Synthetic single domain antibodies for the conformational trapping of membrane proteins.

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles.

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.

Expression and purification of recombinant calpain-derived N-terminal peptides from glycine transporter GlyT2.

Glycine transporter GlyT2 contains an extended N-terminal domain which is about three times longer than the N-termini of its closest family members. We previously found that this domain could be separated from the transporter by proteolysis with calpain resulting in the generation of at least two GlyT2N derived peptides. In this work we analyzed the properties of these peptides using bio-informatics, by expressing them in mammalian cell lines and by overexpressing them in bacteria. When expressed in mammalian cell lines, these peptides show widespread localization in the cytoplasm. Their unusually high number of alanine, proline and glycine residues suggests that they posses significant disorder and conformational flexibility, which is supported by their thermal resistivity. Making use of these phenomena, we developed a simple purification method for obtaining pure recombinant GlyT2N derived calpain fragments without using an affinity tag. This procedure can be used to obtain peptide fragments in large amounts for structural, interaction studies or for determining their potential biological activity.