As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.

Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants, K. The total protein concentration, but not the fraction of labeled protein, needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds "major-minor-mix" liposomes. A Langmuir isotherm model yields K=(2.1±1.1)μM as the single-site dissociation constant. The dcFCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

Fluorescence Correlation Spectroscopy (FCS) has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners however comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a binding model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants D K . The total protein concentration, but not the fraction of labeled protein needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds 'majorminor-mix' liposomes. A Langmuir isotherm model yieldsas the single site dissociation constant. The dual-color FCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

Synthetic model peptides such as GWALP23 (acetyl-GGALW 5 LALALALA LALALW 19 LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membranespanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2 H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2 H-Ala labels within the helical core sequence. Solid-state 2 H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To prevent the acid catalyzed degradation of lipids, we employed ether-linked DOPC bilayers to observe the effect of low pH on the L12H mutant. Under acidic conditions GWALP23-H12 is highly dynamic and exhibits multiple states. Indeed, the multi-state behavior of GWALP23-H12, when His is charged between pH 1.5 and pH 3, resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of GWALP23-H12 indicates a pK a value less than 3 for His near the center of a lipid bilayer. Investigations are in progress to chemically exchange the C2 imidazole hydrogen of His for deuterium in the peptide, toward a goal of enabling direct observation of the His ring by solid-state 2 H NMR over a range of pH and buffer conditions.

G protein-coupled receptors (GPCRs) constitute the most abundant protein family in mammalian genome. Indeed, GPCRs are the target for about 30% of the drugs on the market. In human particularly, there are 22 genes encoding class C GPCRs, which consists of GABAb receptor, calcium sensing receptor, retinoic acid-inducible receptors (orphan), taste receptors, metabotropic glutamate receptors, and a few additional orphan receptors. Metabotropic glutamate receptors (mGluRs) are involved in controlling synaptic transmission, and involved in various CNS disorders including pain, Parkinson's disease, schizophrenia etc... They are naturally homodimers, and each protomer consists of a heptahelical transmembrane domain linked to a bilobate Venus flytrap domain (VFT) by a Cystein-rich domain. Understanding the conformational changes of mGluRs is essential to decipher the allosteric transition associated with their activation. Crystallographic studies suggested that in the inactive and active states the second lobes of the VFTs are distant and close, respectively. However certain ambiguities and discrepancies about these two states have been observed by X-ray crystallography. For the first time in GPCR activation mechanism studies, we proposed structural dynamics investigations by single-molecule FRET (sm-FRET) in solution. Our results show that the isolated mGluR VFTs oscillate between the resting and active states in a time range of 50-100ms (Olofsson et al. Nat. Comm. 2014). Following the success of the powerful sm-FRET methodology on isolated VFTs, we employ here Multi-parameter Fluorescence Detection (MFD) and Pulsed Interleaved Excitation (PIE) to study the full-length receptors in order to gain additional insights into mGluR activation. Our current results confirm the structural dynamics obtained by the mGluRs VFT, and suggest a stabilizing role of the transmembrane domain. Further studies on allosteric modulation and G protein effect are ongoing .