Single-Molecule Protein Conformational Dynamics in Enzymatic Reactions
- H. Peter Lu
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Enzymes involve many critical biological processes, and for some extends, the biological clock of a living cell is often regulated by enzymatic reactions. An enzymatic reaction involves active substrate–enzyme complex formation, chemical transformation, and product releasing, as we know of the Mechalis–Menten mechanism. Enzymes can change the biological reaction pathways and accelerate the reaction rate by thousands and even millions of times. It is the enzyme–substrate interaction and complex formation that play a critical role in defining the enzymatic reaction landscape, including reaction potential surface, transition states of chemical transformation, and oscillatory reaction pathways. Subtle conformational changes play a crucial role in enzyme functions, and these protein conformations are highly dynamic rather than being static. Using only a static structural characterization, from an ensemble-averaged measurement at equilibrium is often inadequate in predicting dynamic conformations and understanding correlated enzyme functions in real time involving in nonequilibrium, multiple-step, multiple-conformation complex chemical interactions and transformations.
Single-molecule assays have revealed static [1, 2, 3, 4, 5] and dynamic [3, 4, 5] disorders in enzymatic reactions by probing co-enzyme redox state turnovers  and enzymatic reaction product formation in real time [4, 5]. Static and dynamic disorders [6, 7, 8, 9, 10] are, respectively, the static rate inhomogeneities between molecules and the dynamic rate fluctuations for individual molecules. Dynamic disorder, which is not distinguishable from static disorder in an ensemble-averaged measurement, has been attributed to protein conformational fluctuations [3, 4, 5, 11]. The protein conformational motions at the enzyme active site, which include enzyme–substrate complex formation, enzymatic reaction turnovers, and product releasing, are mostly responsible for the inhomogeneities in enzymatic reactions [3, 4, 5]. Consequently, direct observations of conformational changes along enzymatic reaction coordinates are often crucial for understanding inhomogeneities in enzymatic reaction systems .
We have applied single-molecule spectroscopy and imaging to study complex enzymatic reaction dynamics and the enzyme conformational changes, focusing on the T4 lysozyme enzymatic hydrolyzation of the polysaccharide walls of Escherichia coli B cells. By attaching a donor–acceptor pair of dye molecules site-specifically to noninterfering sites on the enzyme, we were able to measure the hinge-bending conformational motions of the active enzyme by monitoring the donor–acceptor emission intensity as a function of time. We have also explored a combined approach, applying molecular dynamics (MD) simulation and a random-walk model based on the single-molecule experimental data. Using this approach, we analyzed enzyme–substrate complex formation dynamics to reveal (1) multiple intermediate conformational states, (2) oscillatory conformational motions, and (3) a conformational memory effect in the chemical reaction process . Moving forward to study enzymatic dynamics and enzyme conformational dynamics in living cells, we have developed a single-molecule enzyme delivery approach to place an enzyme specifically to an enzymatic reaction site on a cell membrane.
- Single-Molecule Protein Conformational Dynamics in Enzymatic Reactions
- Book Title
- Single Molecule Spectroscopy in Chemistry, Physics and Biology
- Book Subtitle
- Nobel Symposium
- pp 471-494
- Print ISBN
- Online ISBN
- Series Title
- Springer Series in Chemical Physics
- Series Volume
- Series ISSN
- Springer Berlin Heidelberg
- Copyright Holder
- Springer-Verlag Berlin Heidelberg
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- Editor Affiliations
- ID1. Dept. Biophysics, Stockholm University
- ID3. Dept. Biomolecular Physics, Royal Institute of Technology (KTH)
- H. Peter Lu (1)
- Author Affiliations
- 1. Department of Chemistry Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA
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