Important role of methionine 145 in dimerization of bovine β-lactoglobulin.
β-Lactoglobulin (LG) contains nine β-strands (strands A-I) and one α-helix. Strands A-H form a β-barrel. At neutral pH, bovine LG (BLG) forms a dimer and the dimer interface consists of AB-loops and the I-strands of two subunits. On the other hand, equine LG (ELG) is monomeric. The residues 145-153 of BLG, which compose a dimer interface, are entirely different from those of ELG. The difference in the association states between BLG and ELG can be attributed to the residues 145-153. To confirm this, we constructed a chimeric LG, ImBLG (I-strand mutated BLG), in which the residues 145-153 were replaced with those of ELG. Gel-filtration chromatography and analytical ultracentrifugation revealed that ImBLG existed as a monomer. To identify the residues important for dimerization, we constructed several revertants and investigated their association. This experiment revealed that, in addition to the interface residues (Ile147, Leu149 and Phe151), Met145 is critical for dimerization. Although Met145 does not contact with the other protomer, it seems to be important in determining the backbone conformation of the I-strand. This was supported by the fact that all Met145-containing mutants showed circular dichroism spectra similar to BLG but different from ImBLG.
Department of Bioinformatics, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan.
Hideaki
H
Ohtomo
Kazuo
K
Fujiwara
Masamichi
M
Ikeguchi
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019281:Dimerization; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D007782:Lactoglobulins; D008715:Methionine