β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural system

β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural system

Feilke revisited : 60 Stellenbesuche

Weitere Hrsg.: Thorsten Pohl, Sara Rezat, Torsten Steinhoff, Martin SteinseiferAnlässlich des 60. Geburtstags des Linguisten und Sprachdidaktikers Helmuth Feilke wurden Wegbegleiterinnen und Wegbegleiter gebeten, einzelne Stellen in seinen wissenschaftlichen Schriften erneut zu besuchen. Entstanden sind pointierte Kommentare, kurze wissenschaftliche Abhandlungen und Analysen, Varianten auch des kritischen und kontroversen Nach- und Weiterdenkens und Ansätze zur Neu- oder Re-Kontextualisierung. Je nach wissenschaftlicher Vita der Autorinnen und Autoren kann es sich um Stellen handeln, deren Rezeption zeitlich weit zurückliegt, oder um Passagen, die ganz aktuelle Fragen der eigenen Forschungsarbeit tangieren. Abgesehen davon, dass ein kurzes Format für die Beiträge gewählt und die Autorinnen und Autoren gebeten wurden, die ausgewählte Stelle knapp zu verorten und zu erläutern, war die Bearbeitungsform gänzlich freigestellt. So sind Texte in einer Bandbreite von pointierten Kommentaren, kurzen wissenschaftlichen Abhandlungen und Analysen, Varianten des Nach- und Weiterdenkens, Ansätze zur Neu- oder Re-Kontextualisierung bis hin zu Formen des kritischen Hinterfragens und der kontroversen Auseinandersetzung entstanden

Membrane protein folding on the example of outer

The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded β-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the β-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of β-sheet secondary structure and β-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i. e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of β-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E.coli and FomA of F.nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpAis a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane β-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded β-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of β-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane β-barrels

The Formation of <i>β</i>-Strand Nine (<i>β</i>₉) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers

Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or β-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual β-strands fold. Here, the folding and insertion of the β-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (β9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand β9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the β-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand β9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins