Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy.
The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 A. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 A. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C(4) symmetric particles with edge and diagonal lengths of 65 A and 80 A, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C(4) symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.
Hamada
Daizo
D
Department of Developmental Infectious Diseases, Research Institute, Osaka Medical Center for Maternal and Child Health, 840 Murodo, Izumi, Osaka 594-1011, Japan.
Higurashi
Takashi
T
Mayanagi
Kouta
K
Miyata
Tomoko
T
Fukui
Takashi
T
Iida
Tatsuya
T
Honda
Takeshi
T
Yanagihara
Itaru
I
D001427:Bacterial Toxins / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D053838:Scattering, Small Angle; D014461:Ultracentrifugation; D014736:Vibrio parahaemolyticus / Q000737:chemistry* / Q000472:pathogenicity; D037521:Virulence Factors; D014965:X-Rays
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D037521:Virulence Factors; C068489:thermostable direct hemolysin