Cobratoxina

Eur. J. Biocheni. 193, 789 - 799 (1 990) 0FEBS 1990

Two-dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution
Chin YU, Chang-Shin LEE, Li-Chin CHUANG, You-Rhon SHE1 and Chi Ying WANG Chemistry Department, National Tsing Hua University, Hisnchu, Taiwan (Received February 15/July 6, 1990) - EJB 90 0162

The 'H-NMR spectra of cobrotoxin, a neurotoxic proteinisolated from Formosan cobra Nuju naju atra, have been studied by two-dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple- and double-stranded P-sheet has also beendetermined by observing the NOE. Cobrotoxin is a neurotoxic protein isolated from Formosan cobra Nuju nuju utru [l, 21. It is a small basic single polypeptide consisting of 62 amino acid residues ( M , 6949) cross-linked by four disulfide bonds. This protein binds with high affinity to the nicotinic acetylcholine receptor of the postsynaptic membrane and blocks neuromuscular transmission across cholinergicsynapse [3], eventually stopping muscle contraction. In order to understand the molecular basis of this activity, we have undertaken a detailed study of the structure of cobrotoxin in solution by 'H-NMR spectroscopy. A few 'H-NMR lines of cobrotoxin (mostly due to aromatic and methyl protons) have been previously assigned [4 71. Most assignments were achieved with one-dimensional NMR techniques,and the majority of the resonances were not assigned. Two-dimensional NMR has however been shown to be of considerable value in the spectroscopy of cobrotoxin. In this paper we report a detailed study of the 2D-NMR spectra of cobrotoxin. In addition, our assignments have enabled independent determination of secondary structure of cobrotoxin in solution. at 5°C; the spectrum of such a samplemeasured at 15°C contains resonances from slowly exchanging amide protons. All NMR spectra were recorded on a 400-MHz spectrometer (Bruker AM-400) ; 4,4-dimethyl-4-silapentane-lsulfonate was used as an internal standard. Two-dimensional correlated spectroscopy (COSY) [9, 101 and RELAY [ l l , 121 experiments were performed in the absolute value mode. The solvent peak (H20) was suppressed by constantirradiation. For exchanged samples in D 2 0 solution, double-quantumfiltered COSY (2QF-COSY) [13] was executed in the phasesensitive mode to obtain Jconnectivities. 2D NOE (NOESY) spectra were also performed in the phase-sensitive mode with a mixing period 200ms. Quadrature detection in o1 was achieved using time-proportional phase incrementation [14, 151. All homonuclear 2D experiments wereperformed on a 20-mM protein sample; 768 tl increments were recorded, with 2048 complex points. For each free induction, the decays were Fourier-transformed on co2 using a Kaiser window and an w1 using a 45" phase-shifted sine-bell window. All two-dimensional data were processed on a Computer (pVAX 111, MV 3600), using a version of software provided by Dr Dennis Hare. RESULTS AND DISCUSSION Identifiationof spin systems Spin systems were related to residue types on the basis of COSY, NOESY and RELAY spectra in D 2 0 and H 2 0 . We identified the Trp29 aromatic side-chain spin system by observing C7H-C6H-C5H-C4H connections in the DQF-COSY spectrum. Connections between N I H and C7H of Trp29 were identified using the (NlH,C7H) and (NlH,C2H) cross peaks in the NOESY spectrum. Seven Gly residues werereadily identified by their strong geminal coupling in the DQF-COSY spectrum. Each of eight Thr spin systems was identified by the position and multiplet patterns of its two cross peaks. The CPH,CH3)and (CPH,CaH) cross peaks of Thr are recognized as the typical A3MX spin system. AMX spin systems for 'Tyr, Cys, Asn and Trp amino-acid residues in cobrotoxin have a similar position and cross peak...