a balance between active and inactive neurons. The majority of transporters involved in maintaining the brain’s chemical balance can be grouped into a large gene family SLC6. Part of this family is responsible for transport of serotonin (SERT), dopamine (DAT), and norepenephrine (NET). The focus of our re- search is on the human serotonin transporter. The human serotonin transporter (hSERT) is of particular clinical significance as it is both a common target of psychostimulants, such as cocaine and MDMA (ecstasy), as well as a target for serotonin selective reuptake inhibitors (SSRI) used in the treatment of mood disorders. In our system, the crucial Na1 site is still present but some focus shifts to the Cl- site which is in near proximity. The exact function of the Cl- is un- known, but the human SERT cannot function without it. In our study we are utilizing mutagenesis, in particular of amino acids Asn101 and Ser336, to study why that may be. N101 is in close proximity to the ions as well as the substrate binding site, which makes it a good candidate for an ion co- ordinating residue. S336 functions as a partner to N101 in maintaining this chloride dependence. We utilized our wild-type, N101A, and S336C mutants to observe the coordination of the ions and 5HT. We observed the position- ing of the ions/5HT in each of the mutants relative to the wild-type. We also calculated binding energies of each of the ions and the substrate. This will tell us the effect mutations have on the energetics of binding and coordina- tion, as well as possible clue as to why selectivity and stoichiometry is affected. 2666-Pos Board B436 Carbonate and Tungstate Membranes of Nickel: Synthesis, Characteriza- tion, Diffusive Properties and Electrochemical Behavior Rafiuddin Rafiuddin. Aligarh Muslim University, Aligarh, India. Abstract The high thermal and chemical stability of inorganic-organic composite mem- branes make them desirable for applications in the environment as well as in food, pharmaceutical and many other industries. The permeability properties of inorganic membranes have been explored and their resemblance to biologi- cal systems has been utilized successfully. In the present study, a series of chloride salts (NH 4 Cl, KCl, NaCl and LiCl) were used to study the effect of counter-ion and co-ion species on membrane potential, measured across polystyrene based tungstate and carbonate mem- branes of nickel. The measurements had been carried out in order to evaluate the transport number of ions, mobility ratio, distribution co-efficient and perm- selectivity. The fixed charge densities of the membranes were calculated using Kobatake method. The two limiting forms of Kobatake’s equation were used to evaluate various membrane parameters and the theoretical potentials calculated using this equation were compared with the experimental values and found a good accord between them which proved the applicability of the derived re- lationship to the membrane system. The thermodynamically effective fixed charge densities for the salt electrolytes used were found to be in the order LiCl > NH 4 Cl > KCl > NaCl for the tungstate membrane; while in case of the carbonate membrane, the order is KCl > NaCl > LiCl. The membranes are found to be negatively charged i.e. cation-selective and the selectivity in- creases with dilution. The physico-chemical characterization of the organic-inorganic hybrids was carried out by using X-ray diffraction (XRD), Fourier transformed infrared (FTIR) spectroscopy and Scanning electron microscope (SEM) studies. 2667-Pos Board B437 SecA alone can Promote Protein Translocation and Ion-Channel Activity: SecYEG Increases Efficiency and Specificity Ying-Hsin Hsieh. Georgia State University, Atlanta, GA, USA. SecA is an essential component of Sec translocase through which unfolded pre- proteins cross bacterial cytoplasmic membranes. The current prevailing view depicts SecYEG as the essential core of the protein-conducting channel, while SecA is the peripheral subunit that functions as an ATPase to drive the peptide chain through the SecYEG channel. In a major deviation, we now find that SecA with liposomes alone can promote protein translocation, and can elicit ion-channel activity in the oocyte whole cell recordings and in patch clamp sin- gle channel recordings. We previously reported that SecA forms ring-like pore structures upon binding with anionic phospholipids, providing the biophysical basis of SecA-liposomes channel activities. Further studies show that SecA- liposomes are less efficient and lose signal peptide recognition specificity, re- sembling PrlA suppressor mutants. Addition of purified SecYEG in the SecA-liposomes system restores the efficiency and the signal peptide specific- ity in ion-channel activity and in translocation of proOmpA and ProPhoA. Our data indicate that there is a protein-conducting channel in which SecA plays an important structural role as the core of the channel. Using N-terminal SecA de- letion truncated domains of 901 residues SecA and liposomes in the oocytes re- cordings, we identify two critical SecA domains for the formation of pore channel activity: with phospholipids alone, and for higher activity with SecYEG. The N-terminal region located on amino acid residues #668-828 is required for interaction with liposomes, and #640-649 for interaction with SecYEG, to form functional channels. In a domain reconstitution system, two fragments, N-SecA 1-640 and C-SecA 610-901 together interact with lipo- somes to promote channel activity in oocytes. Our findings establish that SecA forms channel with liposomes, and SecA domains can be identified for intramolecular interaction and with SecYEG for electrophysiological channel activity. 2668-Pos Board B438 Insights about the Translocation Kinetics of Antibiotics through the E. coli Porin OmpC Obtained from Solvation Mapping Que-Tien Tran 1 , Sarah Williams 2 , Ramy Farid 3 , Gul Erdemli 1 , Robert Pearlstein 2 . 1 Novartis Institutes for BioMedical Research, Center for Proteomic Chemistry, Cambridge, MA, USA, 2 Novartis Institutes for BioMedical Research, Global Discovery Chemistry, Cambridge, MA, USA, 3 Schrodinger, Inc., New York, NY, USA. Molecular translocation through the outer membrane of Gram-negative bac- teria is a key step in achieving antibiotic exposure and efficacy at intracel- lular targets. Understanding molecular translocation mechanisms is a prerequisite to the rational design of advanced antibiotics aimed at the treatment of resistant, as well as currently untreatable organisms. The outer membrane of Gram-negative bacteria such as E. coli, contain a large num- ber of channels falling under the broad family of b-barrel membrane-bound porin proteins. Antibiotic penetration has been attributed to such channels. Translocation kinetics measured using electrophysiology was reported for six cephalosporin and fluoroquinolone antibiotic drugs through the E. coli porin OmpC. We previously hypothesized that the rates of association and dissociation between solute partners are determined by the energetic costs of transferring solvent to and from protein surfaces and bulk solvent during binding, respectively. We set about to test whether the measured ki- netics of antibiotic translocation could be explained by the solvation prop- erties of the antibiotic conduction pathway calculated using WaterMap. The results suggest that intra-channel solvation is highly stable relative to bulk water, and as such, the observed rapid entry of substrates requires Tuesday, February 28, 2012 523a