3 research outputs found
Single-Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-Linked Peptidoglycan Substrates
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 exhibited
processive low turnover rates of 20–50 s<sup>–1</sup> and rapid (200–400 s<sup>–1</sup>) nonproductive motions.
The latter nonproductive binding events occupied 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 appeared
to sidestep the peptide cross-links to zigzag through
the wild-type substrate
Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology
Single-molecule techniques can monitor
the kinetics of transitions
between enzyme open and closed conformations, but such methods usually
lack the resolution to observe the underlying transition pathway or
intermediate conformational dynamics. We have used a 1 MHz bandwidth
carbon nanotube transistor to electronically monitor single molecules
of the enzyme T4 lysozyme as it processes substrate. An experimental
resolution of 2 μs allowed the direct recording of lysozyme’s
opening and closing transitions. Unexpectedly, both motions required
37 μs, on average. The distribution of transition durations
was also independent of the enzyme’s state: either catalytic
or nonproductive. The observation of smooth, continuous transitions
suggests a concerted mechanism for glycoside hydrolysis with lysozyme’s
two domains closing upon the polysaccharide substrate in its active
site. We distinguish these smooth motions from a nonconcerted mechanism,
observed in approximately 10% of lysozyme openings and closings, in
which the enzyme pauses for an additional 40–140 μs in
an intermediate, partially closed conformation. During intermediate
forming events, the number of rate-limiting steps observed increases
to four, consistent with four steps required in the stepwise, arrow-pushing
mechanism. The formation of such intermediate conformations was again
independent of the enzyme’s state. Taken together, the results
suggest lysozyme operates as a Brownian motor. In this model, the
enzyme traces a single pathway for closing and the reverse pathway
for enzyme opening, regardless of its instantaneous catalytic productivity.
The observed symmetry in enzyme opening and closing thus suggests
that substrate translocation occurs while the enzyme is closed
Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)
Bioconjugating
single molecules of the Klenow fragment of DNA polymerase
I into electronic nanocircuits allowed electrical recordings of enzymatic
function and dynamic variability with the resolution of individual
nucleotide incorporation events. Continuous recordings of DNA polymerase
processing multiple homopolymeric DNA templates extended over 600
s and through >10 000 bond-forming events. An enzymatic
processivity
of 42 nucleotides for a template of the same length was directly observed.
Statistical analysis determined key kinetic parameters for the enzyme’s
open and closed conformations. Consistent with these nanocircuit-based
observations, the enzyme’s closed complex forms a phosphodiester
bond in a highly efficient process >99.8% of the time, with a mean
duration of only 0.3 ms for all four dNTPs. The rate-limiting step
for catalysis occurs during the enzyme’s open state, but with
a nearly 2-fold longer duration for dATP or dTTP incorporation than
for dCTP or dGTP into complementary, homopolymeric DNA templates.
Taken together, the results provide a wealth of new information complementing
prior work on the mechanism and dynamics of DNA polymerase I