Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin.
We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.
Tame
J
J
Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
Shih
D T
DT
Pagnier
J
J
Fermi
G
G
Nagai
K
K
D004926:Escherichia coli / Q000235:genetics; D005583:Fourier Analysis; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014633:Valine / Q000235:genetics*; D014961:X-Ray Diffraction
D006454:Hemoglobins; D008024:Ligands; D011994:Recombinant Proteins; D005914:Globins; D014633:Valine; D010100:Oxygen