SCIENTIFIC CORES
Bacterial Protein Expression Core
Biochemistry Core Cell Biology and Imaging Core Computation Core EM Crystallography Core EM Tomography Core Eukaryotic Protein Expression Core Fluorescence Spectroscopy Core Protein Interactions Core Protein NMR Spectroscopy Core RNA Structure and Dynamics Core Tissue EM Core Virology Core Virus Imaging Core X-ray Crystallography Core |
Biochemistry Core
Director: Jamie Williamson, PhD The Biochemistry Core provides all essential resources for purification and expression of proteins and nucleic acids, and instrumentation and expertise for quantitative analysis of intermolecular interactions, including protein-protein and protein-RNA interactions. In addition, the Biochemistry Core specializes in structure determination of RNA-protein complexes using X-ray and NMR spectroscopy. Instrumentation and Capabilities The Core has extensive equipment for bacterial cell growth (incubators, centrifuges), and protein purification (3X Biorad LC, Pharmacia AKTA LC, 3X ISCO LC). The Core has a fluorescence plate reader capable of both fluorescence measurements and fluorescence polarization measurements in 96-well and 384-well formats. The Core also has shared access to an isothermal titration calorimeter, an Octet system, and a Biacore system for measuring the kinetics and thermodynamics of intermolecular interactions. During the current grant cycle, we will obtaina Thermo Electron Nanodrop 2000 UV/Vis Spectrophotometer and a Microfluidics Microfluidizer. NMR Instrumentation The Core has access to The Scripps Research Institute (TSRI) Biomolecular NMR facility, which is housed in two separate buildings. There are multinuclear NMR spectrometers operating at 900 MHz, 2 at 800 MHz, 750 MHz, 700 MHz, 4 at 600 MHz, and 2 at 500 MHz, and the Core has access to 15% of the total available spectrometer time (1.5 instruments at any given time, spread across all fields). X-ray Crystallography The Core has access to local X-ray generators and detectors in the laboratory of Prof. Ian Wilson as needed (and see his letter of support). In addition, the Core has access to synchrotron radiation at the TSRI beamline at SSRL. Computation The Core has access to a 1536-CPU Linux high performance computing cluster, which is operated by TSRI free of charge. This includes backup DMF systems for data storage and archiving. Mass Spectrometry The Core has a dedicated Agilent ESI-TOF mass spectrometer specifically for quantitative MS measurements. In addition, the Core has a dedicated Agilent Q-TOF instrument for proteomics measurements, and 50% shared access to a second Q-TOF instrument. Finally, an Applied Biosystems triple-TOF instrument with nano-LC is scheduled for immediate delivery. 
NMR Solution Structures of HIV RNA-ligand Complexes.
A) The HIV-2 TAR-argininamide complex. The UA-U base triple is highlighted in green, the argininamide analog is shown in red, hydrogen bonding to a G-residue in the major groove, while stacking on an A-residue immediately below the bulge (yellow). B) The Rev-RRE complex. The basic region of Rev forms an alpha helix (red). The RRE (blue) consists of two A-form helices interrupted by an internal loop containing G-A and G-G base pairs. Non-Watson Crick pairs open up the normally restricted major groove to admit the bulky, arginine-rich helical basic region of Rev for specific recognition. These structures were determined in the Williamson lab, in collaboration with Alan Frankel and colleagues.  |