Conformationally specific misfolding of an integral membrane protein

Membrane protein misfolding is related to the etiology of many diseases, bu t is poorly understood, particularly from a structural standpoint. This stu dy focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAG K), a 40 kDa homotrimeric protein having nine transmembrane segments. Prepa rations of s-DAGK( sometimes contain a kinetically trapped misfolded popula tion, as evidenced by lower-than-expected enzyme activity (with no accompan ying change in substrate K,) and by the appearance of a second band in elec trophoresis gels. Misfolding of s-DAGK may take place during cellular overe xpression, 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 si ngle additional set of resonances from the misfolded form, indicating a sin gle misfolded conformational state. The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below t he maximum observed for fully folded s-DAGK. Misfolded s-DAGK exhibits a mo dest difference in its far-UV CD spectrum compared to the folded enzyme, co nsistent with a small degree of variance in secondary structural content be tween the two forms. However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-D AGK 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 appea rs to represent the first documentation of conformationally specific misfol ding of an integral membrane protein.