Protease accessibility laddering: a proteomic tool for probing protein structure

Structure
Svetlana DokudovskayaMichael P Rout

Abstract

Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6xHis). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies.

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Citations

Oct 25, 2013·Molecular Biology of the Cell·Mario NiepelCaterina Strambio-De-Castillia
Sep 13, 2008·Biochemistry·Nathaniel C GilbertMarcia E Newcomer
Feb 14, 2009·PloS One·Nicole J CroteauMary Munson
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Apr 2, 2011·Molecular & Cellular Proteomics : MCP·Svetlana DokudovskayaCatherine Dargemont
Feb 15, 2012·The Journal of Cell Biology·Javier Fernandez-MartinezMichael P Rout
Jun 15, 2011·The Journal of Cell Biology·Mark C FieldMichael P Rout
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Jul 31, 2014·Molecular & Cellular Proteomics : MCP·Romain AlgretSvetlana Dokudovskaya
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