ABSTRACT
The effect of detergents and amphiphiles on protein solubility and their use in crystallization solutions was examined for an integral membrane protein, the light-harvesting I complex from Rhodospirillum centenum. Measurement by a centrifugation assay of the solubility of the protein in different detergents and amphiphiles showed high protein-solubility values when either octyl glucoside or lauryldimethylamine-N-oxide was present with heptanetriol or when deoxycholate was present with spermine. The detergent/amphiphile combinations that resulted in high protein solubility were shown to be successful for crystallization of the protein, suggesting that crystallization is favored for detergents and amphiphiles that optimize the solubility of integral membrane proteins. The amphiphiles effective for crystallization were found using laser mass spectrometry to displace the lauryldimethylamine-N-oxide bound to the protein. These results suggest that mass spectrometry can be used for screening of favorable crystallization conditions.
Subject(s)
Detergents/pharmacology , Light-Harvesting Protein Complexes , Rhodospirillum centenum/chemistry , Centrifugation , Crystallization , Deoxycholic Acid/chemistry , Dimethylamines/chemistry , Fatty Alcohols/chemistry , Glucosides/chemistry , Lasers , Mass Spectrometry , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spermine/chemistryABSTRACT
The relationship between the effect of detergents and amphiphiles on protein solubility and their use in crystallization solutions was examined for the reaction center from Rhodobacter sphaeroides. Measurement by a centrifugation assay of the solubility of the reaction center as a function of ionic strength revealed dramatic differences in the intrinsic solubility at zero ionic strength in the presence of various detergents and amphiphiles. High protein-solubility values were found for beta-octyl glucoside and for lauryldimethylamine-N-oxide with heptanetriol. The solubility differences are interpreted in terms of fundamental properties such as the polarity of the detergent molecules. Conditions that resulted in high protein solubility correspond to conditions that have been shown to be successful for crystallization of the reaction center. These results suggest that crystallization is favored for detergents and amphiphiles that optimize the solubility of integral membrane proteins.
Subject(s)
Detergents/chemistry , Membrane Proteins/chemistry , Photosynthetic Reaction Center Complex Proteins/chemistry , Osmolar Concentration , Rhodobacter sphaeroides/chemistry , SolubilityABSTRACT
The effect of the amphiphile heptanetriol on the properties of solutions containing several detergents commonly used for crystallization of membrane proteins was characterized. The critical micelle concentration was found to be relatively unchanged by the presence of the amphiphile. In contrast, the addition of heptanetriol to solutions containing both detergent and polyethylene glycol exhibited significant shifts in the clouding behavior, with the largest shifts being for lauryl dimethylamine oxide. These results suggest that conditions favorable for crystallization of integral membrane proteins can be inferred from the properties of the detergents and amphiphiles.
