Oligomerization of the SPP1 scaffolding protein |
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Authors: | Poh Siew Lay el Khadali Fatima Berrier Catherine Lurz Rudi Melki Ronald Tavares Paulo |
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Affiliation: | 1 Unité de Virologie Moléculaire et Structurale, UMR CNRS 2472, UMR INRA 1157 and IFR 115, Bât. 14B, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France 2 Laboratoire d'Enzymologie et Biochimie Structurales UPR 3082, Bât. 34, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France 3 Groupe Canaux Ioniques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR-CNRS 8619, Bât 430, Université de Paris-Sud 11, 91 405 Orsay Cedex, France 4 Max-Planck-Institut für Molekulare Genetik, Ihnestraße 73, 14195 Berlin, Germany |
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Abstract: | 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 (∼ 90%) and a smaller fraction of 2.4 S dimers (∼ 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. |
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Keywords: | f/f0, frictional ratio value SEC, size-exclusion chromatography s20,w, sedimentation coefficient MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight |
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