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A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallization

2002; Wiley; Volume: 59; Issue: 1 Linguagem: Inglês

10.1107/s0907444902019236

1399-0047

Corrie J.B. daCosta, John E. Baenziger,

thermodynamics and calorimetric analyses

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 µl 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.