Placing single-molecule T4 lysozyme enzymes on a bacterial cell surface: toward probing single-molecule enzymatic reaction in living cells

Biophysical Journal
Dehong Hu, H Peter Lu

Abstract

The T4 lysozyme enzymatic hydrolyzation reaction of bacterial cell walls is an important biological process, and single-molecule enzymatic reaction dynamics have been studied under physiological condition using purified Escherichia coli cell walls as substrates. Here, we report progress toward characterizing the T4 lysozyme enzymatic reaction on a living bacterial cell wall using a combined single-molecule placement and spectroscopy. Placing a dye-labeled single T4 lysozyme molecule on a targeted bacterial cell wall by using a hydrodynamic microinjection approach, we monitored single-molecule rotational motions during binding, attachment to, and dissociation from the cell wall by tracing single-molecule fluorescence intensity time trajectories and polarization. The single-molecule attachment duration of the T4 lysozyme to the cell wall during enzymatic reactions was typically shorter than the photobleaching time under physiological conditions. Applying single-molecule fluorescence polarization measurements to characterize the binding and motions of the T4 lysozyme molecules, we observed that the motions of wild-type and mutant T4 lysozyme proteins are essentially the same whether under an enzymatic reaction or not. The changing...Continue Reading

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Citations

Jan 14, 2012·Journal of the American Chemical Society·Yongki ChoiGregory A Weiss
May 2, 2013·Journal of the American Chemical Society·Tivoli J OlsenGregory A Weiss
Apr 12, 2005·Biophysical Journal·Mark E BowenAxel T Brunger
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Dec 7, 2013·Chemical Society Reviews·H Peter Lu
Mar 8, 2020·Nature Communications·Hugo SanabriaClaus A M Seidel
Jun 12, 2013·Physical Chemistry Chemical Physics : PCCP·Yongki ChoiPhilip G Collins
Nov 21, 2020·International Journal of Molecular Sciences·Seung-Gyun WooDae-Hee Lee
Oct 28, 2008·The Journal of Physical Chemistry. B·Xuefei Wang, H Peter Lu
Dec 17, 2009·The Journal of Physical Chemistry. B·Zuojia LiuErkang Wang
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Feb 28, 2014·The Journal of Physical Chemistry. B·Shirsendu GhoshKankan Bhattacharyya
May 9, 2014·The Journal of Physical Chemistry. B·Shyamtanu ChattorajKankan Bhattacharyya

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