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.

The key roles contributed to biological processes by enzymes make correlating enzyme motions with their catalytic functions an important and challenging problem.^1,2 The spatial and temporal heterogeneity of enzymes in bulk solution prevents ensemble measurements from examining an enzyme’s conformational dynamics along its reaction coordinates.^3–5 However, single molecule studies, typically using fluorescence resonance energy transfer (FRET), can characterize conformational dynamics,^6,7 and also reveal the static and dynamic disorders inherent to enzyme activities.^2,8

Lysozyme offers a particularly good model protein to elucidate detailed enzyme dynamics and conformational motions from single molecule observations.⁹ An antibiotic component of innate immunity, lysozyme digests the peptidoglycan of bacterial cell walls. The enzyme catalyzes the hydrolysis of the glycosidic bonds connecting the repeating subunits of the cell walls between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

During catalysis, lysozyme undergoes hinge bending motions of 8 Ź⁰ that, with the addition of fluorescent labels, enable single molecule FRET experiments.³ As visualized by FRET on bacterial surfaces, lysozyme adheres to the peptidoglycan for long periods of time.³ Such observations suggest, but do not prove, that lysozyme processively catalyzes the hydrolysis of a large number of glycosidic bonds before dissociation. FRET also proves that lysozyme interrupts its catalytic glycosidic hydrolysis with periods of rapid movements that do not result in bond hydrolysis.¹¹ However, the degree of processivity, the reason for the rapid nonproductive motions, and the effects of substrate cross-linking remain incompletely understood for two main reasons. First, the peptidoglycan is highly heterogeneous in size, and features a heavily cross-linked structure due to pentapeptides connecting the NAM subunits of the polysaccharides chains.^3,12 Second, FRET and other optical techniques are limited by fluorophore bleaching, which prevents long-term measurement of the same individual molecule. Such considerations motivate development of new methods for examining single proteins.

To address these issues, two developments are reported here. First, we synthesized a linear peptidoglycan substrate for lysozyme that includes non-cross-linked tripeptides (Figure 1(a)). Second, we have developed nano-circuits comprising individual lysozyme molecules attached to single-walled carbon nanotube (SWNT) field effect transistors (FETs). The dynamic motions of the attached lysozyme induce fluctuations in the SWNT-FET conductance through a charge gating effect, similar to previous work with SWNT-FET sensors.^13–15. This electronic, rather than optical, transduction allows monitoring of the dynamic interactions of individual lysozyme molecules over long periods of time. Combining these two advances provides new insights into lysozyme processing of peptidoglycans.

FIGURE 1.

(a) The chemical structure of the synthesized peptidoglycan substrate. (b) Schematic diagram of a single molecule T4 lysozyme SWNT-FET circuit. (c) AFM topography of a SWNT FET after coating with the pyrene linker, lysozyme incubation, and washing to reduce nonspecific binding. The arrow indicates a site of lysozyme attachment.

To examine the dynamics of lysozyme, individual lysozyme molecules were attached to the sidewalls of SWNT-FETs as shown in Figure 1(b). Atomic force microscopy images confirmed attachment of one lysozyme within a window opened by electron beam in an insulating poly-methyl methacrylate layer placed over the entire device (Figure 1(c)).¹⁶ Used throughout this report, an S90C variant of pseudo-wild-type T4 lysozyme¹⁷ provided a single thiol for conjugation to a pyrene-linked maleimide.^13,18 The aromatic pyrene of this linker can strongly adhere to the SWNT sidewall via π-π interactions.^13,19 Then, multiple washing steps tailored the density of the lysozyme attachments to yield an average of one attachment per device.

As the enzyme moves, charged surface functionalities near the SWNT attachment site can modulate the source-drain conductance G(t) of the underlying device. Electrical measurements were performed with the device submerged in electrolyte (phosphate buffer, pH 7.5) under an applied source-drain voltage of 0.1 V and an electrolyte-drain bias of 0 V (controlled by a Pt reference electrode). Devices were exposed to excess substrate and measured for 600 s; then, the same device was thoroughly rinsed with water to remove surface-bound substrate before being probed a second time with a different substrate. The two substrates were tested in alternating orders on different devices, in order to protect against systematic bias. Our analysis filters the DC and lowest frequency (<10 Hz) components of G(t) in order to focus on time-varying fluctuations and transients of ΔG(t).

Three distinct behaviors are observed and categorized in Figure 2. The most common behavior is two-state G(t) fluctuation that switches asymmetrically at an average rate of 30.0 s⁻¹ (Figure 2(a), colored green). A second behavior involves fluctuations between the same two G(t) levels, but at the much faster average rate of 287 s⁻¹ (Figure 2(b), blue). A relatively featureless, inactive behavior also occurs (Figure 2(c), black), though this is primarily observed when measurements are performed in the absence of the lysozyme substrate peptidoglycan. Previous FRET measurements have proven that lysozyme motions are absent or disorganized in the absence of substrate, but that, when present, substrate can drive one dimensional, hinge bending motions at the same two rates we observe.¹¹ The slower rate is understood to be caused by productive processing of substrate by the enzyme, while the faster rate is associated with nonproductive, catalytically ineffective motions of the lysozyme.¹¹

FIGURE 2.

Source-drain conductance fluctuations of a lysozyme device in the presence of the cross-linked substrate. The color differentiates the three types of signal behavior observed, which include (a) slow switching with catalytic turnover (green), (b) rapid, nonproductive switching (blue), and (c) inactive (black).

Long duration measurements on single lysozyme molecules are possible with the SWNT-FET architecture. Five example measurements are shown in Figure 3 with data points colored to correspond to the three behaviors defined above. Figures 3(a), (b), and (c) are obtained from a single lysozyme device measured either without substrate (Figure 3(a)) or with one of two different substrates described in more detail below. Figures 3(d) and (e) show control measurements from devices incorporating two catalytically inactive lysozyme variants,²⁰ T26E and E11H. The two mutated residues, Thr and Glu, play key roles in the lysozyme mechanism for the catalysis of glycoside hydrolysis. The T26E variant produces a covalent adduct with the peptidoglycan substrate, and thus provides a constitutively substrate-bound version of the lysozyme. The E11H variant is also catalytically inoperable, but does not form a covalent bond to the substrate. The absence of two-level fluctuations when substrate is absent (Figure 3(a)) or when the two control variants are probed with substrate (Figures 3(d) and (e)) confirms that the two level ΔG(t) dynamics are caused by substrate-lysozyme interactions and catalysis, and not merely the presence of one substrate or the other.

FIGURE 3.

Long-duration, source-drain conductance fluctuations. (a) In the absence of substrate, no conductance fluctuations are observed, as demonstrated here for a single lysozyme device incubated in phosphate buffer. Addition of (b) linear substrate or (c) the cross-linked substrate results in the switching described in the text. Control experiments using (d) an E11H or (e) a T26E catalytically inactive T4 lysozyme variant show no activity when probed with the cross-linked (shown) or the linear substrate (not shown).

The main focus of this report is to compare lysozyme processing of two different peptidoglycan substrates, a linear and a cross-linked version. The linear lysozyme substrate was obtained through chemical synthesis as described in the Supporting Information. Designed to mimic bacterial cell walls, the linear substrate features an extended (NAG-NAM)_n polysaccharide. Appended to each NAM subunit, tripeptides are linked to lactic acid, but do not cross-link polysaccharide fragments (Figure 1(a)). An Alexa Fluor 647 dye was incorporated at a density of approximately 5% on the peptides; the fluorophore was not used in the present experiments, and is not expected to alter enzyme dynamics. The commercially obtained cell walls of peptidoglycan from Micrococcus luteus provide a second substrate to examine the effects of substrate cross-linking on enzyme catalysis. As shown by fluorescence-based assays with ensemble or bulk enzyme, T4 lysozyme can hydrolyze both substrates (Figures S3 and S4).

Figures 3(b) and 3(c) compare the catalytic activities of lysozyme processing linear or cross-linked substrates, respectively. Both substrates allow processive catalysis by lysozyme (green segments), but with different proportions of the nonproductive (blue) and inactive (black) behaviors. T4 lysozyme does not require, nor is it hindered by, the peptide cross-links of the substrate for its processive movement. Nevertheless, the linear substrate fundamentally alters the distribution of the lysozyme dynamics. Figure 4 summarizes the analysis of 24 independent measurements performed using 14 different devices, of which Figure 3 is representative. On average, lysozyme spends 88% of the total time processing the linear substrate, and makes few rapid, nonproductive motions (7% of its time).

FIGURE 4.

Percentages of lysozyme activities in the presence of linear (left) or cross-linked (right) peptidoglycan substrates. The standard deviation values for the activities are indicated in parentheses (n = 6 for the linear and n = 8 for the cross-linked substrates).

This processing of linear substrate contrasts sharply with observations made in the presence of the cross-linked substrate. The lysozyme increases the amount of time spent in rapid, nonproductive motions from 7% to 43% in the presence of the cross-linked peptidoglycan substrate (Figure 4). This difference in activity was very reproducible, regardless of whether the cross-linked or linear substrate was tested first. Furthermore, no degradation of lysozyme activity was observed during the reported experiments. Monitoring the same lysozyme molecule serially processing the two different types of substrates proves that the peptide cross-links trap the enzyme in this rapidly oscillating, nonproductive state. We hypothesize that lysozyme catalysis stalls before attempting to transit a cross-link to reach a neighboring polysaccharide. Thus, lysozyme could zigzag across the cell wall, as it processively catalyzes glycoside hydrolysis and passes across peptide cross-links.

Despite the major differences in the distribution of enzyme activities, the two substrates have chemically identical glycosidic bonds, and the two result in remarkably similar kinetic rates. The fluctuation in the rates of the catalytic processing in Figure 2(a) and the nonproductive motion in Figure 2(b) change only slightly, if at all, when one substrate is substituted for another. Ambiguity in this comparison is caused by the static disorder of individual enzymes and the local environment of the enzyme tethered to the SWNT-FETs, which both contribute to broad rate distributions. For example, the instantaneous, single molecule rates of catalytic turnover with the cross-linked substrate range from 17 to 59 s⁻¹. Nevertheless, quantitative comparisons have been made by averaging the mean rates, reported here with one standard deviation, from 100-s segments across multiple devices. Accordingly, the rate of catalytic turnover decreases 16%, from 35.9 ± 17.6 s⁻¹ (n = 9) for the linear substrate to 30.0 ± 14.5 s⁻¹ (n = 15) for the cross-linked substrate. The rate of nonproductive motions decreases 13%, from 329 ± 167 s⁻¹ for the linear substrate to 287 ± 184 s⁻¹ for the cross-linked substrate. Although the two substrates have overlapping ranges, the cross-linked substrate does appear to result in slightly slower rates. As might be expected, the presence of cross-links appears to slow catalytic processing. The additional slowing of the nonproductive motions is consistent with a model of the enzyme transiting from one polysaccharide to another at cross-linked points.

Both the linear and the cross-linked peptidoglycan exhibit a similar percentage of inactive times (black data in Figures 3 and 4). During these inactive periods, which have an average duration of 1.06 s, no switching was observed by the lysozyme device and G(t) remained in its low state. This low G(t) state is identical to the value observed when no substrate is present (Figures S5(b) and (d)). Furthermore, the inactive periods occur exclusively during periods of catalytic processing (green), and are never observed during the enzyme putatively transits at peptide cross-links (blue). These observations suggest that the inactive period is caused by dissociation that occurs when lysozyme processes to the end of a poysaccharide substrate. In this interpretation, lysozyme proceeds along the polysaccharide backbone, catalyzing the hydrolysis of multiple glycosidic bonds before dissociation occurs. Upon association of new substrate, the inactive period concludes. We observe that the enzyme always returns to a substrate-processing state (green).

Finally, we note a fourth type of behavior observed only with the cross-linked substrate and colored yellow in Fig. 3(c). These segments correspond to a novel behavior in which rapid, nonproductive motion at the peptide cross-links is interrupted for 0.5 to 3.0 s with G(t) stuck in its high value. Whereas the inactive periods discussed above are associated with dissociation events because of their low G(t), these pauses at high G(t) imply the enzyme is stuck in an enzyme-closed, substrate-bound configuration (Figures S5 (c) and (e)). This state occurs randomly and with a low probability, and it appears to be independent of the return to catalytic processing.

The results presented here demonstrate the tremendous potential of molecular electronics to uncover fundamental knowledge in biophysics. Lysozyme orthologs have been studied for over a century, and yet the reported nano-circuits can unveil new information about T4 lysozyme’s activities and dynamics including its processivity and the ability to potentially transit peptide cross-links. Lysozyme proceeds linearly to the ends of the linear substrate, but could sidestep over peptide cross-links of the cross-linked substrate. This study providing a relationship between lysozyme dynamics and functions will allow deeper understanding of the precise lysozyme motions for digesting the bacterial cell wall, and could guide the design of new enzymes for antibacterial and other applications.