Purification, N-terminal sequencing, crystallization and preliminary structural determination of atratoxin-b, a short-chain alpha-neurotoxin from Naja atra venom.
Atratoxin-b, a short-chain alpha-neurotoxin purified from Naja atra (mainland Chinese cobra) venom using a three-step chromatography procedure, has an apparent molecular mass of 6950 Da with an alkaline pI value (>9.5) and consists of one single polypeptide chain as estimated by MALDI-TOF mass spectrometry and SDS-PAGE. The protein is toxic to mice, with an in vitro LD(50) of about 0.18 mg kg(-1). Its N-terminal amino-acid sequence, LECHNQQSSQTPTIT, displays a very high homology to those of other alpha-neurotoxins. The overall three-dimensional structure of atratoxin-b is very similar to that of the homologous erabutoxin-a, as shown by the crystallographic molecular replacement and preliminary refinement results, with an R factor and R(free) of 27 and 29%, respectively. The microcrystal slowly grew to dimensions of approximate 0.1 x 0.1 x 0.15 mm over eight months using hanging-drop vapour-diffusion method. It gave a set of diffraction data to 1.56 A resolution using X-rays of wavelength 1.1516 A generated by the X-ray Diffraction and Scattering Station of beamline U7B at the National Synchrotron Radiation Laboratory (Hefei, China); this is the first example of the use of this beamline in protein crystallography. The crystals belong to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 49.28, c = 44.80 A, corresponding to one molecule per asymmetric unit and a volume-to-mass ratio of 1.96 A(3) Da(-1).
Lou
Xiaohua
X
Key Laboratory of Structural Biology, Chinese Academy of Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People's Republic of China.
Tu
Xiongying
X
Pan
Guoqiang
G
Xu
Chaoyin
C
Fan
Rong
R
Lu
Wanhua
W
Deng
Wenhan
W
Rao
Pingfan
P
Teng
Maikun
M
Niu
Liwen
L
D000595:Amino Acid Sequence; D000818:Animals; D003460:Crystallization; D004546:Elapid Venoms / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006860:Hydrogen Bonding; D019476:Insect Proteins; D051379:Mice; D008958:Models, Molecular; D009498:Neurotoxins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014961:X-Ray Diffraction
D004546:Elapid Venoms; D019476:Insect Proteins; D009498:Neurotoxins; C480725:atratoxin-b