No abstract

The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membranemimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid-and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.Keywords: NMR spectroscopy; membrane-mimetic; protein-membrane interactions; protein-lipid interactions tgcqzviations: DihepPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPC, dodecylphosphocholine; GB1, B1 immunoglobulin binding domain of streptococcal protein G (56 residues); GB1-Xa, GB1 followed by a thrombin and a factor Xa recognition site (¼ LVPRGS-IEGR); (m)TOR, (mammalian) target of rapamycin; min, minute(s); SI, supplementary information; y1FATC, residues 2438-2470 of S. cerevisiae TOR1.

Edited by Sandro SonninoThe conserved C-terminal FATC domain of the kinase 'target of rapamycin' is important for its regulation and was suggested to contain a peripheral membrane anchor. Here, we present the characterization of the interactions of the yeast TOR1 FATC domain (2438-2470 = y1fatc) and 15 mutants with membrane mimetic micelles, bicelles, and small unilamellar vesicles (SUVs) by NMR and CD spectroscopy. Replacement of up to 6-7 residues did not result in a significant abrogation of the association with micelles or bicelles. However, replacement of only one residue could result in an impairment of the interaction with SUVs that are usually used at low concentrations. Some mutants not binding liposomes may be introduced in full-length TOR for future functional and localization studies in vivo.

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

FAT C-terminal (FATC) is a circa 33 residue-long domain. It controls the kinase functionality in phosphatidylinositol-3 kinase-related kinases (PIKKs). Recent NMR- and CD-monitored interaction studies indicated that the FATC domains of all PIKKs can interact with membrane mimetics albeit with different preferences for membrane properties such as surface charge and curvature. Thus they may generally act as membrane anchoring unit. Here, we present the H,N, and C chemical shift assignments of the DPC micelle immersed FATC domains of the human PIKKs ataxia-telangiectasia mutated (ATM, residues 3024-3056) and DNA protein kinase catalytic subunit (DNA-PKcs, residues 4096-4128), both fused to the 56 residue long B1 domain of Streptococcal protein G (GB1). Each fusion protein is 100 amino acids long and contains in the linking region between the GB1 tag and the FATC region a thrombin (LVPRGS) and an enterokinase (DDDDK) protease site. The assignments pave the route for the detailed structural characterization of the membrane mimetic bound states, which will help to better understand the role of the proper cellular localization at membranes for the function and regulation of PIKKs. The chemical shift assignment of the GB1 tag is useful for NMR spectroscopists developing new experiments or using GB1 otherwise for case studies in the field of in-cell NMR spectroscopy or protein folding. Moreover it is often used as purification tag. Earlier we showed already that GB1 does not interact with membrane mimetics and thus does not disturb the NMR monitoring of membrane mimetic interactions of attached proteins.