Structurally Guided In Vivo Crosslinking
The focus of modern molecular biology on protein structure and function has reached unparalleled levels. Whether interacting with nucleic acids or other proteins, protein contacts are the basis for fine-tuning all cellular processes. It is for this reason imperative that protein interactions are studied in ways that reflect actual events taking place inside living cells.
Here, we describe in detail a method that combines the residue-level resolution provided by structural biology with physiological studies inside living cells, with the overall goal of explaining the contribution of protein–protein interactions in cellular processes. We use as a powerful example our experience with the DNA exit gate interface of the chromosomal cohesin complex, and we argue that this methodology may be followed to address similar questions within any protein complex and in various model systems.
Key wordsCohesin Protein–protein interaction Cysteine Immunoprecipitation Yeast Crosslinking BMOE dBBr
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