Changing single side-chains can greatly enhance the resistance of a membrane protein to irreversible inactivation.
The thermal inactivation rates of a set of 20 cysteine-substituted variants of the integral membrane protein diacylglycerol kinase were measured. Two of the mutations, I53C and I70C, were found to significantly prolong the half-life of the enzyme in detergent solution. By combining the single mutants to create a double mutant, I53C/I70C, the half-life of the enzyme was improved from less than a minute at 70 degrees C to 51 minutes. These results demonstrate that individual side-chain substitutions can significantly improve the properties of membrane proteins in detergent solution.
Lau
F W
FW
Department of Chemistry and Biochemistry and Laboratory of Structural Biology and Molecular Medicine, UCLA, 405 Hilgard Avenue, Los Angeles, CA, 90095-1570, USA.
Nauli
S
S
Zhou
Y
Y
Bowie
J U
JU
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003545:Cysteine / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D013816:Thermodynamics
D004075:Diglycerides; D004220:Disulfides; D008565:Membrane Proteins; D019852:Diacylglycerol Kinase; D003545:Cysteine