RESUMO
Observations of the jet-cooled infrared spectrum of CH(3)O in the CH stretching region have been extended, down to 2756 cm(-1) and up to 3003 cm(-1). In the lower frequency extension, a single vibronic band has been assigned. In the higher frequency region, the spectrum becomes complex above 2900 cm(-1) and remains so until near 2970 cm(-1) where it rapidly becomes sparse. Including the single vibronic band previously reported, a total of four bands have been assigned. Two bands including the original one follow a perpendicular DeltaP = +1 rotational selection rule and the other two bands follow a parallel DeltaP = 0 selection rule. In addition to these in the congested region between 2900 and 2970 cm(-1), ten isolated sub-bands (two P'' = -1/2, two P'' = +1/2, and six P'' = +1.5) have been assigned, but it has so far not been possible to connect these together to form bands. Taken together these observations suggest that there are strong vibronic couplings between the two CH stretching vibrations and the overtone and combination levels in the region.
RESUMO
Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain alpha-neurotoxins preferentially inhibit muscle-type nAChRs, whereas long-chain alpha-neurotoxins block both muscle-type and alpha7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of alpha- and kappa-neurotoxins is essential for their action on the alpha7 and alpha3beta2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfide-containing loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain alpha-neurotoxins and kappa-neurotoxins. The correct folding and structural stability for both chimeras were shown by (1)H and (1)H-(15)N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I blockalpha7 nAChRs with similar potency (IC(50) 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain alpha-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for alpha7 nAChR (IC(50) 126 nM) but did not convey activity at alpha3beta2 nAChRs. Docking of both chimeras toalpha7 andalpha3beta2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of kappa-neurotoxins underlie their selectivity for alpha3beta2 nAChRs.
