The Escherichia coli biotin repressor, BirA, is an allosteric transcription regulatory protein to which binding of the small ligand corepressor, biotinyl-5'-AMP, promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo-or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212-234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate in the BirA adenylate structure, is referred to as the adenylate binding loop or ABL. Although residues in the loop do not directly interact with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side chain in the cluster with alanine and analysis of the variant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor.
Novel methods are required for site-specific, quantitative fluorescence labeling of G protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb native structure and function as little as possible. We evaluated bioorthogonal reactions to label genetically encoded p-acetyl-l-phenylalanine (AcF) or p-azido-l-phenylalanine (azF) residues in receptors heterologously expressed in mammalian cells. We found that keto-selective reagents were not truly bioorthogonal, possibly due to posttranslational protein oxidation reactions. In contrast, the strain-promoted [3+2] azide–alkyne cycloaddition (SpAAC) with dibenzocyclooctyne (DIBO) reagents yielded stoichiometric conjugates with azF-rhodopsin while undergoing negligible background reactions. As one useful application, we used Alexa488-rhodopsin to measure the kinetics of ligand uptake and release in membrane-mimetic bicelles using a novel fluorescence-quenching assay.
Background: Unnatural amino acids can be genetically incorporated into 7-transmembrane receptors. Results: A photoreactive amino acid introduced into the neurokinin-1 receptor cross-links substance P to the N-terminal and extracellular loop II domains of the receptor.
Conclusion:The extracellular domain of the neurokinin-1 receptor possesses multiple potential binding sites for substance P. Significance: A photocross-linking methodology reveals novel interaction sites in the neurokinin-1-receptor-substance P complex.
We developed a general strategy for labeling expressed membrane proteins with a peptide epitope tag and detecting the tagged proteins in native cellular membranes. First, we genetically encoded the unnatural amino acid p-azido-L-phenylalanine (azF) at various specific sites in a G protein-coupled receptor (GPCR), C-C chemokine receptor 5 (CCR5). The reactive azido moiety facilitates Staudinger ligation to a triarylphosphine-conjugated FLAG peptide. We then developed a whole-cell-based enzyme-linked immunosorbent assay approach to detect the modified azF-CCR5 using anti-FLAG mAb. We optimized conditions to achieve labeling and detection of low-abundance GPCRs in live cells. We also performed an accessibility screen to identify azF positions on CCR5 amenable to labeling. Finally, we demonstrate a preparative strategy for obtaining pure bioorthogonally modified GPCRs suitable for single-molecule detection fluorescence experiments. This peptide epitope tagging strategy, which employs genetic encoding and bioorthogonal labeling of azF in live cells, should be useful for studying biogenesis of polytopic membrane proteins and GPCR signaling mechanisms.
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