Abstract
In this paper, we demonstrate that single enzyme molecules of β-galactosidase interconvert between different activity states upon exposure to short pulses of heat. We show that these changes in activity are the result of different enzyme conformations. Hundreds of single β-galactosidase molecules are trapped in femtoliter reaction chambers and the individual enzymes are subjected to short heating pulses. When heating pulses are introduced into the system, the enzyme molecules switch between different activity states. Furthermore, we observe that the changes in activity are random and do not correlate with the enzyme's original activity. This study demonstrates that different stable conformations play an important role in the static heterogeneity reported previously, resulting in distinct long-lived activity states of enzyme molecules in a population.
Highlights
Enzymes exhibit both fast changes and long-lived differences in activity [1,2,3,4,5,6,7]
We developed a platform to study changes in the kinetics of single non-immobilized enzyme molecules by introducing short heating pulses to the system (Figure S1A in File S1)
The assay consists of an array of surface-passivated sealed microwells containing a single active enzyme and a high concentration of the fluorogenic substrate resorufin b-D-galactopyranoside (RBG) (Figure 1A)
Summary
Enzymes exhibit both fast changes and long-lived differences in activity [1,2,3,4,5,6,7]. Previous experiments indicate that enzymes can exhibit long-lived activity differences at room temperature resulting in a distribution of activities between ostensibly identical molecules, termed ‘‘static heterogeneity’’ [2,3,12]. This static heterogeneity arises either from different conformations of the enzyme and/or different primary sequences between molecules, with the latter possibility resulting from errors in transcription and translation [11,13,14,15]. We demonstrate that static heterogeneity of an enzyme population is caused by the existence of many different yet stable conformations
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