Sequential assembly of the wedge of the baseplate of phage T4 in the presence and absence of gp11 as monitored by analytical ultracentrifugation.
The baseplate wedge of bacteriophage T4 consists of seven gene products, namely, gp11, gp10, gp7, gp8, gp6, gp53, and gp25, which assemble strictly in this order with an exception that gp11 can bind to gp10 at any stage of the assembly. In this study, all the seven corresponding genes are expressed as recombinant proteins and all the possible combinations of the gene products are tested for interactions by analytical ultracentrifugation. No interactions among gene products that violate the strict sequential binding are observed except that gp6, gp53, and gp25 interact with each other weakly, but significantly. However, when gp6 is previously bound to the precursor complex, only gp53 binds to gp6 strongly and then gp25 binds to complete the wedge formation. This result indicates that the strict sequential association is based on the conformational change of the complex upon addition of each gene product. The binding constant between subunits in the intermediate complexes is too high to be measured. In fact, the binding of gp11 to gp10 is so tight that the binding constant could not be determined by trace sedimentation equilibrium. Also, no indication of dissociation of the intermediate complexes is found in sedimentation velocity, which indicates that other subunit interactions in the intermediate complexes are also strong. The 43.7 S complex, which formed upon addition of gp53, is a hexamer of the wedge complex and resembles the star-shaped baseplate. The s-value of the baseplate-like complex decreased to 40.6 S upon association with gp11 in spite of the increased molecular weight, which is reflected in the sharper edges of the baseplate-like structure which would have a higher friction.
Yap
Moh Lan
ML
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
Mio
Kazuhiro
K
Ali
Said
S
Minton
Allen
A
Kanamaru
Shuji
S
Arisaka
Fumio
F
D017122:Bacteriophage T4 / Q000378:metabolism*; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D014461:Ultracentrifugation / Q000379:methods*; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins