A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallization.
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.
daCosta
Corrie J B
CJ
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada.
Baenziger
John E
JE
D000577:Amides / Q000737:chemistry; D003460:Crystallization / Q000379:methods*; D005374:Filtration / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry*; D008823:Micelles; D009113:Muramidase / Q000032:analysis / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000032:analysis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013501:Surface-Active Agents / Q000032:analysis / Q000737:chemistry*
D000577:Amides; D008565:Membrane Proteins; D008823:Micelles; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; D009113:Muramidase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine