In Vivo Disulfide-Bond Crosslinking to Study β-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates

Membrane-embedded β-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of β-barrels. One such method is “in vivo disulfide-bond crosslinking” which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of β-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between β-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.