The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.
The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. S]GTP␥S-bound G␣ q/11 revealed additional coupling to G q/11 , which also was homologously desensitized. Although G␣ q/11 coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G i in addition to G q/11 in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G s -and G q/11 -mediated signaling steps and desensitization and another leading to G i -mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.The hypothalamic peptide corticotropin-releasing factor (CRF) 1 not only regulates the stress response in mammals by activation of the pituitary adrenal axis (1) but is also involved in the control of the immune response, cardiovascular, reproductive, and cognitive function, ingestive behavior, pregnancy and labor (for a review, see Refs. 2 and 3). The multiple actions of CRF are mediated by two classes of specific CRF receptors, CRFR1 (4 -6) and CRFR2 (7,8), which are encoded by unique genes and of which some variants exist, produced by alternative processing of the transcripts from each of the genes (for
The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N-and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ⑀-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC 50 : 100 -300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC 50 : 0.6 -50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.
Background and purpose: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF 1 ), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N-and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs-and Gi-protein coupling of CRF 1 by a peptide (a-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF 1 states for Gs and Gi coupling are similar or different. Experimental approach: We studied the inhibitory effect of a-helical CRF(9-41) and antalarmin on the coupling of CRF 1 to Gs-and Gi-protein in human embryonic kidney cells, using the [ 35 S]-GTPgS binding stimulation assay. Key results: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin-and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. Conclusions and Implications:The results allow us to extend the two-domain model of CRF 1 activation by assuming that CRF 1 agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF 1 .
Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge. By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.