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Journal Article

Oligomerization of the SPP1 scaffolding protein


Lurz,  Rudi
Max Planck Society;

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Poh, S. L., el Khadali, F., Berrier, C., Lurz, R., Melki, R., & Tavares, P. (2008). Oligomerization of the SPP1 scaffolding protein. Journal of Molecular Biology, 378(3), 551-564. doi:scaffolding protein; circular dichroism; chemical cross-linking; procapsid assembly; protein association.

Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-804E-0
Viral scaffolding proteins direct polymerization of major capsid protein subunits into icosahedral procapsid structures. The scaffolding protein of bacteriophage SPP1 was engineered with a C-terminal hexahistidine tag (gp11-His6) and purified. The protein is an α-helical-rich molecule with a very elongated shape as found for internal scaffolding proteins from other phages. It is a 3.3 S tetramer of 93.6 kDa at micromolar concentrations. Intersubunit cross-linking of these tetramers generated preferentially covalently bound dimers, revealing that gp11-His6 is structurally a dimer of dimers. Incubation at temperatures above 37 °C correlated with a reduction of its α-helical content and a less effective intersubunit cross-linking. Complete loss of secondary structure was observed at temperatures above 60 °C. Refolding of gp11-His6 thermally denatured at 65 °C led to reacquisition of the protein native ellipticity spectrum but the resulting population of molecules was heterogeneous. Its hydrodynamic behavior was compatible with a mix of 3.3 S elongated tetramers (not, vert, similar 90%) and a smaller fraction of 2.4 S dimers (not, vert, similar 10%). This population of gp11-His6 was competent to direct polymerization of the SPP1 major capsid protein gp13 into procapsid-like structures in a newly developed assembly assay in vitro. Although native tetramers were active in assembly, refolded gp11-His6 showed enhanced binding to gp13 revealing a more active species for interaction with the major capsid protein than native gp11-His6.