Background

During the past 5 years, we have seen a stunning increase in the number of novel technologies applied to structural biology. These developments have increased the rate of protein structure deter mination and lowered the average cost, mainly for prokaryotic soluble proteins. Most of these technologies are now commercially available and the cost is within the affordable range for the single academic user laboratory, similar to what occurred with the introduction of the ABI 3700 for gene sequencing. However, production of structure-grade proteins remains the most challenging technical problem in biology, particularly for integral membrane proteins. To develop novel technologies for integral membrane protein expression and sample preparation, we have assembled a consortium of investigators that have extensive experience in both technology development for structural biology as well as for membrane protein production and structure determination by NMR, EM, and X-ray.

Aim 1: Expression. Develop and validate novel methods and technologies for the reliable expression of integral membrane proteins. Secondary goal is to reduce the cost of membrane protein expression. Conduct a systematic expression survey with a representative number of integral membrane proteins and then develop novel technologies to increase the reliability of the system, to reduce the complexity of the processes and/or and lower the cost of expression through miniaturization

• Cell free expression
• Baculovirus expression
• Mammalian expression

Aim 2: Stabilization. Develop novel methods, sample preparation reagents (including small molecules, polymeric compounds, antibodies, rafts, etc.) and tools for the stabilization of integral membrane proteins for structural biology studies.

• Lipid Chemistry
• Protein Stabilization Reagents
• Nanofabrication

Aim 3: Biophysical Analysis. Adapt the available high throughput biophysical characterization techniques for integral membrane proteins, evaluate the quality of membrane protein production and sample preparation, and correlate data.

• Electron Microscopy
• Nuclear Magnetic ResonanceSpectroscopy
• X-ray Crystallography