MPI für molekulare Genetik / Microscopy |
|Oligomerization of the SPP1 scaffolding protein|
|Authors:||Poh, Siew Lay; el Khadali, Fatima; Berrier, Catherine; Lurz, Rudi; Melki, Ronald; Tavares, Paulo|
|Date of Publication (YYYY-MM-DD):||2008-02-23|
|Title of Journal:||Journal of Molecular Biology|
|Journal Abbrev.:||J Mol Biol.|
|Issue / Number:||3|
|Copyright:||© 2008 Elsevier Ltd All rights reserved.|
|Review Status:||not specified|
|Abstract / Description:||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.|
|Free Keywords:||scaffolding protein; circular dichroism; chemical cross-linking; procapsid assembly; protein association|
|External Publication Status:||published|
|Communicated by:||Rudi Lurz|
|Affiliations:||MPI für molekulare Genetik|
|External Affiliations:||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.
DOI:scaffolding protein; circular dichroism; chemical ...
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