Functional reconstitution of prokaryote and eukaryote membrane proteins.
A new strategy for the functional reconstitution of membrane proteins is described. This approach introduces a new class of protein stabilizing agents--osmolytes--whose presence at high concentration (10-20%) during detergent solubilization prevents the inactivations that normally occur when proteins are extracted from natural membranes. Osmolytes that act in this way include compounds such as glycerol and higher polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and certain amino acids (glycine, proline, betaine). The beneficial effects of osmolytes are documented by reconstitution of a variety of prokaryote and eukaryote membrane proteins, including several proton- and calcium-motive ATPases, cation- and anion-linked solute carriers (symport and antiport), and a membrane-bound hydrolase from endoplasmic reticulum. In all cases, the presence of 20% glycerol or other osmolyte during detergent solubilization led to 10-fold or more increased specific activity in proteoliposomes. These positive effects did not depend on use of any specific detergent for protein solubilization, nor on any particular method of reconstitution, but for convenience most of the work reported here has used octylglucoside as the solubilizing agent, followed by detergent-dilution to form proteoliposomes. The overall approach outlined by these experiments is simple and flexible. It is now feasible to use reconstitution as an analytical tool to study the biochemical and physiological properties of membrane proteins.
Maloney
P C
PC
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Ambudkar
S V
SV
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology; D003902:Detergents; D004926:Escherichia coli / Q000502:physiology*; D005990:Glycerol; D013294:Lactococcus lactis / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008862:Microsomes, Liver / Q000378:metabolism / Q000502:physiology*; D009994:Osmolar Concentration; D051381:Rats; D012995:Solubility
D001426:Bacterial Proteins; D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins; D005990:Glycerol