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Comparative Study
. 2003 Jan;59(Pt 1):77-83.
doi: 10.1107/s0907444902019236. Epub 2002 Dec 19.

A Rapid Method for Assessing Lipid:protein and Detergent:protein Ratios in Membrane-Protein Crystallization

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Comparative Study

A Rapid Method for Assessing Lipid:protein and Detergent:protein Ratios in Membrane-Protein Crystallization

Corrie J B daCosta et al. Acta Crystallogr D Biol Crystallogr. .

Abstract

A simple procedure for rapidly measuring lipid:protein ratios and detergent concentrations at different stages of the solubilization, purification and crystallization of membrane proteins has been developed. Fourier-transform infrared spectra recorded from 10 micro l aliquots of solution using a single-bounce diamond-attenuated total reflectance apparatus exhibit characteristic bands arising from the vibrations of lipid, protein and detergent. Lipid:protein molar ratios as low as 5:1 (for a protein with a molecular weight of 300 kDa) are determined by comparing the ratio of the integrated intensity of the lipid ester carbonyl band near 1740 cm(-1) with the protein amide I band near 1650 cm(-1). Detergent concentrations at levels well below the critical micellar concentration of most detergents are determined by comparing the integrated intensities of the detergent vibrations, particularly in the 1200-1000 cm(-1) region, with a standard curve. Protein amide I band-shape analysis provides insight into the effects of detergents on protein secondary structure. The importance of monitoring detergent concentration changes during simple procedures, such as the concentration of a membrane protein by ultrafiltration, is demonstrated. This analytical tool has been used to rapidly establish protocols for minimizing lipid and detergent levels in solubilized membrane-protein samples.

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