title: Conformationally specific misfolding of an integral membrane protein.
abstract: Membrane protein misfolding is related to the etiology of many diseases, but is poorly understood, particularly from a structural standpoint. This study focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAGK), a 40 kDa homotrimeric protein having nine transmembrane segments. Preparations of s-DAGK sometimes contain a kinetically trapped misfolded population, as evidenced by lower-than-expected enzyme activity (with no accompanying change in substrate K(m)) and by the appearance of a second band in electrophoresis gels. Misfolding of s-DAGK may take place during cellular overexpression, but can also be reproduced using the purified enzyme. TROSY NMR spectra of s-DAGK as a 100 kDa complex with detergent micelles exhibit a single additional set of resonances from the misfolded form, indicating a single misfolded conformational state. The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below the maximum observed for fully folded s-DAGK. Misfolded s-DAGK exhibits a modest difference in its far-UV CD spectrum compared to the folded enzyme, consistent with a small degree of variance in secondary structural content between the two forms. However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-DAGK support the notion that they represent very different structural states. Cross-linking experiments indicate that both the correctly folded enzyme and the kinetically trapped misfolded form are homotrimers. This work appears to represent the first documentation of conformationally specific misfolding of an integral membrane protein.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
authors.forename: K
authors.initials: K
authors.lastname: Oxenoid
authors.forename: F D
authors.initials: FD
authors.lastname: Sönnichsen
authors.forename: C R
authors.initials: CR
authors.lastname: Sanders
mesh_terms: D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics; D004198:Disease Susceptibility; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005976:Glutaral / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019852:Diacylglycerol Kinase; D005976:Glutaral