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          Document History for Document ID 412083

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Document Version Version Comment Date Status
412083.0 [No comment] 18.03.2009 14:57 Released

ID: 412083.0, 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
Language:English
Date of Publication (YYYY-MM-DD):2008-02-23
Title of Journal:Journal of Molecular Biology
Journal Abbrev.:J Mol Biol.
Volume:378
Issue / Number:3
Start Page:551
End Page:564
Copyright:© 2008 Elsevier Ltd All rights reserved.
Review Status:not specified
Audience:Experts Only
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
Document Type:Article
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.
Identifiers:URL:http://www.sciencedirect.com/science?_ob=ArticleUR...
DOI:scaffolding protein; circular dichroism; chemical ...