Effect of Surface Crowding and Surface Hydrophilicity on the Activity

Jun 27, 2017 - Increasing surface hydrophobicity progressively decreased enzyme activity, but had no effect on thermal stability. Surface-sensitive su...
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Effect of Surface Crowding and Surface Hydrophilicity on the Activity, Stability and Molecular Orientation of a Covalently Tethered Enzyme McKenna M. Schroeder,† Qiuming Wang,† Somayesadat Badieyan,† Zhan Chen,*,† and E. Neil G. Marsh*,†,‡ †

Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States

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ABSTRACT: We have investigated two surface properties that are generally thought to have an important influence of enzyme activity and stability: surface hydrophobicity and surface crowding. Here two variants of an engineered bacterial nitro-reductase were covalently tethered to orient the protein’s pseudo-2-fold symmetry axis either parallel or perpendicular to the surface. The surface hydrophobicity was systematically varied by changing the ratio of methyl- to hydroxyl-groups displayed on the SAM surface, and the effects on enzyme activity, thermal stability, and structure investigated. Increasing surface hydrophobicity progressively decreased enzyme activity, but had no effect on thermal stability. Surface-sensitive sum frequency generation and attenuated total reflectance Fourier transform IR spectroscopies indicated that the enzyme is not denatured by the more hydrophobic surface, but is more likely trapped in less active conformations by transient hydrophobic interactions. In contrast, increasing enzyme surface concentration increased the specific activity of the parallel oriented enzyme, but had no effect on the activity of the perpendicularly oriented enzyme, suggesting that crowding effects are highly context dependent.

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INTRODUCTION Surface-immobilized proteins and enzymes find widespread use in applications diverse as industrial catalysis,1,2 biofuel cells,3 antibacterial and/or antifouling coatings,4−7 drug delivery,8 and biosensors.9,10 Immobilization of an enzyme on a solid support allows the catalyst to be easily recovered from a reaction and reused. Immobilization can also substantially improve enzyme stability under reaction conditions.11−15 This is thought to be a consequence of reducing enzyme mobility that in turn disfavors unfolding or aggregation, although the molecular mechanisms underlying the changes in stability and activity are not well understood.16,17 However, one drawback of immobilization is that the activity of the immobilized enzyme is often significantly lower, although there are certainly examples in which increases in activity are observed.18 We have previously investigated the properties of surfaceimmobilized enzymes that are covalently tethered through a unique cysteinyl residue to a chemically well-defined supporting surface comprising a self-assembled monolayer (SAM) of short poly ethylene glycol chains19 terminated with maleimide groups.20−24 The maleimide groups react specifically with the cysteine residue so that the enzyme is tethered to the SAM surface through a chemically precise linkage. This results in a population of enzyme molecules that is homogeneous with respect to their surface attachment point. This homogeneity facilitates the use of surface-sensitive vibrational spectroscopic techniques to obtain information on the structure and orientation of the immobilized enzymes. We have shown that altering the location of the attachment point, and thereby the © XXXX American Chemical Society

surface orientation of the enzyme, can have pronounced effects on both the specific activity and the thermal stability of the enzyme.20,21 Studies by other groups using different experimental systems, e.g., enzymes conjugated to gold nanoparticles,25 have documented similar links between enzyme orientation and stability and activity. As a model enzyme for our studies we have used an engineered version of the FMN-dependent enzyme nitroreductase, NfsB.20 NfsB catalyzes the NADH-linked reduction of a wide range of substrates including quinones and aromatic compounds containing nitro-groups. Its broad substrate range and convenient spectroscopic assay make it a good model system to examine the effects of surface interactions on enzyme stability and activity. Although NfsB is naturally a homodimeric enzyme, we have previously engineered a monomeric version of NfsB by designing a fusion protein in which the two monomers are linked with a ten-residue glycine sequence.20 The enzyme was coupled to the surface through cysteine residues introduced at one of two positions, 424 and 360, that were chosen to immobilize the enzyme with its pseudo-2-fold rotational symmetry axis either parallel or perpendicular to the surface normal (Figure 1). These variants were designated NfsB-424C and NfsB-360C respectively.20 This allowed us to examine the effect of enzyme surface orientation on the specific activity and thermal stability of NfsB. Here we extend our studies by Received: February 25, 2017 Revised: June 8, 2017 Published: June 27, 2017 A

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Figure 1. Immobilization of NfsB of SAMs. The two variants studied here were tethered on SAMs though engineered cysteinyl residues positioned to orient the enzyme with either the 2-fold axis either perpendicular (V424C, left) or parallel (H360C, center) to the surface. The molecular structure of the tethering group used to immobilize the enzyme is shown on the right. hours at 4 °C to ensure that cysteine residues were in the thiol form. The reduced enzyme was reacted with EG3−maleimide-derivatized surfaces (prepared as described above) at a final concentration of 5 μM in PBS at 4 °C, with shaking overnight. After reaction, excess enzyme was removed with 3 rinses of PBS, followed by 3 rinses with 5% Tween-20 in PBS to eliminate nonspecific binding. Finally, Tween20 was removed with 3 more PBS rinses. Control experiments using enzymes that lack surface-exposed cysteine residues were performed under identical protocols to evaluate the extent of nonspecific physisorption. In all cases, the amount of protein adsorbed was less than 10% that obtained with the chemically tethered enzymes and the enzymatic activity was negligible, suggesting that this represents a small amount of denatured adhering to the surface. SFG and ATR-FTIR Spectroscopy and Protein Orientation Determination. Detailed experimental procedures have been reported previously.15,20,21 Briefly, a commercial SFG system (EKSPLA Inc., Lithuania) was equipped with right angle CaF2 prisms to realize the near total reflection geometry for data collection. The incident angles for the input visible and IR beams are 59.0° and 54.5° with respect to the Z axis in the lab coordinate system. All the spectra were collected from 1500 to 1800 cm −1 with different polarization combinations of ssp (s-polarized SFG signal, s-polarized visible input, and p-polarized IR input) and ppp (p-polarized SFG signal, ppolarized visible input, and p-polarized IR input) at room temperature. A Nicolet 6700 FTIR spectrometer was used to collect s- and ppolarized protein amide I spectra from 1550 to 1750 cm −1. D2O buffer was used to avoid spectral confusion.20 The conformation and orientation of immobilized proteins were determined by fitting the spectral data using the in-house-developed programs reported previously.21 The NfsB crystal structure (PDB 1DS7) was used as a model for orientation determination. Activity Assay. Enzyme-functionalized beads were rinsed 3 times with 5 mL of PBS. The assay solution (2 mL) containing NADH (160 μM), 4-nitrobenzenesulfonamide (500 μM), in PBS, pH 7.5 was then added to the bead sample. A zero time point sample was immediately removed and absorbance measured at 340 nm. The sample was replaced into the reaction and the reaction mixture shaken vigorously so the beads remained suspended. Samples were removed every 30 s over the course of 2 to 4 min, and the absorbance due to NADH was measured. After the activity assays were complete, the bead samples were then washed with PBS and the protein concentration was measured using the micro-BCA reaction, as described previously.20 Thermal Stability Measurements. Thermal stability assays were performed both in free solution and on immobilized enzyme samples. For NfsB samples in solution, the enzyme was exchanged into PBS buffer and its concentration adjusted to 10 μM. FMN (final concentration 100 μM) was added to enzyme samples, and they were heated to a temperature between 20 to 65 °C for 5 min in a thermocycler. The samples were then cooled and held at room temperature for 10 min. The remaining activity was then measured as

investigating two parameters that have an important influence on the activity of immobilized enzymes: the hydrophobicity/ hydrophilicity of the supporting surface and the surface concentration of the enzyme molecules.13,14

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MATERIALS AND METHODS

NfsB Variant Design and Expression. Wild-type NfsB is a homodimer; however these experiments used the fusion protein described previously20 in which the two subunits are genetically linked by a string of ten-glycine residues. In this enzyme, a native cysteine residue at position 85 was mutated to an alanine and surface cysteines were introduced at position 424 (NfsB-V424C) and or 360 (NfsBH360C). The NfsB fusion genes were commercially synthesized by GenScript (New Jersey) and were cloned in the pET28b bacterial expression vector, which introduced an N-terminal His-tag. The enzymes were expressed and purified as previously described.20 Functionalization of Glass Beads. 75 μm glass beads (Supelco, acid-washed) were prepared for functionalization by cleaning in piranha solution (3:1 v:v concentrated sulfuric acid and 30% hydrogen peroxide) overnight. The beads were rinsed with water until neutral pH. Water was removed from the beads by rinsing with DMSO 4 times followed by 3 rinses with toluene. The beads were silanized by shaking overnight in anhydrous toluene containing 0.1% v/v, O(propargyl)-N-(triethoxysilylpropyl) carbamate in which the liquid volume was 4 times the volume of beads being functionalized. Excess silane-alkyne reagent was removed by rinsing 3 times with toluene. Azide-functionalized EG3 linkers, terminated with −OH, −OMe or −maleimide groups were reacted with the alkyne-functionalized glass bead surfaces by a click reaction in the presence of 1 mM copper sulfate, 50 mg·mL−1 sodium ascorbate in 50% DMSO and water. Reactions were incubated overnight with shaking at room temperature after the addition of appropriate azido linkers. Azido-EG3−maleimide was prepared by dissolving azido-EG3−amine 0.075 mmol in 1 mL dry DMSO, then adding maleimide−NHS ester 0.075 mmol, and shaking at room temperature for 1 h. The various EG3 SAM surfaces were constructed by reacting alkynefunctionalized 75 μm glass beads with a total of ∼12 nmol of azidoEG3 linkers per gram of beads. To construct EG3 SAM surfaces with mixed functionality, the appropriately functionalized azido-EG3 linkers were mixed in the desired molar ratios (total amount 12 nmol/g beads) and allowed to react with the alkyne-functionalized beads as described above. For example, an SAM surface containing a 1:20 mol ratio of EG3−maleimide to EG3−OH would have 0.57 nmol/g beads EG3−maleimide and 11.4 nmol/g beads EG3−OH. Similar protocols were used to functionalize SiO2-coated CaF2 prisms for SFG spectroscopy and supporting surfaces for ATR-FTIR spectroscopy.21 Enzyme Immobilization. Enzyme samples were prepared for immobilization by dialysis against phosphate-buffered saline solution (PBS), pH 7.2, followed by reduction with 1 mM TCEP for several B

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Figure 2. Specific activity of NsfB variants as a function of mole ratio of EG3−maleimide. Enzymes were immobilized on either EG3−OH-terminated or EG3−CH3-terminated SAMs containing EG3−Mal at molar ratios of 1:1, 1:10, or 1:20. described above. For NsfB immobilized on beads, samples were resuspended in PBS containing 100 μM FMN. Samples were then heated to a temperature between 20° to 65 °C using a thermocycler for 5 min. After heating, samples were cooled and held at room temperature for 10 min. The remaining specific activity was then measured as described above. Surface Density of Immobilize dNfsB. Parameters for the definition of a monolayer were calculated based on a diameter of ∼50 Å for NfsB and assuming the glass beads had an atomically flat surface. A diffuse monolayer was defined as one enzyme per 10 000 Å2, which is equivalent to 26 ng enzyme per mg of beads. A tightly packed monolayer was defined as 4 enzymes per 10 000 Å2, which is equivalent to 102 ng enzyme per mg of beads. Beads for surface coverage experiments were prepared as described above with alkyne functionalization followed by a click reaction with azido-EG3−Mal and azido-EG3−OH. Enzyme surface coverage was changed by increasing or reducing the enzyme concentration in the enzyme-binding step. The enzyme was covalently bound to functionalized beads in PBS at 4 °C with shaking overnight.

screen to determine the optimal surface density of maleimide functional groups, EG3−Mal was introduced into EG3−CH3 and EG3−OH SAMs at mole fractions ranging from 0.5 to 0.05. The NfsB-V424C and NfsB-H360C variants were immobilized on these surfaces, as described in the Materials and Methods section, and the specific activity of the enzymes determined (Figure 2). For both enzyme variants, immobilized on either the EG3−OH or EG3−CH3 SAM surfaces, the highest specific activities were obtained at a mole fraction of 0.1 EG3−Mal. Therefore, this mole fraction of EG3−Mal was used in the SAMs synthesized for all subsequent experiments in which the ratio of EG3−OH to EG3−CH3 was varied. The hydrophobicity of the surface was found to have a significant effect on enzyme activity. The activities of NfsBV424C and NfsB-H360C on the EG3−OH functionalized SAM surface were 5.7 ± 0.6 and 5.8 ± 0.4 μM/min/μg, respectively, whereas on the more hydrophobic EG3−CH3 functionalized SAM the activities dropped to 2.6 ± 0.1 and 2.8 ± 0.3 μM/ min/μg, respectively. To investigate the influence of the surface on enzyme activity in more detail, we selected one enzyme variant, NfsB-V424C, and immobilized it on mixed EG3−OH/ EG3−CH3 SAMs in which the mole fraction of EG3−CH3 was varied from 0.0 to 0.9 (the mole fraction of EG3−Mal was held constant at 0.1). The specific activity of NfsB-V424C immobilized on these surfaces deceased monotonically with increasing mole fraction of EG3−CH3 (Figure 3A). This suggests that the decrease in activity arises from multiple, nonspecific hydrophobic protein−surface interactions that are increased as the proportion of methyl-terminated SAM increases. Effect of Surface Hydrophobicity on Orientation and Mobility of NfsB. To gain more information on the interactions between NfsB and the surface, we investigated the orientation of the two NfsB variants on the EG3−OH and EG3−CH3 SAM surfaces using a combination of SFG and ATR-FTIR vibrational spectroscopies. These surface-sensitive techniques allow the overall orientation of the surface immobilized enzymes with respect to the surface normal to be determined using protein backbone amide I signals. pppand ssp-polarized SFG spectra, together with p- and s- polarized ATR-FTIR spectra were recorded for both NfsB-V424C and NfsB-H360C immobilized on EG3−OH and EG3−CH3 SAM surfaces containing 0.1 mole fraction EG3−Mal. Details of the data analysis methods have been published previously20,21,27 and so are summarized on briefly here. Using the protein crystal structure, the ratio of the ppp- to ssp-polarized SFG signals can be calculated as a function of the protein orientation, which is

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RESULTS In previous studies, we focused on examining the effects of orientation on the activity of NsfB immobilized on SAM surfaces assembled exclusively from maleimide-terminated oligo-ethylene glycol chains.20 In this study, we aimed to undertake a more detailed examination of the effects of surface hydrophobicity and protein surface density on enzyme activity and stability. These factors are often cited as important determinants of enzyme activity13,14 but quantitative measurements on well-defined systems are lacking. Effect of Surface Hydrophobicity on Activity of NfsB. To modulate the hydrophobicity of the supporting surface, we synthesized SAM surfaces assembled from a triethylene glycol unit terminated with either a free hydroxyl group, denoted EG3−OH here, or terminated with a methoxy- group, denoted EG3−CH3 here. Whereas these surfaces may both be considered broadly hydrophilic, the EG3−CH3 surface is significantly more hydrophobic as evidenced by the difference in contact angles, θC = 53° and 73°, measured for the pure EG3−OH and EG3−CH3 surfaces, respectively. By synthesizing SAMs with varying ratios of EG3−OH to EG3−CH3 the hydrophobicity of the SAM surface could be modulated in a fine-grained manner. Highly hydrophobic surfaces can cause nonspecific adsorption and unfolding of proteins16,26 and were therefore avoided in these studies. To facilitate the covalent attachment of enzymes to the surfaces we introduced EG3−maleimide-terminated (EG3− Mal) into the EG3−CH3 and EG3−OH SAMs. In an initial C

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angles orientations with any degree of certainty because all the possible deduced orientations have less than 10% probability. It should be noted that to determine protein orientation by the above methods, one has to assume that all the proteins at the interface adopt the same orientationor a delta orientation distribution. Here, it appears that none of the orientation angles (tilt/twist angles) can satisfy the measured SFG and ATR-FTIR data. This is most readily explained if the tethered proteins adopt many different orientations with respect to the surface. This result implies that the proteins are much more conformationally labile when attached to this more hydrophilic surface, so that they do not adopt unique orientations. Influence of Surface on Enzyme Stability. To examine how the hydrophobicity of the surface may affect the stability of the enzyme, we compared the thermal denaturation of the surface-immobilized enzymes with that of NfsB in free solution. Representative plots of the fraction of activity remaining as a function of temperature for NfsB in free solution, and NfsBV424C and NfsB-H360C immobilized on EG3−Mal/EG3−OH (1:10 ratio) SAM surface are shown in Figure 3B. From these plots, the melting temperature, T1/2, for NfsB in free solution was determined as 45 ± 2 °C. The T1/2 for immobilized NfsBV424C was 48 ± 1 °C whereas T1/2 for immobilized NfsBH360C was 50 ± 2 °C. The small differences in T1/2 values are not considered significant. Interestingly, the thermal denaturation curves for the surface-immobilized NfsB enzymes are significantly sharper than that for NfsB in solution, indicating that the unfolding transition becomes more cooperative in the immobilized enzymes. We also investigated the time-dependent thermal inactivation of both NfsB-V424C and NfsB-H360C immobilized on 1:10 ratio EG3−Mal/EG3−OH surfaces. At 45 °C, the half-life of each enzyme variant was very similar at ∼3 min. This again suggests the orientation of the enzyme with respect to the surface has little impact on its thermal stability. To examine how the hydrophobicity of the surface influences the thermal stability of the enzyme in more detail, we measured the T1/2 of one NfsB variant, NfsB-V424C, as a function of SAM composition. This variant was immobilized on SAM surfaces comprising 0.1 mole fraction EG3−Mal and varying mole fractions of EG3−OH and EG3−CH3; the enzyme loading was maintained constant at 1.2 nmol of protein per gram of beads. The results are summarized in Figure 3A. Somewhat surprisingly, although the specific activity was dependent on the ratio of EG3−OH to EG3−CH3, as discussed above, T1/2 for the enzyme was, within error, independent of surface hydrophobicity and varied little from an average value of 47 °C. Effect of Enzyme Surface Density on Activity and Stability. The density at which enzymes are immobilized on supporting surfaces has been shown to affect both enzyme activity and stability.10,13,14 We therefore examined the effects of changing the surface density (loading) on the activity and stability of both NfsB-V424C and NfsB-H360C. The surface density of the enzyme was varied by changing the concentration of enzyme in the solutions used to derivatize the SAM surfaces, as described in the Material and Methods. For each sample, enzyme activity and protein surface densities were independently measured. It proved possible to vary enzyme surface density over a ∼4-fold range. The upper density limit approached that estimated for a monolayer of immobilized enzyme molecules, assuming a footprint of ∼2500 Å2 for NfsB. The lowest surface density that could be examined was limited by the ability to accurately measure enzyme activity and surface protein concentrations.

Figure 3. Effect of surface hydrophobicity on specific activity and thermal stability (T1/2) of NfsB-V424C. (A) Variation of specific activity and T1/2 as a function of SAM composition. (B) representative thermal stability curves for NfsB in solution and immobilized on EG3− OH-terminated SAMs.

defined by two angles, tilt, θ, and twist, ψ, relative to the surface normal. By fitting the experimentally measured SFG signal strengths to the calculated signal strengths, the combinations of tilt and twist angles most compatible with the data can be determined. Similarly, the ratio of p to s-polarized signals for ATR-FTIR measurements can also be calculated as a function of protein tilt and twist angles and fitted to experimental data. Because SFG and ATR-FTIR are independent measurements, the final deduced orientation angle regions should be those that overlap between the two techniques. Such overlapping regions are plotted using a heat map (see Figure 4) to represent the most probable combinations of the tilt and twist angles that are compatible with the experimental data.21 Figure 4A shows the ppp and ssp polarized SFG spectra and p and s polarized ATR-FTIR spectra obtained for NfsB-V424C immobilized on a EG3−Mal/EG3−CH3 (1:10 ratio) mixed SAM. The heat map of deduced possible orientations using SFG and ATR-FTIR (ψ and θ angles, Figure 4A right panel) indicates that the most probable orientation for NfsB-V424C is described by a tilt angle, θ = 28° and twist angle, ψ = 30 o with a ∼30% probability. A similar set of experiments was performed on NfsB-H360C immobilized on the same EG3−Mal/EG3− CH3 (1:10 ratio) mixed SAM (Figure 4C). The deduced orientations indicated that the most probable orientation for NfsB-H360C is described by a tilt angle, θ = 27° and twist angle, ψ = 60 o with an ∼80% probability. These orientations are similar to those measured previously on a pure EG4−Malterminated SAM.20 The spectroscopic studies were repeated for NfsB−V424C and NfsB-H360C immobilized on a EG3−Mal/EG3−OH (1:10 ratio) mixed SAM surface (Figure 4B,D). The proteins gave well resolved vibrational spectra, but on this more hydrophilic surface it proved impossible to fit the spectra to specific ψ and θ D

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Figure 4. Orientation of NsfB variants on different surfaces deduced by SFG and ATR-FTIR spectroscopies. (Left) SFG spectra; (center) ATRFTIR spectra; and (right) heat maps showing possible orientation angle regions deduced from SFG and ATR-FTIR dichroic ratios. (A) NfsB-V424C on EG3−CH3-terminated SAMs (B) NfsB-V424C on EG3−OH-terminated SAMs (C) NfsB-H360C on EG3−CH3-terminated SAMs (D) NfsB− H360C on EG3−OH-terminated SAMs. The bar on the right of each heat map shows the percentage probability that the experimental data matches the calculated value for each combination of tilt and twist angles that define the protein orientation. For details see the text. E

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Figure 5. Variation of specific activity and T1/2 of the immobilized NfsB variants as a function of the surface density of the immobilized enzymes. (A) specific activity (left) and T 1/2 (right) for NfsB-V424C; (B) specific activity (left) and T1/2 (right) for NfsB-H360C.

noteworthy that even the over the fairly narrow range of hydrophilicity studied (contact angles, θC, between 53° and 73°) enzyme activity varied by a factor of 2. This suggests that for practical catalyst design, enzyme activity will, in general, be maximized by immobilization on the most hydrophilic polymer support compatible with the application. One common explanation for the reduced activity of enzymes on hydrophobic surfaces is that localized denaturation of the protein occurs on the surface.14 However, our data do not support this as an explanation for the lower activity of NfsB on EG3−CH3 surfaces because the strong SGF and ATR-FTIR signals obtained on both surfaces indicate that the protein retains its native structure. Furthermore, the composition of the surface appears to have little if any influence on the thermal stability of the enzyme as judged by the T1/2. It is interesting that whereas on the more hydrophobic EG3− CH3 surface the tilt (ϕ) and twist (ψ) angles describing the protein’s orientation could be located for both NfsB variants, albeit with low probability for NfsB-V424C, on the hydrophilic EG 3 −OH surface there appeared to be no preferred orientation. This suggests a model in which transient “sticky” hydrophobic interactions between the surface of the enzyme and the SAM may result in the protein preferring certain orientations. As the surface becomes more hydrophilic such interactions are diminished, and the protein freely rotates about the tether point sampling a wide range of tilt and twist angles. Increasing hydrophobicity may decrease the activity of the enzyme by a number of mechanisms, including locking the protein in a less active conformation and/or perturbing protein dynamic motions that are important for catalysis. We note that our initial experiments on NfsB were conducted on 100% maleimide-terminated SAM surfaces that were more hydrophobic than the surfaces studied here. The enzyme adopted a better-defined range of ϕ and ψ angles on this more hydrophobic surface, consistent with our hypothesis. Molecular crowding has been suggested as mechanism for increasing protein stability, based on the observation that an unfolded protein occupies a greater volume than a folded

The results of these measurements are summarized in Figure 5. In this experiment some clear differences between the behavior of NfsB-V424C and NfsB-H360C were apparent. For NfsB-V424C, the specific activity and thermal stability remained, within error, constant over the range of surface densities studied; the mean specific activity was 5.6 μM/min/ μg and the mean T1/2 was 49 °C. In contrast, the specific activity for NfsB-360C, whereas similar to that for NfsB-V424C at high densities, decreased by ∼25% at the lowest surface density studied (Figure 5). The lowest specific activity value was for the lowest surface coverage (30 ng/mg beads) at 4.4 ± 0.3 μM/min/μg enzyme. The T1/2 measurements on NfsBH360C suggest that the enzyme stability may decrease at lower enzyme surface densities, although the larger experimental errors associated with these measurements (a consequence of the low enzymatic activity) do not allow definitive conclusions to be drawn.

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DISCUSSION It is generally accepted that factors such as the chemical nature of the supporting surface, the method of surface immobilization, and the density of catalyst loading play an important role in determining the activity and stability of surface-supported enzyme catalysts.12−14 However, such observations remain largely empirically based because, for practical applications, the chemical structure of the surface support and the methods used to immobilize enzymes on the surface result in systems that are too complex and heterogeneous to study in molecular detail. Our approach has been to immobilize enzymes on chemically well-defined, atomically flat SAM surfaces through unique covalent linkages.15,20−22 This yields relatively homogeneous populations of immobilized enzymes that are amenable to detailed spectroscopic and kinetic analysis, and for which parameters such as surface hydrophobicity and enzyme loading can be systematically varied. Our experiments show clear correlation between the hydrophilicity of the surface and the activity of the enzyme. This observation is in accord with expectations, but it is F

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Langmuir protein.28,29 Crowding may stabilize proteins in living cells, which contain very high concentrations of proteins and other macromolecules.28,29 Similarly, the local concentration of surface-immobilized protein molecules can be very high and the volume available for the protein to unfold is further reduced by its proximity to the surface.14 Our experiments find some evidence for crowding effects stabilizing NfsB, but, interestingly, this is orientation dependent. Thus, for NfsB-V424C neither specific activity nor thermal stability appeared to depend on surface concentration. However, NfsB-H360C clearly demonstrated a decrease in activity at lower surface concentrations. It may also be less thermally stable at low concentrations, although the error in these measurements do not allow definitive conclusions to be drawn.

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CONCLUSIONS Although hydrophobic surfaces are well-known to reduce enzyme activity due to nonspecific adsorption and protein denaturation, we have shown here that even small changes in surface hydrophobicity/hydrophilicity can exert a significant effect on enzyme activity. This does not appear to be due to surface denaturation, but rather to nonspecific sticky interactions between the enzyme and surface that trap the enzyme in less active conformations. These observations indicate that maximizing surface hydrophilicity is likely to be generally beneficial in the design of surface-supported enzyme catalysts. We found the enzyme surface concentration to exert a less significant effect on NfsB activity and stability than surface hydrophobicity, but interestingly, this depended on which residue was used to attachment the enzyme to the surface. This suggests that it may be possible to significantly improve the performance of immobilized enzyme catalysts by judicious choice of the tethering point.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.C.). *E-mail: [email protected] (E.N.G.M.). ORCID

E. Neil G. Marsh: 0000-0003-1713-1683 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Defense Threat Reduction Agency (HDTRA-11-1-0019 and HDTRA-1-16-0004) and the Army Research Office (W911NF-11-1-0019).

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DOI: 10.1021/acs.langmuir.7b00646 Langmuir XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.langmuir.7b00646 Langmuir XXXX, XXX, XXX−XXX