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

Abstract

Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure-function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H 2 O 2-amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzymesubstrate interactions, enzyme-substrate active complex formation, and protein folding-binding interactions.enzyme | single molecule TIRF-magnetic tweezers microscopy | manipulate the conformation | enzymatic conformational dynamics | conformational fluctuation

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…For that purpose we used magnetic tweezers applying a pulling force of ∼2 pN. Although the applied force is much weaker than the force needed to break a hydrogen bond (6-9 pN), previous reports suggest that the 1-to 2-pN external pulling force can deform or unfold an enzyme by 30-100%, thereby weakening the enzyme-substrate interaction (20,44) (see Supporting Information for the force calibration curve). Fig.…”

“…Additionally, fluorescent product molecules are produced continuously in turnover cycles and this eliminates the negative impact of photobleaching, and thereby long time trajectories from a single enzyme can be recorded (8,9). In this work, we extend the SM fluorogenic assay and imaging analysis to a new dimension by combing the total internal reflection fluorescence microscopy (TIRFM)-guided confocal time-resolved SM photon time-stamping spectroscopic approach and the SM fluorogenic assay combined with magnetic tweezers force manipulation of molecular conformations: We used the nascent formed fluorescent product as an in situ probe to study the active site conformational fluctuation dynamics during the enzymatic reaction from the moment the product incipiently formed to the releasing of the fluorescent product from the active site (9,20,25). It is a conceptual and technical advancement to use a nascent-formed fluorogenic product molecule serving as a probe for enzymatic reaction active site with perfectly fitted position with the critical molecular interactions without perturbing the active site as in the case of site-specific labeling.…”

“…It is a conceptual and technical advancement to use a nascent-formed fluorogenic product molecule serving as a probe for enzymatic reaction active site with perfectly fitted position with the critical molecular interactions without perturbing the active site as in the case of site-specific labeling. Furthermore, magnetic tweezers give us the edge over chemical denaturation by introducing conformational perturbation in a noninvasive and controlled way (20,26), that is, we are able to deform the enzymatic conformations under enzymatic conditions, which is beyond the conventional protein structure denature assays that are carried out under chemical conditions, such as pH, electrolytes, and chemical denaturants, which are not physiological enzymatic reaction conditions.…”

“…We recently reported that horseradish peroxidase (HRP) can involve multiple intermediate conformational states and pathways during product releasing, and a significant pathway is that the product molecules are spilled out from the enzymatic reaction site; however, releasing the product molecules through a loosely bound enzyme-product state and an open active site remain parallel product releasing pathways (9). Further, very recently our group has reported that HRP retains a significant amount of its activity even under conformational perturbations (20). Here, we aim to systematically characterize not only the mechanism and dynamics involved in product release of HRP but also the complex conformational dynamics involved in each step of a catalytic cycle by directly manipulating the reaction coordinates.…”

Single-molecule fluorescence resonance energy transfer in molecular biology

“…Presently, smFRET coupled with optical tweezers, magnetic tweezers, or AFM has shown very promising results. 58,128,142 Potential future directions could involve the combination of smFRET with biomembrane force probe (BFP) 131 and lattice light-sheet microscopy. 133 Also, resolution of smFRET efficiency in 3D orientation will also be a major future advancement.…”