Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

Significance It is highly informative to actively manipulate the conformations of an enzyme molecule by an external force and simultaneously observing the responses of the enzymatic activity changes. However, one of the challenges for a conventional approach is that an enzyme cannot be denatured under an enzymatic reaction condition, which prevents a simultaneous conformational manipulation and activity measurements. Using our single-molecule total internal reflection (TIRF)-magnetic tweezers microscopic approach to manipulate the conformations of enzymes and simultaneously probing the enzymatic reactivity changes under enzymatic reaction conditions, we identified that enzymatic activity can be manipulated by external pulling force and that enzyme molecules with deformed conformation are still capable of showing significant activities, involving the enzymatic active site conformational fluctuations and substrate binding-induced folding-binding conformational changes.