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Maintaining membrane proteins at the right place at the right time is crucial to cellular function. To support such organization a broad spectrum of regulating proteins from the cytosol bind, recruit and arrange the membrane proteins into specific structures. PICK1 is an abundant scaffolding protein interacting with more than 30 different receptors, transporters and ion channels, embedded in lipid membranes. PICK1 contains a single N-terminal PDZ domain and forms homodimers via its central membrane bending and curvature sensing N-BAR domain thus forming a functional unit with two PDZ domains. The PICK1 PDZ domain binds many different PDZ peptide ligands such as GluA2, mGluR7 and the dopamine transporter with no apparent conserved binding motif. The wide span of affinities seen for such proteins raises the question of the importance of the affinity for the isolated domain interactions in context of the overall avidity and the functional effects of these interactions.We have established a system based on the previously published supported membrane sheet system to study the binding of PICK1 to a cell membrane expressed ligand to determine the avidity for “on membrane interactions”. Secondly, we address the functional effects of lowering the affinity of the PDZ domain interactions on the functional effects of PICK1.We demonstrate a dramatic increase in the binding Kd for the oligomeric interaction compared to affinities previously reported in non-native condition binding assays. Furthermore, we show that the interaction is only facilitated by functional PDZ domain. We also observe a significant change in Bmax for lower affinity ligands indicating that the increase of the PDZ affinities might rely on a cooperative binding mechanism of the PICK1 homodimer, which is consistent with results showing that the homodimer binds tighter than the monomer.

It is widely recognized that curved membranes play a major role in the regulation and control of cell functions. It is, however, less clear how exactly biological cells generate and maintain membrane curvature in different organelles as well as during vesicle budding.In the present study, we investigate the peripheral membrane nBAR (Bin-Amphiphysin-RVS) class of proteins, which is known to generate and stabilize membrane structures. These proteins are characterized by a banana-shaped scaffolding domain, an amphipathic helix, and may have additional amphipathic motifs. Recent investigations by Boucrot et al. (Cell 2012) suggest that BAR domain proteins may promote either tubulation or fission of vesicles, as the BAR domains limits the vesicle fission caused by hydrophobic insertions of amphipathic motifs. In contrast, our studies show that the Arfaptin class of proteins displays a high deformation capability despite few amphipathic motifs pr. BAR domain.Using a combination of biochemical and biophysical techniques, the activity and function of several proteins are investigated in vitro, both in quantitative bulk measurements and in qualitative single particle studies.Specifically, we use a bulk assay to determine the deformation efficiency of proteins in a solution containing vesicles and protein.This is supplemented by the Single Liposome Curvature Sensing (SLiC) assay, which uses fluorescently labeled, immobilized vesicles on a glass coverslip to achieve single vesicle resolution and determine size dependent effects and protein/vesicle colocalization, i.e. fraction of vesicles with bound protein.Supporting these quantitative methods, we use TEM (Transmission Electron Microscopy) to directly image vesicles and other membrane structures.

PICK1 (Protein Interacting with C-kinase 1) is a functionally important protein, which is distributed mainly in testis, pancreas and brain. It has been shown to play a central role in regulation of dense core vesicles from the golgi apparatus and trafficking of ionotropic glutamate receptors.PICK1 contains a N-terminal PDZ-domain, which we have earlier demonstrated to be important for interaction with a large number of proteins, including several important receptors and transporters. In addition, it has a BAR (Bin/Amphiphysin/Rvs) domain in the C-terminal end. BAR domains are generally believed to either recognize or induce curvatures of lipid membranes, but as we have demonstrated, proteins of the N-BAR family (incl. PICK1) binds lipids and recognizes membrane curvature (MC) through an associated amphipathic helix (AH) rather than through the BAR domain itself.Here we show that the lipid binding AH of PICK1 contains a phosphorylation-site, which, through PKC activation, is responsible for an altered cellular distribution of PICK1. To investigate whether the altered cellular distribution results directly from a change in the lipid binding capacities of the AH, we employ a Single Liposome Curvature Sensing (SLiC) assay. We use quantitivative fluorescence microscopy to evaluate the binding of the phospho-mimicking mutants to nanosized liposomes in terms of MC-sensing, lipid affinity and membrane deformation.Intriguingly, we find that this single phospho-mimicking mutation in the AH is sufficient to change the lipid binding capacities of the entire protein, likely causing the altered cellular distribution of the phosphorylated protein seen in the cells. As MC-sensing has been shown to be dependent on the AH of N-BAR proteins in general, we speculate that the finding may apply generally to phospho-regulation of N-BAR proteins.

PDZ domains are scaffolding proteins that assemble and regulate many cellular signaling pathways by recognizing specific C-terminal type II peptide sequences. Consequently, these domains are associated as well with human disease and represent putative targets for new pharmacotherapeutics. The PICK1 (Protein Interacting with C Kinase 1) contains a N-terminal PDZ for which the first small molecule inhibitor (FSC231) was identified from fluorescent polarization assay screening in the lab of Ulrik Gether (Thorsen TS, Madsen KL, Rebola N, Rathje M, Anggono V, Bach A, Moreira IS, Stuhr-Hansen N, Dyhring T, Peters D, Beuming T, Huganir R, Weinstein H, Mulle C, Stromgaard K, Ronn LCB, Gether U - submitted). To identify the binding modes of FSC231 in both the wild type and a K83H mutant for which FSC231 exhibited higher affinity, we undertook computational docking of the compound to the crystal structure of the PDZ domain and subsequent refinement by Molecular Dynamics simulations. Based on the structure of FSC231, a library of over 1000 novel small molecules were designed by employing Ligbuilder GROW strategy and were docked to the same PICK1 PDZ domain. The top-ranked molecules from the docking results were subjected to binding free energy calculation using a potential of mean force (PMF) simulation method (Woo and Roux, PNAS 2005) with restraining potentials. This method samples the physical path of the protein-ligand binding, involving the decomposition of the binding process to several stages, and had proven successful in predicting the binding affinity for a variety of peptide-PDZ domains complexes. The results from the modeling and free energy simulation work characterized the molecular interaction network of PICK1 PDZ domain and guide the efficient rational design of new lead compounds.

Conformational changes are an essentially prerequisite for the function of transmembrane(TM) proteins. Ensemble studies typically average dynamics of the conformational changes that consequently become obscure. Therefore an approach for revealing the dynamics of conformational changes is to study each protein at the single molecule (SM) level. At present only two reports have addressed the conformational dynamics of any TM protein at SM level1,2 reflecting the difficulties and importance of these experiments. Proteins in these experiments were solubilized in detergent micelles. Here we investigate conformational dynamics of the Leucine transporter(LeuT) reconstituted in lipid vesicles, to understand the influence of the membrane on the transporter. We are focusing on single vesicle and SM microscopy measurements of allosteric transitions and oscillations between different states of the sixth TM helix (TM6) of LeuT.We have developed a unique strategy3,4,5,6 for immobilization of TM proteins under conditions that minimize non-specific interactions with the surface and thus minimize denaturation. We reconstitute membrane proteins into vesicles, which are anchored on a Neutravidin coated surface with biotinilated lipids. In this manner vesicle thus serves as a 3D scaffold that minimizes protein-surface interactions. By employing this method we have successfully reconstituted LeuT. The protein is labeled with the tetramethylrhodamine (TMR) dye on TM6 (at position 192C), which is quenched by Histidine (position 7H) when LeuT is in an inactive conformation. Variation in distance or orientation between the quencher and TMR, which are induced by the conformational changes of the protein during binding of substrate affect fluorescence signal intensity of the TMR. The dynamics of conformational changes are monitored by Total Internal Reflection microscopy at the single vesicle and the SM level.