Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein |
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Authors: | Padilla-Meier G Pauline Teschke Carolyn M |
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Institution: | 1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA2 Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA |
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Abstract: | Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60-300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini. |
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Keywords: | dsDNA double-stranded DNA FRET fluorescence resonance energy transfer WT wild type NEM N-ethyl maleimide |
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