Quantitative Cross-Linking Via Engineered Cysteines to Study Inter-Domain Interactions in Bacterial Collagenases

Inter-domain movements act as important activity modulators in multi-domain proteins. Here, we present a protocol for inter-domain cross-linking via engi-neered cysteines. Using collagenase G (ColG) from Hathewaya histolytica as a model, we describe steps for the design, expression, purification, and cross -link-ing of the target protein. We detail a system to monitor the progress of the cross -linking reaction and to confirm the structural integrity of the purified cross-linked proteins. We anticipate this protocol to be readily adaptable to other multi -domain enzymes. For complete details on the use and execution of this protocol, please refer to Serwanja et al.1