Single-Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-Linked Peptidoglycan Substrates

Abstract

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field effect transistors through pyrene linker molecules. The substrate driven, hinge bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibits processive slow turnover rates of 20 – 50 s−1 and rapid 200 – 400 s−1 non-productive motions. The latter, nonproductive binding events occupy 43% of the enzyme’s time in the presence of the cross-linked peptidoglycan, but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate, but appears to sidestep the peptide cross-links to zigzag through the wild-type substrate.

Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)

“…29,30 Nucleotide incorporation occurs after closing of the fingers subdomain; 12 in X-ray crystal structures, both template and complementary dNTP binding are required for KF to access its closed conformation. 17 Therefore, we assign the low- and high-current states of the FET to correspond to the fingers-closed and fingers-opened conformations of KF, respectively.…”

“…This variant of KF was designed to have a single cysteine at residue 790 near the highly mobile fingers subdomain for bioconjugation to the SWNT, following reaction with pyrene-maleimide as described previously. 29,30 This position places the SWNT close to a region undergoing dramatic conformational changes during the opening and closing of the enzyme. 17 (Figure 1b).…”

Real-time monitoring of conformational transitions of single-molecule histone deacetylase 8 with nanocircuits

“…15,16 To systematically monitor the dynamic interaction between HDAC8 and ligands, two configurations of nanocircuits are prepared (Fig. 1).…”

Protein Engineering with Biosynthesized Libraries from Bordetella bronchiseptica Bacteriophage

“…Our lab uses this protein in single molecule studies [18], [19], and its binding partners and inhibitors could provide useful tools for biophysical studies. The Bvg – - SMPL was biopanned against biotinylated lysozyme in solution before capture with streptavidin-coated magnetic beads.…”