Tail-anchored (TA) proteins are involved in cellular processes including trafficking, degradation, and apoptosis. They contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase interacting with the hetero-oligomeric Get1/2 receptor. We have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states at 3.0, 3.2, and 4.6 angstrom resolution. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. These data suggest a molecular mechanism for nucleotide-regulated delivery of TA proteins.

No abstract

A small 63-residue membrane protein, p7, has essential roles in the infectivity of the Hepatitis C virus (HCV) in humans. This hydrophobic membrane protein forms homo-oligomeric ion channels in bilayers, which can be blocked by known channel blocking compounds. In order to perform structural studies of p7 by NMR spectroscopy, it is necessary to produce milligram quantities of isotopically labeled protein; as is the case for most membrane-associated proteins, this is challenging. We describe the successful expression of full-length p7 and two truncated constructs in E. coli using a fusion partner that directs the over-expressed protein to the inclusion bodies. Following isolation of the fusion proteins by affinity chromatography, they were chemically cleaved with cyanogen bromide. The p7-polypeptides were purified by size exclusion chromatography. Solution NMR two-dimensional HSQC spectra of uniformly 15 N-labeled p7-polypeptides in DHPC isotropic micelles are fully resolved, with a single resonance for each amide site. The solid-state NMR spectra of the same polypeptides in magnetically aligned 14-O-PC/6-O-PC bicelles demonstrate their reconstitution into planar phospholipid bilayers.

An extension to HN(CO-α/β-N,C(α)-J)-TROSY (Permi and Annila in J Biomol NMR 16:221-227, 2000) is proposed that permits the simultaneous determination of the four coupling constants (1) J (N'(i)Cα(i)), (2) J (HN(i)Cα(i)), (2) J (Cα(i-1)N'(i)), and (3) J (Cα(i-1)HN(i)) in (15)N,(13)C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the (2) J (CαN') coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both (2) J (CαN') and (1) J (N'Cα) (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.