A method for assessing the stability of a membrane protein.
The integral membrane protein diacylglycerol kinase (DGK) from Escherichia coli has been reversibly unfolded in a protein/detergent/mixed micelle system by varying the molar ratio of n-decyl beta-D-maltoside (DM) and sodium dodecyl sulfate (SDS). Unfolding was monitored by circular dichroism (CD) and ultraviolet (UV) absorbance spectroscopy. When unfolding is monitored by measuring changes in absorbance at 294 nm, two distinct denaturation phases are observed, indicative of a stable intermediate. When CD is used as a conformational probe, the resulting denaturation curve contains only one major transition, which corresponds to the first unfolding phase observed by absorbance changes. The unfolding behavior of several mutant proteins in which the tryptophan residues were selectively replaced made it possible to assign the first unfolding phase to a denaturation event in a cytoplasmic domain and the second phase to denaturation of the membrane-embedded portion of the protein. The denaturation curves fit well to a model which assumes two cooperative transitions and a linear relationship between unfolding free energy and SDS concentration. Extrapolation back to zero denaturant indicates an unfolding free energy of 6 kcal/mol for the cytoplasmic domain and 16 kcal/mol for the transmembrane domain. The high apparent stability of the transmembrane domain could explain the high degree of tolerance to amino acid substitutions seen for DGK and other membrane proteins. The approach described in this paper may be applicable to other membrane protein systems.
Lau
F W
FW
Department of Chemistry and Biochemistry, University of California at Los Angeles, 90095-1570, USA.
Bowie
J U
JU
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D019852:Diacylglycerol Kinase; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D019265:Spectroscopy, Near-Infrared
D008565:Membrane Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase