Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.

Abstract

Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1.