Simultaneous Observation of the Orientation and Activity of Surface

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Interfaces: Adsorption, Reactions, Films, Forces, Measurement Techniques, Charge Transfer, Electrochemistry, Electrocatalysis, Energy Production and Storage

Simultaneous Observation of the Orientation and Activity of Surface-Immobilized Enzymes Joshua Jasensky, Kyle L. Ferguson, Maximillian Baria, Xingquan Zou, Ryan McGinnis, April Kaneshiro, Somayesadat Badieyan, Shuai Wei, E. Neil G. Marsh, and Zhan Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01657 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Simultaneous Observation of the Orientation and Activity of SurfaceImmobilized Enzymes

Joshua Jasensky1, Kyle Ferguson1, Maximillian Baria1, Xingquan Zou1, Ryan McGinnis1, April Kaneshiro2, Somayesadat Badieyan1, Shuai Wei1, E. Neil. G. Marsh*,1,2, and Zhan Chen*,1

Department of Chemistry1 and Department of Biological Chemistry2, University of Michigan, 930 N. University Avenue, Ann Arbor MI, 48109, USA

Corresponding Author: Zhan Chen, [email protected]

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Abstract Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes’ structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using β-glucosidase (β-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented towards the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin β-D-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring β-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface bound enzymes.

KEYWORDS: Sum Frequency Generation Spectroscopy, Beta-Glucosidase, Fluorescence Activity, Protein Orientation

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Introduction Enzymes provide a “greener”, more environmentally sustainable, alternative to industrial catalysis, and only require aqueous reactions at standard temperature and pressure.1,2 Current enzyme production costs are typically high, therefore to enhance the efficiency and reusability of such biocatalysts, they are often adsorbed or immobilized on solid supports.2-8 This can be accomplished in a variety of ways, including encapsulation of enzymes into a porous material or immobilization of enzymes through chemical tethering or physical adsorption.9-11 Physical adsorption is commonly employed because of its simplicity, however this method often results in leaching of enzyme from the surface over time. Leaching can be prevented by covalent crosslinking, for example by using lysine-reactive reagent glutaraldehyde.2,12,13 However, because proteins typically have multiple lysine residues, this leads to random surface orientation of enzymes, and often enzyme unfolding. To improve both the activity and stability of surface bound enzymes, researchers have developed approaches in which enzymes are immobilized through defined chemical linkages.14-16 Enzymes that are chemically tethered to a solid support in a controlled and predictable way often exhibit activities that are similar to enzymes in solution, and can easily be reused.17 In order to optimize the performance of surface immobilized enzymes, it is crucial to understand the interaction between the surface and the enzymes and the factors that govern favorable interactions between enzymes and immobilized surfaces. In this work, we have used β-Glucosidase (β-Glu) as a model system. This enzyme has been extensively used in industrial processes such as the production of ethanol from lignocellulosic feedstocks for biofuel.18 β-Glu converts short chain oligosaccharides into glucose monomers, which is considered to be the rate-limiting step in the cellulase complex.18 Optimizing the

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function and reusability of this enzyme is, therefore, of practical concern. Sum frequency generation (SFG) vibrational spectroscopy is a valuable tool to elucidate the structure and orientation of surface bound enzymes.19-22 SFG is a second-order nonlinear optical spectroscopy that is intrinsically surface sensitive.23-43 SFG examines the secondary structures and orientations of various enzymes adsorbed/immobilized on surfaces in situ.19,21,22 Measuring surface activity is as equally important as understanding enzyme surface interactions. Typically, surface activity is measured separately, using a different experimental set up from that used for the determination of immobilization orientation;19,22 however, given the sensitivity of some enzymes to surface conditions, this approach may not accurately correlate changes in enzymesurface interactions observed spectroscopically with changes in activity. To address this, we have developed an instrument that incorporates fluorescence microscopy into an existing SFG spectrometer, which we call a SFG-µScope.44,45 This allows the sequential acquisition both of molecular structural information (via SFG), and enzymatic activity using a fluorogenic substrate. In this work, β-Glu was covalently tethered to maleimide-terminated SAM surfaces and the ability of SFG-µScope to accurately measure both enzyme orientation and immobilized enzyme activity was demonstrated. The results of this work will aid efforts to design more active surface-immobilized enzymes by allowing researchers to better correlate changes in immobilized orientation and activity to surface chemistry.

Materials and Methods All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification, unless stated otherwise.

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Transformation of β-Glucosidase. The gene from C. cellulovorans (CcbglA; NCBI accession number AY268940), synthesized and codon optimized for usage for E. coli by Genescript, was cloned into a Pet21a vector with a Hisx6 extension to the C-terminal. The constructs were transformed into E. coli BL-21 (DE3) chemical competent cells for protein expression.

Expression and Purification of β-Glucosidase. Six 10 mL overnight cultures were used to inoculate 6 liters of fresh lysogeny broth (LB) medium containing 100 µg/mL ampicillin at 30°C, and was subsequently incubated until the A600 OD was 0.8-1.0. The temperature was allowed to cool to 13 °C, and the bacteria were allowed to cool for one hour before adding the inducer, isopropyl B-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. After further growth for 16 hrs, the bacteria were harvested by centrifugation at 4000 g and re-suspended in 100 mL of a lysis buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 500 mM NaCl, and 10 mM imidazole, pH 7.5. A single protease inhibitor tablet (Sigma) 100 mg lysozyme (Sigma) and 2 µl of benzonase nuclease (Sigma) were added to the lysis mixture. The cells were sonicated by using a 550 Sonic Dismemberator System (Fischer Scientific) and the cell debris was removed by centrifugation at 18500 rpm for 45 minutes. The cell free extract was loaded onto a Ni2+-nitrilotriacetic acid column, which had been equilibrated with the lysis buffer. The column was washed with the same buffer and the His6-tagged protein was subsequently eluted by a linear gradient of 10-300 mM imidazole. After checking the purity of the protein by SDS-PAGE, the proteins were buffer exchanged into a 100 mM citrate buffer of pH 6.5 with 10% glycerol and flash frozen. About 50 mg/L of β-Glucosidase was obtained with greater than 95% purity, as measured at the A280 via a nanodrop instrument.

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Transformation, Expression, and Purification of the C226S β-Glucosidase Mutant. Primers were designed

for

gene

cloning

using

the

Agilient

(https://www.genomics.agilent.com/primerDesignProgram.jsp)

Online for

Primer

Design

optimization

with

tool the

Agilient QuikChange II tool kit. Protocols regarding the expression and purification of this βGlucosidase mutant are identical to the wild type form, and can be found in the section above. Similar to wild type β-Glucosidase, about 50 mg/L of C226S mutant β-Glucosidase was obtained with greater than 95% purity, as measured at the A280 via a nanodrop instrument.

Ellman’s Reagent Assay. A reaction buffer containing 100 mM sodium phosphate, pH 8.0 with 1 mM EDTA was made. A stock solution of Ellman’s reagent (4 mg of 5,5’-Dithio-bis-(2nitrobenzoic acid) 396.35 g/mol) was dissolved in 1 mL of reaction buffer. Samples were made with 25 µM of β-Glu in 2.45 mL total volume of reaction buffer and 50 µL of Ellman’s Reagent buffer. The mixture was allowed to incubate at room temperature for 15 minutes. The absorbance of the solution was measured at 412 nm (E = 14,150 M-1cm-1). 25 µM β-Glu was then dissolved in a denaturing reaction buffer containing 100 mM sodium phosphate, pH 8.0 with 1 mM EDTA and 8 M Urea. The reaction was allowed to incubate at room temperature for 15 minutes and then the absorbance at 412 nm was measured (E = 14,290 M-1cm-1).

Native Gel. 2.5 µM, 5 µM, and 10 µM of β-Glu was loaded with NativePAGE (4x) sample buffer and diluted to a final volume of 50 µL. The Native gel was then loaded into a NativePAGE system and ran with 1 L of NativePAGE Running Buffer. The gel was then ran at 4°C at 30 V overnight. After that, the gel was stained using the InstaBlue protein gel stain and analyzed using the ImageLab Software.

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Surface Functionalization and Enzyme Immobilization. Due to the presence of cysteine in the primary sequence of this enzyme, maleimide-thiol chemistry can be used to chemically immobilize β-Glu to a solid support. The construction of this support, as demonstrated in previous publications,22,46 requires the assembly of a two-step self-assembled monolayer (SAM) in order to functionalize an otherwise non-reactive surface, allowing for a chemical reaction that will covalently bind enzyme to the surface. The steps required to prepare samples for use in SFG-µScope are summarized here: First, right-angle CaF2 prisms, purchased from Altos Photonics (Bozeman, MT, USA), were soaked in toluene for 24 h and sonicated in 1% Contrex AP solution (Decon Laboratories, King of Prussia, PA, USA) for 10 min. These prisms were then thoroughly rinsed with Millipore water (18.2 MΩ cm), dried under gaseous nitrogen, and coated with 100 nm of SiO2 via an electron-beam deposition process using an SJ-26 evaporator system at a pressure below 10−5 Torr with a deposition rate of 5 Å/s. Newly coated CaF2 prisms were treated with O2 plasma for 1 min in a benchtop plasma cleaner (PE-25-JW, Plasma Etch, Carson City, NV, USA), then were immediately placed into a freshly prepared 1.0 mM alkyne-silane solution in anhydrous toluene for 24 h at room temperature. The functionalized prisms were rinsed with copious amounts of toluene and methanol, and were then dried under nitrogen gas. Azido-PEG3-maleimide linker was prepared according to the manufacturer’s instructions. The alkyne-functionalized prisms were placed into an aqueous solution containing azido-PEG3-maleimide linker (1 mM), sodium ascorbate (0.2 M), and copper sulfate (0.5 mM), and reacted overnight. The prisms were rinsed with deionized water, ethylenediaminetetraacetic acid (EDTA), and deionized water again in order to remove

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any residual copper ions. It is worth mentioning that the linker containing a PEG3 segment ensures good enzyme surface coverage while maintaining high resistivity to physical adsorption.47 Immobilization of β-Glu onto maleimide-functionalized prisms requires the use of tris(2carboxyethyl)phosphine (TCEP) in order to reduce any disulfides that may form in solution between neighboring proteins. 5 µM β-Glu and 0.1 mM TCEP were added to a 20 mL reaction vial containing 4 mL of 100 mM citrate buffer (pH 6.5). Functionalized prisms were introduced to this reaction solution and incubated in enzyme solution for 2 hours. They are subsequently rinsed with additional citrate buffer and Tween-20 detergent to remove any unreacted or physically adsorbed enzyme from the surface.

SFG Microscope (SFG-µScope) and Enzymatic Activity. The design and implementation of SFGµScope has been described in a previous publication.44,45 Its construction is based around an EKSPLA SFG spectrometer. Details of the SFG system can be obtained from the manufacturer, have been described in previous publications,48-53 and will not be repeated here. SFG is a secondorder nonlinear optical process and SFG spectroscopy is an ideal tool for probing the structure (e.g., conformation and orientation) of surface immobilized proteins due to its intrinsic surfacesensitivity.19,22 This, in combination with fluorescence detection, allows for the sequential measurements of both enzyme orientation and activity of the same sample under the same conditions. SFG-µScope has been designed to use an inverted prism geometry with near-total reflection (opposite of the traditional SFG sample geometry), and a schematic of this can be found in Figure 1. Two input laser beams were directed onto a CaF2 prism with immobilized β-Glu onto a

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maleimide terminated SAM. SFG spectra with polarization combinations of ssp (s-polarized sum frequency output, s-polarized visible input, and p-polarized IR input) and ppp were collected and used for orientation analysis. Simultaneously, enzyme activity was measured on resorufin β-Dglucopyranoside using a broadband fluorescence lamp (U-HGLGPS, Olympus, Japan), and a corresponding filter set (excitation: 559±17 nm bandpass filter; emission: 630±35 nm bandpass filter) designed to match the excitation and emission profiles of resorufin, the enzymatic product. 100 µL of 25 µM enzyme substrate was introduced to the β-Glu surface and enzymatic activity was measured as an increase in fluorescence intensity via a mounted CCD camera.

Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR). ATR-FTIR experiments were carried out with a Nicolet Magna 550 FTIR spectrometer using a detachable germanium total-internal-reflection crystal (Specac Ltd., Slough, England). Germanium was chosen due to the fact that its surface can undergo similar silane chemistry to the surface of silica (evaporated onto calcium fluoride). The germanium crystal surface was cleaned with methanol, 1% Contrex AP solution, and Millipore water and then treated in the O2 plasma chamber for 2 min immediately before SAM growth. After the SAM was formed on the crystal top surface, 1.6 mL of pH 6.5 citrate buffer and TCEP solution in D2O were added onto the top surface of the crystal to reach final concentrations of 5 and 0.1 mM, respectively. D2O was used to avoid possible signal confusion between the O−H bending mode and the peptide amide I mode and to ensure a better signal-to-noise ratio in the peptide amide I band region. After the background spectra were recorded, the appropriate volume of an enzyme stock solution (incubated with 1 mM TCEP to reduce potential disulfide bonds as described above) was injected into the subphase

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to achieve the desired enzyme concentration of 5 µM. The s- and p-polarized ATR-FTIR spectra of immobilized β-Glu were recorded for orientation analysis.

Results and Discussion Biochemical Characterization of β-Glucosidase. β-Glucosidase (PDB: 3AHX) was chosen as a model enzyme because of its extensive helical content, which facilitates SFG and ATR-FTIR spectroscopic analysis,52,53 and its ease of purification and assay. The wild type enzyme has two cysteine residues at positions 55 and 226 that allow the enzyme to be covalently immobilized on maleimide-terminated SAM surfaces. Accurate calculation of surface orientation requires the immobilization via a single cysteine. Of the two cysteines present in the primary sequence of βGlu, it is important to determine which, if any, are solvent accessible and have the ability to covalently bind to a maleimide terminated SAM surface. As a first step, the solvent accessible surface area54 (SASA) was calculated for β-Glucosidase and was used to determine the accessibility of water molecules to each residue of the protein crystal structure. This calculation indicated that only C226 is solvent accessible. To further confirm this hypothesis, a single-residue mutation of β-Glucosidase was constructed at position C226 to remove the suspected solvent-accessible cysteine. This residue was modified to serine, as to not significantly impact the chemistry of that amino acid position, and will be identified as C226S from this point forward. An Ellman’s reagent assay was performed on this mutant in order to assess the number of solvent accessible cysteines and confirm the results from SASA. As shown in Figure 2, the absorbance of the Ellman’s product is minimal in 30 µM of protein indicating that there are no available cysteines on surface of β-Glucosidase C226S. When exposed to 8 M Urea, the protein

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unfolds, and the Ellman’s product is formed solely from C55. From a generated calibration curve, absorbance of this product is calculated to be 28 µM of product, and correlates to about one cysteine per protein. Therefore, both SASA and an Ellman’s reagent assay with C226S have demonstrated that native β-Glucosidase has a single solvent-exposed cysteine, C226. Upon inspection of the PDB file, the structure of β-Glu is shown as a tetramer. SFG analysis of protein structure requires a monomeric form of the protein, and raises the question of whether or not this enzyme remains in its monomeric form in a low concentration range used for SFG spectroscopy and measurement of its surface immobilized activity. Gel electrophoresis experiments were performed at three different enzyme concentrations, 10 µM, 5 µM, and 2.5 µM. Results, shown in Figure 3, indicate that between 2.5-10 µM, greater than 95% of the β-Glu exists in the monomeric form calculated using the integrate band function within BioRad Image Lab (BioRad, Hercules CA).

Analysis of β-Glucosidase Orientation using SFG-µScope. Methods for determining protein orientation by SFG rely on measuring the ratio of the effective second-order nonlinear optical susceptibility tensor components detected in the ppp and ssp polarizations (χzzz(2)/χxxz(2); under the near total reflection experimental geometry which we adopted, other χ(2) terms (e.g., χxxz(2), χzxx(2), χxzx(2) terms) in ppp can be ignored. These methods are by now fairly standard and the details have been reported previously.55 Additional details regarding how to calculate orientation have been included in the Supplementary Information. Generally, analysis of SFG spectra alone is insufficient to determine the tilt and twist angles that define the orientation of the protein with respect to the surface normal. Therefore, polarized ATR-FTIR spectra are also recorded; the

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different selection rules associated with this technique allow the protein orientation to be further narrowed down. β-Glucosidase immobilized on SAM (on CaF2 prism) was measured using SFG spectroscopy. Insuring that the surface only contained covalently bonded enzyme, the surface was washed with a mild detergent, Tween-20. This detergent is important in removing any physically bound enzyme. Figure 4 shows SFG ssp- and ppp-polarized spectra collected from immobilized β-Glu with corresponding fits to the data. All SFG spectra were normalized according to the intensities of the input IR and visible beams. From the fitting, the ratio between ssp and ppp was calculated to be 1.76. After correcting for variations in the Fresnel coefficients, the measured χzzz/χxxz ratio was determined to be 1.98. Based on the crystal structure of the enzyme, a heat map of possible orientation regions of immobilized β-Glu was computed, that can satisfy the SFG data (Figure 4) In deducing this orientation, the enzyme reference position [(tilt angle, twist angle) = (0°, 0°)] was defined using the initial position referenced in the crystal structure. Further details of how to determine orientation of β-Glu via SFG can be found in the Supplemental Information.

ATR-FTIR measurements of β-Glucosidase. To narrow down the possible orientations for βGlu with respect to the surface, polarized ATR-FTIR measurements were performed. Similar to samples prepared for SFG spectroscopy, β-Glucosidase immobilized to ZnSe ATR-FTIR crystals was first washed with Tween-20 to ensure that only covalently bound enzyme was on the surface. Figure 5 shows the measured P and S components of surface immobilized β-Glu. From this plot, the dichroic ratio RATR was calculated and a corresponding heat map generated in a manner similar to that for SFG data (Figure 5). By combining the heat maps generated from the

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SFG and ATR-FTIR, the overlapped heat map which contains both the SFG and ATR-FTIR results shown in Figure 6 was generated.

The most probable orientation of the enzyme,

characterized by tilt = 105° and twist = 80°) is consistent with residue C226 being close enough to the surface to form a covalent attachment. When a separate area is considered where probability is also high (tilt = 75° and twist = 260°), the location of the cysteine group is far away from the maleimide surface. Therefore we believe that this orientation (tilt = 75° and twist = 260°) is unlikely. The above determined orientation (tilt = 105° and twist = 80°) places the active site at 90° to the surface normal, which may cause substantial blocking of the active site thereby impeding diffusion of the substrate and reducing activity. This suggests that re-orienting the enzyme by engineering a cysteine attachment point remote from the active site may improve activity. A similar approach on another model enzyme, β-Galactosidase, showed that re-orientation can improve the activity of surface-immobilized enzymes.22

Surface and Solution Activity of β-Glucosidase measured using SFG-µScope.

Using the

fluorescence microscopy capability of this instrument, enzymatic activity was measured in situ on the same sample of immobilized β-Glu used to record SFG spectra. These measurements employed a fluorogenic substrate, resorufin-β-D-glucopyranoside, allowing the activity of βglucosidase be monitored by the release of the fluorophore resorufin. To determine rate of product formation as a function of fluorescence intensity recorded by the CCD camera of the SFG-µScope, calibration curve was constructed with of known concentrations of resorufin. In order to calculate specific activity, full surface coverage of immobilized enzyme is assumed. With a surface of 1 cm2 and an enzyme diameter of 10 nm (and occupies an area of 100 nm2), we

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can then estimate the specific activity of β-Glucosidase to be 5.27x104 µM/min/mg. This value was determined within the linear regime of this curve in order to make sure enzymatic substrate was in excess and the intensity is only a factor of enzymatic rate. While the measurement of enzymatic activity of surface immobilized protein is an important step in determining structure vs. function relationships, solution measurements must be performed as a comparison to the effect of immobilization of this enzyme to this solid support via C226. Figure 7 shows the comparison of solution and immobilized enzymatic activity of βglucosidase. An identical concentration of enzyme was matched in solution by using the concentration of enzyme on the prism surface determined in the previous step. The same enzyme substrate concentration was used in this solution enzyme activity study and the above immobilized enzyme activity study. Specific activity for this solution enzyme using SFG-µScope was calculated to be 1.12x105 µM/min/mg, and agrees with previously reported activities of this enzyme.56 From this, we can determine that there was a loss of 53% of enzyme activity upon surface immobilization. Additionally, the enzymatic activity of C226S was measured via SFG microscope. This mutant of β-Glucosidase measured no detectable product, which is likely due to its inability to successfully immobilize to the maleimide SAM surface. The detergent washing step mentioned previously had removed all physically adsorbed enzyme which left an untracable amount on the surface. Upon inspection of the orientation measured via SFG and ATR-FTIR, we can see that the active site is not readily solvent accessible, which can ultimately affect the surface bound activity. In addition, previous research investigated the dependence of enzyme activity on the location of the active site relative to the surface. Chemical linkages both close to57 and far away22

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from the active site of β-Glactosidase were investigated. Results from this study have shown that on the same maleimide terminated SAM surface (the one used in the current research), enzymatic activity can greatly be improved when the active site is readily solvent accessible. In contrast, active sites that are close to the maleimide SAM substrate they are immobilized to show a significant reduction in enzymatic activity. However, when a more hydrophilic substrate was used for enzyme immobilization, the activity reduction can be prevented due to the “wrong” enzyme orientation.56 Therefore it is likely that the original activity loss of the enzyme is due to the strong enzyme active site-surface interaction. Although every enzyme is different and the trend of increasing activity with increasing distance away from the substrate cannot be guaranteed to be linear, it is our best assumption that the activity reduction shown in βGlucosidase studied in this research on maleimide terminated SAM surface can be attributed to its orientation as compared to solution. When considering other mechanisms of enzymatic activity loss, factors such as restriction of essential enzymatic motions, reduced accessibility of the substrate to the catalytic active site, and/or alteration of the conformational integrity of the enzyme may need to be considered.58 With such a densely packed surface, we cannot exclude the possibility that one or all of these factors may also play a role in the observed reduced activity.

Conclusion In summary, the development of SFG-µScope has allowed for the simultaneous measurement of both molecular orientation, as well as enzymatic activity, thereby bridging the gap between understanding both structure and function on the same system. Unlike measurements done with different sample environments and immobilization supports, the use of SFG-µScope eliminates

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the possible systematic error associated with different sample environments, and variations in immobilization substrate material and geometry. β-Glucosidase, a model enzyme for this investigation, was an ideal candidate for this system. Its two native cysteines allowed for no further genetic modification of the enzyme, simplifying the sample preparation process and reducing the sample cost. Its rich α-helical structure also allowed for SFG to easily probe this immobilized enzyme. Overall, SFG has demonstrated that this enzyme is covalently attaching to the maleimide surface (where the cysteine residue is facing towards the solid support), and a fluorescence assay via SFG-µScope has determined an activity measurement of 53% relative to solution activity. An assessment of the enzyme orientation reveals an active site that is not easily solvent accessible, which is a likely factor in the significant decrease in surface bound activity. To improve the activity of surface immobilized enzyme, a more hydrophilic surface (e.g., using a mixed maleimide terminated and hydroxyl terminated SAM) could be used.46 Understanding the structure-function relationship of surface immobilized enzymes is important for rational design of immobilized enzyme systems with improved functions. The construction and application of the SFG-µScope to measure structure and activity of surface immobilized enzyme on the same sample under the same sample environment could greatly facilitate the understanding of the structure-function relationship at surfaces.

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Supporting Information The following content has been added to the Supporting Information: Figure S1 and S2 Further Introduction of SFG Orientation Studies This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.

Acknowledgments This research is supported by the National Science Foundation (CHE-1505385) and Defense Threat Reduction Agency (HDTRA1-16-1-0004). We would like to thank the Lurie Nanofabrication Facility at the University of Michigan for SiO2 deposition.

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Figure 1: SFG-µScope schematic. A combination of mirrors and stages allow for an inverted prism geometry. This allows for monitoring of a sample via CCD camera where fluorescence imaging can be performed.

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0.30 0.25

absorbance

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C226 mutant denatured C226 mutant

0.20 0.15 0.10 0.05 0.00 400

450

500

550

600

wavelength / nm

Figure 2: Ellman’s reagent assay. An Ellman’s agent assay was run to test the solvent accessibility of the C226S mutant of β-Glucosidase. No Ellman’s product was formed in the folded state of the protein. Upon addition of concentrated urea, the enzyme unfolds and Ellman’s product is formed. From an initial concentration of 30 µM enzyme, a calculated thiol concentrated was determined to be 28 µM.

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840 kDa

480 kDa

Figure 3: Native gel of β-Glu for 10 µM (Lane 1), 5 µM (Lane 2), and 2.5 µM (Lane 3) concentrations. Using the reference ladders on the leftmost lanes, we see that monomeric form is dominant in this expression at these concentrations (tetrameric β-Glucosidase has a molecular weight of 844 kDa). Band analysis indicates that greater than 95 percent of β-Glu is monomeric at a concentration range of 2.5-10 µM

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Figure 4: β-Glu orientation as measured by SFG. Left: SFG ssp and ppp spectra collected from surface immobilized β-Glu. Right: The ratio measured between ppp and ssp polarization combinations allows for the deduction of orientation. A specific heat map can be generated based on the native crystal structure of this enzyme and the experimentally deduced ratio.52

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Figure 5: Deduction of β-Glu orientation using ATR-FTIR as a supplemental technique. Left: ATR-FTIR spectra collected from surface immobilized β-Glu. Right: Similar to SFG, a heat map can be generated using PDB crystal structure and measured dichroic ratio.

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Figure 6: Combined heat map from both SFG and ATR-FTIR results. a) Overlay of the previous two heat maps generates the most likely orientations that satisfy both measurements. b) Regions of only the highest ranking (top 10 percent) values from the heat map in (a) have been colored in red, indicating only the most likely orientations. c) Top: Initial (0,0) position of β-Glucosidase as referenced by the crystal structure and plotted from the protein data bank. Bottom: Using this initial position, the coordinates of highest overlap between SFG and ATR-FTIR are found to be (105,80); the orientation at this coordinate is plotted. This puts C226 close to the maleimide surface.

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Figure 7: Enzymatic activity of native β-glucosidase measured in solution and immobilized to a surface as measured by SFG-µScope. Activity of surface immobilized mutant β-glucosidase C226S was also measured. Product generated (y-axis) has been normalized to protein concentration against both solution and surface immobilized enzyme. Results show a 53% reduction in activity as indicated by the value of the slope for each fitted curve. Mutant βglucosidase, with its lack of a surface cysteine, was unable to surface immobilized and produced no measureable activity.

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Simultaneous Observation of the Orientation and Activity of Surface

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