University Of Chicago Work Values

Daily Duties at University Of Chicago:

1. Cation selective pathway of OmpF porin revealed by anomalous X-ray diffraction The OmpF porin from the Escherichia coli outer membrane folds into a trimer of β-barrel, each forming a wide aqueous pore allowing the passage of ions and small solutes. A long loop (L3) carrying multiple acidic residues folds into the β -barrel pore to form a narrow “constriction zone”. A strong and highly conserved charge asymmetry is observed at the constriction zone, with multiple basic residues attached to the wall of the β -barrel (Lys16, Arg42, Arg82 and Arg132) on one side, and multiple acidic residues of L3 (Asp107, Asp113, Glu117, Asp121, Asp126, Asp127) on the other side. Several computational studies have suggested that a strong transverse electric field could exist at the constriction zone as a result of such charge asymmetry, giving rise to separate permeation pathway for cations and anions. To examine this question, OmpF was expressed, purified and crystallized in the P63 space group and two different data sets were obtained at 2.6 Å and 3.0 Å resolution with K+ and Rb+, respectively. The Rb+ soaked crystals were collected at the rubidium anomalous wavelength of 0.8149 Å and the cation positions were determined. The crystal structure with the Rb anomalous scattering data reveal the cation selective pathway across the constriction zone. Interestingly, a PEG molecule was observed in the pore region for both the K+ and Rb+ soaked crystals, where it is interacting with the loop L3. 2. Structural Studies of the KcsA-Shaker chimera There are about 80 different potassium channels classified into voltage-gated(Kv,6TM),inward rectifying (Kir, 2TM), Calcium dependent (BK,6TM) and prokaryotic (KcsA, 2TM). Voltage sensitive channels have a voltage sensor domain, trans membrane segments S1-S4, that responds to changes in membrane potential which triggers the pore domain to undergo the conformational change required to allow passage of ions. KcsA is a ligand gated potassium channel that opens on stimulus by intracellular protons. KcsA is a tetramer, each subunit composed of two membrane spanning helices connected by a short pore loop. Generally the ion channel “gating” refers to the conformational changes involved in the opening and closing of the conduction pathway. As for now, the crystal structure reveals only the closed state and the expectation is like most of the pore architecture remains intact in the open state. Shaker channel is a voltage gated K+ channel, composed of four subunits, each containing 6 trans membrane segments. An ion-pore module (S5-S6 equivalent to that of M1-M2 in KcsA) is surrounded by the voltage gating modules. So far the studies suggest that mutations in S4 segments affect channel gating, wherein, the S4 segments function as voltage sensors. In Shaker channel, the channel gate appears to be tightly coupled to the voltage sensors. In this experiment our aim is to study the structural and functional aspects of the KcsA-Shaker chimera. 3. Anion selective pathway of OmpF porin revealed by anomalous X-ray diffraction To examine the permeation pathway of anions in the constriction zone, OmpF was expressed, purified and crystallized in three different space groups viz., C2, P3 and P622 space group. Three different data sets were obtained at 1.9Å(space group C2), 1.7Å(Space group P622) and 2.0Å(space group P3) resolution with 0.3M, 0.2M and 0.1M KBr concentrations respectively. The Br soaked crystals were collected at the Bromide anomalous wavelength of 0.928Å and the anion positions were determined. The crystal structure with the Br anomalous scattering data reveal the anion selective pathway across the constriction zone. 4. Structural Studies of bacterial membrane protein, ZntB - a Zinc transport protein Zinc plays an essential role in cells, but excess free zinc ions are toxic to cells and these are removed by the Zinc transporters. One such type of zinc transporter from E.coli (ZntB) was cloned from the E.coli Genomic DNA and inserted between restriction enzyme sites SphI and HindIII in pQE32 vector system. The ZntB protein was expressed in XL10 Gold Ultra competent cells and purified to homogeneity using 1mM DDM as detergent. The final purified protein was analyzed by western blot method and SDS-PAGE. Crystallization is in progress to reveal the structural mechanism of flow of zinc ions across the cell membrane. 5. Structural Studies of bacterial NaK type ion channels All K+ channels contain the highly conserved signature sequence TVGYG, which is essential for K+ ion selectivity. In a search of the microbial genome, we identified four two-trans membrane(2TM) channels from 4 different bacillus species, that have sequences very similar to NaK and KcsA channels, except for the selectivity filter. These channels can either be non-selective to Na/K or may conduct divalent metal ions. In order to understand the detailed functional analysis the 4 different bacterial 2TM domain channels were cloned into pQE60 vector system from their respective genomic DNA. Expressed in XL-1 Blue cells and purified using DM and DDM as detergents and confirmed by western blot. Initial crystallization trails yielded crystals for 2 different species and diffracted only up to 6Å resolution. Further crystallization attempts are made in order to improve the resolution.

What they like about University Of Chicago:

You highly value a work environment built on a formal structure, rules, and regulations. You do not enjoy the unpredictability of shifting priorities and deadlines that upset your routine. You require and enjoy direction, input and accountability as part of your work environment. You have a strong need to participate in making key decisions and feel left out if your superiors or co-workers do not seek your input when making decisions. You thrive on providing good customer service to both internal or external customers, and doing so makes you feel good.

Tags

biochemistry, structural biology, scientist, membrane protein, crystallography, protein purification, protein crystallization, synchrotron data collection, molecular biology

Skills

membrame protein crystallography, Protein purification using HPLC, AKTA purifier, protein crystallization, Genomic DNA purification, Molecular Cloning, Protein Expression, purification and characterization of recombinant proteins, including selenomethionine-substituted proteins. , Membrane protein expression, purification and crystallization., Characterization of proteins using electrophoresis (native, non-reduced and Western blotting) and spectroscopic (UV/VIS and fluorescence) techniques., DNA-protein, RNA-protein crystallization., X-ray diffraction experiment and data collection (PF-KEK, Tsukuba, Japan & APS - Chicago, USA ). , Expertise in data collection techniques using mar345 image plate detectors and data reduction software such as DENZO, Scalepack, HKL2000, MOSFLM and AUTOMAR., Protein Phasing by MR and SAD/MAD technique using crystallographic programs, SHARP, CCP4, SHELXD and Solve/Resolve., Proficient in using structure refinement and model building software namely CNS, CCP4, PHENIX, SHELXE, ARP/wARP and analyze the structure using software O, CCP4mg and Coot., Proficient in small molecule structure analysis packages (SHELXS, SHELXL, ORTEP and PLATON)., Expertise in molecular modeling and graphics software such as Insight II- Biosym software (Silicon Graphics), Pymol, Swiss-PDB viewer, RasMol, Molscript, Bobscript, O, Coot, XtalView etc., Experience in using Insight II Biosym Softwares for drug docking and Molecular Mechanics studies and Homology modeling. , Expertise in operating system Windows, Linux and Unix, MALS ( Multiple angle dynamic light scattering), electrophysiology - patch clamp

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