Labeling studies of photolabile philanthotoxins with nicotinic acetylcholine receptors: mode of interaction between toxin and receptor

AbstractBackground: The nicotinic acetylcholine receptors (nAChRs) and glutamate receptors are ligand-gated cation channels composed of five separate polypeptide chains. A 43 kDa protein of unknown function is noncovalently associated with the cytoplasmic side of nAChR in vivo. The -venoms of many wasps and spiders ccntain toxins that block the activity of these channels. Philanthotoxin-433 (PhTX-433) is a non-competitive channel blocker found in the venom of the wasp Philanthus. We have used a photolabile derivative to investigate how PhTX-433 interacts with nAChRs.Results: A radiolabeled PhTX analog, containing a photolabile group substituted on one of its aromatic rings, photocrosslinked to all five subunits (α, α′, β, γ, δ) of purified nAChR in the absence of the 43 kDa protein. In the presence of the 43 kDa protein, the a subunit was preferentially labeled. Proteolysis of the receptor after crosslinking indicated that the hydrophobic end (head) of the PhTx-433 analog bound to the cytoplasmic loop(s) of the a-subunit. Binding is inhibited by other non-competitive channel blockers such as the related polyamine-amide toxins from spiders and chlorpromazine.Conclusions: These results, coupled with previous structure/ activity studies, lead to a putative model of the binding of PhTx and related polyamine toxins to nAChRs in vitro. The 43 kDa protein appears to influence the orientation of toxin binding. Further binding studies are necessary to confirm the model and to define how toxins enter the receptor and how they are oriented within it. A precise understanding of ligand/receptor interaction is crucial for the design of drugs specific for a particular subtype of receptor

Synthesis of both enantiomers of akolactone B and (+)-ancepsenolide

The syntheses of (+)- and (−)-akolactone B and (+)-ancepsenolide were accomplished using a Pd-catalyzed carbonylation. As to the absolute configuration of akolactone B, making a comparison of the specific rotation of both enantiomers of synthetic akolactone B and the natural compound suggests that the absolute configuration at the 4-position of akolactone B is (R).ArticleTETRAHEDRON-ASYMMETRY. 25(20-21):1367-1371 (2014)journal articl

Engineering a Hyper-catalytic Enzyme by Photoactivated Conformation Modulation

Enzyme engineering for improved catalysis has wide implications. We describe a novel chemical modification of Candida antarctica lipase B that allows modulation of the enzyme conformation to promote catalysis. Computational modeling was used to identify dynamical enzyme regions that impact the catalytic mechanism. Surface loop regions located distal to active site but showing dynamical coupling to the reaction were connected by a chemical bridge between Lys136 and Pro192, containing a derivative of azobenzene. The conformational modulation of the enzyme was achieved using two sources of light that alternated the azobenzene moiety in <i>cis</i> and <i>trans</i> conformations. Computational model predicted that mechanical energy from the conformational fluctuations facilitate the reaction in the active-site. The results were consistent with predictions as the activity of the engineered enzyme was found to be enhanced with photoactivation. Preliminary estimations indicate that the engineered enzyme achieved 8–52 fold better catalytic activity than the unmodulated enzyme