Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination.
Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.
Columbus
Linda
L
The Joint Center for Structural Genomics and The Scripps Research Institute, Department of Molecular Biology, La Jolla, California 92037, USA. columbus@scripps.edu
Lipfert
Jan
J
Klock
Heath
H
Millett
Ian
I
Doniach
Sebastian
S
Lesley
Scott A
SA
D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D003902:Detergents / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008823:Micelles; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D020124:Thermotoga maritima / Q000737:chemistry*; D014965:X-Rays
D003432:Cross-Linking Reagents; D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles