Design, construction, crystallization, and preliminary X-ray studies of a fine-tuning mutant (F133V) of module-substituted chimera hemoglobin.
A chimera betaalpha-subunit of human hemoglobin was crystallized into a carbonmonoxy form. The protein was assembled by substituting the structural portion of a beta-subunit of hemoglobin (M4 module of the subunit) for its counterpart in the alpha-subunit. In order to overcome the inherent instability in the crystallization of the chimera subunit, a site-directed mutagenesis (F133V) technique was employed based on a computer model. The crystal was used for an X-ray diffraction study yielding a data set with a resolution of 2.5 A. The crystal belongs to the monoclinic space group P21, with cell dimensions of a = 62.9, b = 81.3, c = 55.1 A, and beta = 91.0 degrees . These dimensions are similar to the crystallographic parameters of the native beta-subunit tetramers in three different ligand states, one of which is a cyanide form that was also crystallized in this study.
Shirai
T
T
Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Japan. i45282a@nucc.cc.nagoya-u.ac.jp
Fujikake
M
M
Yamane
T
T
Inaba
K
K
Ishimori
K
K
Morishima
I
I
D000595:Amino Acid Sequence; D017076:Computer-Aided Design; D018360:Crystallography, X-Ray; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D006454:Hemoglobins; D011993:Recombinant Fusion Proteins