Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791-2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain ␦-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist (ϳ10 nM); the lowest when receptor was bound to an antagonist (ϳ500 nM); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of ϳ60 nM. Furthermore, GTP binding to the receptor⅐G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the G␣ o (K d ϳ 20 nM) and lowest affinity toward the G␣ i2 (ϳ590 nM) subtypes, whereas the agonist-bound state had highest affinity for the G␣ o and G␣ i2 subtypes (K d ϳ 9 nM and ϳ 7 nM, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.Opioid receptors belong to the superfamily of GPCRs.1 Their predicted topology is that of a polytopic integral membrane protein with seven membrane-spanning helical segments, an extracellular N terminus and an intracellular C terminus. These receptors and their endogenous ligands, opioid peptides such as endorphins and enkephalins, form a neuromodulatory system that is involved in stress-induced analgesia, that affects locomotive activity and regulates neuroendocrine physiology and autonomic functions such as respiration, blood pressure, and gastrointestinal motility. The opioid system has also been shown to play a role in learning and memory and possibly in the modulation of the immune system (1) and is an important factor in pain modulation and drug abuse. Studies to determine the G-protein subtypes that mediate the intracellular signaling of the opioid receptor systems have shown that functional coupling occurs to the G i -G o family of G-proteins (2-4). The human brain ␦-opioid receptor has been cloned (5), stably transfected in Chinese hamster ovary (CHO) cells, and characterized (6).Due to their integral membrane nature and their low cellular concentrations, there is little information on the structures and functional mechanisms of the opioid receptors. Furthermore, classic pharmacological methods only give indirect information about the interaction of GPCRs with G-protein...

The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.