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May 6, 2014 - ABSTRACT: We demonstrate the control of enzyme orientation for enzymes chemically immobilized on surfaces. Nitro-reductase (NfsB) has th...
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Surface Orientation Control of Site-Specifically Immobilized Nitroreductase (NfsB) Lei Shen,† McKenna Schroeder,‡ Tadeusz L. Ogorzalek,‡ Pei Yang,† Fu-Gen Wu,† E. Neil G. Marsh,*,†,‡ and Zhan Chen*,† †

Department of Chemistry and ‡Chemical Biology Graduate Program, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: We demonstrate the control of enzyme orientation for enzymes chemically immobilized on surfaces. Nitro-reductase (NfsB) has the ability to reduce a broad range of nitro-containing compounds and has potential applications in a broad range of areas including the detection and decomposition of explosives. The enzyme was tethered through unique surface cysteine residues to a self-assembled monolayer (SAM) terminated with maleimide groups. One cysteine was introduced close to the active site (V424C), and the other, at a remote site (H360C). The surface-tethered NfsB variants were interrogated by a combination of surfacesensitive sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) to determine how the mode of attachment altered the enzyme’s orientation. The activities of the two immobilized NfsB variants were measured and can be well correlated to the deduced orientations. The relationships among enzyme engineering, surface immobilization, enzyme orientation, and enzyme activity were revealed.

1. INTRODUCTION

on the orientation and secondary structure of enzymes on surfaces in situ. We have developed a systematic way to investigate the molecular orientation of proteins at solid/liquid interfaces in situ using a combination of surface-sensitive vibrational spectroscopies: sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance-Fourier transformation infrared spectroscopy (ATR-FTIR).18,19 As a secondorder nonlinear optical process, SFG is sensitive to interfacial ordered chromophores under the electric dipole approximation.20−26 SFG probes the second-order nonlinear optical susceptibility χ(2) of the sample. Using different polarization combinations of the input and output beams in the SFG experiment, we can measure different components of the χ(2) tensor, from which orientation information on the interfacial molecules or functional groups can be deduced. Polarized ATRFTIR measurements provide independently measured orientation information for SFG results. Combining SFG and ATRFTIR data, we can determine more details about protein interfacial orientations.17,27−32 Previously, we applied the combined SFG and ATR-FTIR approach to study the orientation of a model enzyme, βgalactosidase, immobilized on a surface.33 Here, using a similar approach, we investigated the orientation of nitroreductase (NfsB) after surface immobilization. NfsB is a flavin-containing

Immobilized enzymes are used in a variety of applications, including antifouling coatings,1,2 biosensors,3−5 and industrial catalysts.6 Immobilizing the enzyme facilitates its reuse, and in many cases it has been observed that enzyme immobilization increases stability, leading to increased productivity.7−9 Enzymes can be surface immobilized by either physical adsorption or covalent tethering.7,9,10 Physical adsorption relies on a combination of electrostatic and hydrophobic interactions, but the simplicity of this approach makes it common and the relatively weak protein−surface interaction typically leads to the leaching of the catalyst over time.7 Covalently tethering the enzyme to the surface prevents leaching.7,8 However, the most common approachcross-linking the protein to the surface through lysine residuesis nonspecific and leads to randomly oriented and possibly unfolded proteins.8,11 Whereas increases in stability are often observed when enzymes are immobilized, activity is frequently decreased. Unfavorable orientation leading to the occlusion of the active site and surface-induced unfolding of the protein are among the hypotheses proposed to explain this phenomenon.5,12,13 A number of techniques have been used to examine protein orientation and morphology at surfaces, including atomic force microscopy,14 quartz crystal microbalance,15 time-of-flight secondary ion mass spectrometry (TOF-SIMS),16,17 and nearedge X-ray absorption fine structure (NEXAFS).16,17 These techniques, however, fail to provide molecular-level information © 2014 American Chemical Society

Received: May 5, 2014 Published: May 6, 2014 5930

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NADH and 300 μM ferricyanide in 1× PBS with varying amounts of surface-bound enzyme. The enzyme concentration was measured by the bicinchoninic acid assay,38 and the amount of enzyme in each reaction was controlled by the mass of beads added to the reaction. The reaction mixture was mechanically shaken to suspend glass beads while the NADH oxidation was measured by removing aliquots of reaction solution and measuring the absorbance at 340 nm. 2.4. Deposition of Maleimide SAM onto a CaF2 Prism Surface for SFG Study. Right-angle CaF2 prisms (Altos Photonics, Bozeman, MT) were used in the SFG experiments. A 100-nm-thick SiO2 thin film was coated onto one right-angle surface of each CaF2 prism; the detailed procedure was described in a previous publication.33 The SiO2 surface was plasma treated for several minutes to generate hydroxide groups. The prisms were then placed in a toluene solution dissolved in maleimide-EG4-silane (NanoCS, USA). The molecular formula for maleimide-EG4-silane is shown in Scheme 1. The silane headgroup can react with the OH groups on the prism

enzyme that catalyzes the reduction of a broad range of nitrocontaining compounds, including TNT, PETN, and other xenobiotic compounds, which can be used for sensing and decontaminating chemical warfare agents.34 NfsB has also been used to activate prodrugs as part of a cancer treatment in clinical trials.35 The crystal structure of this enzyme is known,36,37 and the mechanism of reduction is well understood, making this enzyme an attractive system to examine. In this study we engineered NfsB to contain a single surfaceexposed cysteine residue at two different locations (V424C and H360C) that allows the enzyme to be tethered to a maleimideterminated self-assembled monolayer (SAM) assembled on a silica surface in a chemically well-defined manner. For the first time, we have examined how the locations of mutation affect its activity when tethered to the surface and investigated the orientation of the enzyme with respect to the surface using SFG and ATR-FTIR to elucidate the structure−function relationship of immobilized NfsB enzymes.

Scheme 1. Molecular Formula for Maleimide-EG4-silane

2. MATERIALS AND METHODS 2.1. NfsB Constructs and Expression of Modified NfsB. To allow a single cysteine to be introduced per NfsB dimer, a fusion protein was designed in which the two subunits of NfsB were linked by a 10 glycine spacer unit. Each of the cysteine residues in the native protein was replaced by alanine. Valine 424 and histidine 360 were mutated to cysteine. A synthetic gene, codon-optimized for expression in E. coli, encoding this “fused” NfsB protein (PDB 1DS7) was synthesized commercially (Genscript, NJ) and subcloned into the expression vector pET28b. Two NheI restriction sites were introduced flanking the 10 Gly linker and the second tandem gene, allowing us to excise the linker and second gene and express NfsB as an unlinked dimer containing no surface cysteine residues. Expression vectors containing the NfsB gene were transformed into E. coli BL21(DE3). Cells were grown in YT media containing 50 μg/ mL kanamycin to an OD of 0.6 at 600 nm, at which point the temperature was lowered to 18 °C. Protein expression was induced 30 min after the temperature was lowered by the addition of 100 μM IPTG. The cell culture was harvested 18 h postinduction by centrifugation at 5000g at 4 °C for 20 min. 2.2. Purification of Recombinant NfsB. Cells (10 g damp weight) were resuspended in 50 mL of 100 mM Tris-Cl buffer, pH 8.0, containing 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM tris(2-carboxyethyl)phosphine (TCEP), and a complete EDTA-free protease inhibitor cocktail tablet (Roche). Resuspended cells were sonicated using a 2 s on/8 s off pulse sequence for a total pulse time of 5 min. The lysate was centrifuged at 15 000g at 4 °C for 20 min. The supernatant from the lysate was incubated with 4 mL of Ni-NTA resin at 4 °C for 1 h. The Ni-NTA resin was then decanted into a chromatography column and washed with 50 mL of 20 mM imidazole dissolved in 100 mM potassium phosphate, pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM TCEP. NfsB was eluted from the column using 10 mL of 200 mM imidazole dissolved in 100 mM potassium phosphate, pH 8.0, NaCl, 10% glycerol, 1 mM TCEP. Fractions containing pure enzyme were collected and dialyzed into buffer containing 100 mM potassium phosphate, pH 7.6, 10% glycerol, 1 mM TCEP. The enzyme was then concentrated using Amicon Ultra-15 centrifugal filters to a concentration of 50−100 μM and stored frozen at −80 °C. 2.3. Enzyme Assay. The NfsB activity was assayed by following the oxidation of NADH using potassium ferricyanide as an electron acceptor. The assay buffer contained 160 μM NADH and 300 μM ferricyanide in 1× PBS. The NfsB concentration was varied between 20 and 5 nM. The oxidation of NADH to NAD+ was followed by the decrease in absorption at 340 nm. It was found that the activities of various NfsB constructs studied here are similar. The surface-bound enzyme activity was also measured. NfsB V242C and H360C were bound to the surface of 75 μm glass beads (Supelco) via a maleimide-EG4 linker. The reaction solution contained 160 μM

surface, leading to the formation of a self-assembled monolayer (SAM). Ethylene glycol (PEG) groups in the maleimide-EG4-silane chain are used to prevent the nonspecific bindings of the enzymes, and its length can be controlled during the synthesis. 2.5. SFG Experiments. SFG theory has been extensively published and will not be repeated here.20−23 It has been widely applied to study biological molecules on various surfaces.16,17,32,39−51 In this study, all of the SFG spectra were collected using a commercial SFG system from EKSPLA, Inc. The details of the SFG experimental procedures have been described in our previous publications.29,30,32,52,53 Briefly, SFG spectra in the amide I frequency region were collected using both ssp (s-polarized SFG signal, s-polarized visible input, and p-polarized input IR) and ppp polarization combinations to measure the immobilized enzyme orientation. Prior to the SFG spectral collection, 100 μL of 0.1 mM TCEP solution was added to the enzyme stock solution and incubated for at least 1 h to avoid the formation of the disulfate bonds between enzyme molecules. SFG spectra were first acquired when the prism coated with SAM was in contact with a phosphate-buffered saline (PBS) solution. The enzyme solution was then added to the PBS solution, and the SFG signal at 1655 cm−1 from enzyme molecules immobilized at the interface was detected as a function of time. This time-dependent measurement permits the observation of enzyme adsorption/immobilization kinetics and the adsorption/immobilization equilibrium. SFG ssp and ppp spectra from interfacial enzyme molecules in the amide I mode frequency region (i.e., 1500−1800 cm−1) were acquired after the adsorption/ immobilization reached equilibrium. To ensure that the collected SFG spectra are contributed by chemically immobilized enzyme molecules, the enzyme solution in contact with the prism surface was replaced with PBS solution (pH 7.2) three times. SFG spectra were collected again from the prism/PBS solution interfaces. 2.6. ATR-FTIR Experiments. Details of the polarized ATR-FTIR experiments were reported in previous publications.27,31,32 The maleimide-terminated SAM was deposited on the ATR-ZnSe crystal using the same method as for the SFG experiment. In this research, a Nicolet 6700 FTIR spectrometer was used. ATR-FTIR spectra were collected from the SAM/PBS solution interface and used as background signals. ATR-FTIR spectra were then collected after the addition of the enzyme solution to the PBS solution and the enzyme immobilization reached equilibrium. To avoid spectral confusion, D2O instead of H2O was used in the ATR-FTIR study. Both s- and ppolarized ATR-FTIR spectra in the amide I frequency region were collected to determine the enzyme orientation. 5931

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2.7. Data Analysis. Using polarized SFG spectra and ATR-FTIR spectra, we can deduce the orientation of interfacial protein molecules. Details of the orientation determination method were published previously,18,19 and some related introductions/discussions are presented in the Supporting Information. We used the NfsB dimer crystal structure (PDB 1DS7) in the orientation determination.

3. RESULTS AND DISCUSSION 3.1. Engineering NfsB. Native NfsB is a dimeric protein that contains a single cysteine residue per subunit (Cys85) that

Figure 1. Crystal structure of NfsB V424C (left) and NfsB H360C (right). NfsB holds a dimer structure as depicted. Here in NfsB V424C the amino acid valine 424 was replaced by a cysteine residue, while in NfsB H360C the amino acid histidine 360 was replaced by cysteine. The thiol group in the cysteine residue can bind to the maleimide group on the maleimide-EG4-silane SAM.

is not required for activity and therefore was mutated to alanine. The symmetrical dimeric nature of the enzyme poses a problem for engineering a unique surface attachment point into the structure. This was overcome by genetically engineering a “duplicated” version of NfsB in which the two subunits were linked by a 10-glycine connector sequence. The construct was encoded by a single gene containing a tandem repeat of the two NfsB genes. This allows mutations to be introduced independently into either the first or second domain of the fusion protein. The specific activity of NsfB was indistinguishable from the wild-type enzyme, indicating that the linker causes minimal perturbation to the dimeric structure. Using this engineered enzyme, we introduced a single cysteine into the second domain of NfsB, replacing Val-424. This position is on a surface loop and was chosen to minimize the potential disruption to the secondary structure of the enzyme upon covalent attachment to the surface. As shown in Figure 1, this mutation should orient the protein so that one active site faces the bulk solution and the second faces the surface. A second construct was engineered in which a cysteine replaced His-360 in the second domain of NfsB, as shown in Figure 1. Placing the cysteine at position 360 should orient the active sites so that they are parallel to the surface and both are equally exposed to bulk solution. The introduction of cysteine mutations was confirmed by DNA sequencing. As a control, an NfsB construct was synthesized that retained the wild-type dimeric subunit structure but contained no cysteine residues, termed NsfB-Δcys. The NsfB-Δcys, NfsB-H360C, and NfsBV424C enzymes were overexpressed and purified from E. coli by standard methods. The presence of a single reactive cysteine residue in NfsB-V424C or NfsB-H360C was confirmed using 5,5′-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent). The specific activities for NfsB-Δcys, NfsB-V424C, and NfsB-H360C variants were determined as described in the Materials and Methods section and were found to be 28 ± 0.7,

Figure 2. (Top) ssp SFG spectra collected from the interfaces between maleimide-EG4 SAM and buffer solution before (squares) and after adding NfsB V424C to the buffer (triangles) and washing with PBS buffer (circles). (Middle) ppp and ssp SFG spectra collected from the immobilized NfsB V424C on maleimide-EG4 SAM. (Bottom) ppp and ssp SFG spectra collected from the immobilized NfsB H360C on maleimide-EG4 SAM.

24 ± 0.5, and 27 ± 0.7 μM/min/μg enzyme, respectively. The difference in the specific activities between the enzyme with no cysteine and the cysteine mutants is minimal, indicating that the addition of the glycine linker and the introduction of the 5932

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Figure 5. Left: Dependence of the ATR-FTIR p/s intensity ratio on the tilt/twist angles of NfsB calculated using the newly developed computer package.18 An orientation with the V424C close to the surface was chosen as the reference orientation (0, 0 orientation). Right: Possible orientation angle regions deduced on the basis of the experimentally measured dichroic ratio of NfsB V424C.

Figure 6. Left: Possible orientation angle regions deduced for immobilized NfsB V424C based on both the SFG and ATR-FTIR measurements. Right: Schematic showing the deduced orientation (25, 30) of surface-immobilized NfsB V424C (highlighted in black). Figure 3. ATR-FTIR spectra of NfsB(V424C) (top) and NfsB (H360C) (bottom) immobilized on maleimide-EG4 SAM (p and s polarizations).

these NfsB variants, which have very similar activities in solution, demonstrates the importance of the surface attachment point in the activity of the surface-bound enzyme. In one case, NfsB-H360C, we expect that one active site will be held in close proximity to the surface, limiting access to substrates. However, in the case of NfsB-V424C, both active sites would be equally accessible to substrates when it is coupled to the surface. To confirm this hypothesis, the SFG and AT-FTIR studies described below were undertaken to determine the orientation of these variants. We note that in both cases there is a significant decrease in activity compared to that of the enzymes in free solution. The

surface cysteine residues do not affect the folding of the enzyme. The NfsB-V424C and NfsB-H360C variants were immobilized on silica surfaces coated with maleimide-terminated EG4silane SAMs. The protein surface density was approximately 1 molecule/100 A2. The specific activities of the immobilized enzymes were found to be 14 ± 1 μM/min/μg enzyme for the NfsB-H360C mutant and 7 ± 2 μM/min/μg enzyme for the NfsB-V424C mutant. The 2-fold difference in the activities of

Figure 4. Left: Dependence of the SFG χzzz/χxxz ratio on the tilt/twist angles of NfsB calculated using the newly developed computer package.18 An orientation with the V424C close to the surface was chosen as the reference orientation (tilt θ = 0, twist ψ = 0). The coordinate system and the defined orientation angles are shown above the protein structure. They remain the same for the following figures. Right: Possible orientation angle regions deduced on the basis of the experimentally measured χzzz/χxxz ratio of NfsB (V424C). 5933

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Figure 9. Most likely orientations of NfsB V424C (25, 30) (left) and NfsB H360C (145, 280) (right). V424C is labeled in black, and H360C is labeled in red.

surface cysteine group) at the maleimide-terminated SAM surface. No SFG signal was detected from the maleimide SAM/ PBS solution interface in the amide I frequency region, as shown in Figure 2, top panel. After the addition of the solution of NfsB with no surface cysteine to the PBS solution, still no SFG signal was detected in the amide I frequency region (spectrum not shown). We believe that very likely this is due to the lack of NfsB adsorption/immobilization onto the maleimide SAM surface. This NfsB has no surface cysteine group; therefore, it cannot react with the maleimide group on the SAM surface for chemical immobilization. Due to the ethylene glycol segments in the SAM, very likely no (or a very small amount of) physical adsorption occurred. It has been extensively reported that the ethylene glycol functionality can resist nonspecific protein adsorption.54

Figure 7. Time-dependence measurements at two frequencies (1650 and 1800 cm−1, ppp). NfsB V424C was initially added to the buffer in contact with the SAM, and CP1c was added to the NfsB solution later.

reasons for this are unclear. Many factors may potentially contribute to the decrease in activity, including the reduced local concentration of substrates at the solution−surface interface, local small structural changes, and changes in the dynamics of the protein that may be important in promoting catalysis. 3.2. SFG and ATR-FTIR Results. 3.2.1. SFG Studies on NfsB Immobilized on Maleimide SAM. SFG ssp and ppp spectra were collected to study the interfacial orientation of the three NfsB enzymes (V424C, H360C, and the one without a

Figure 8. (Top left) Possible orientation angle regions of NfsB (H360C) deduced by SFG. (Top right) Possible orientation angle regions of NfsB (H360C) deduced by ATR-FTIR. (Middle) Possible orientation angle regions of NfsB (H360C) deduced by using both SFG and ATR-FTIR measurements. (Bottom) Schematic showing the possible orientations of NfsB (H360C highlighted in red): left (145, 280) and right (35, 100). 5934

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second-order nonlinear optical susceptibility of all of the αhelical components as a function of the tilt and twist angles of any protein with a structure in the protein data bank. Using this program and the crystal structure of NfsB, we calculated χzzz and χyyz of all of the α-helices as a function of the NfsB orientation (tilt and twist angles), from which the orientationdependent χzzz/χyyz ratio can be calculated, as shown in Figure 4, left panel. According to our experimentally measured NfsB χzzz/χyyz and the calculated orientation-dependent ratio, we can deduce the possible orientation ranges (tilt and twist angle ranges) of the immobilized NfsB. We developed a method to use a heat map to present the possible orientation ranges.18 For the immobilized NfsB V424C construct, the possible orientations are shown in Figure 4, right panel. It is clear that many different combinations of the tilt and twist angles are possible results which satisfy the polarized SFG measurements. We also developed a similar software package to calculate the polarized ATR-FTIR signal of α-helical components in a protein as a function of protein orientation. Using this program, we can calculate p- and s-polarized ATR-FTIR signals as a function of NfsB orientation, from which we can deduce the p/ s signal strength ratio as a function of NfsB orientation, as shown in Figure 5, left panel. Using the measured p/s signal strength ratio, we can use a heat map to show the possible regions of the NfsB V424C orientation which satisfy the polarized ATR-FTIR measurements (Figure 5, right panel). Similar to SFG data, Figure 5, right panel, shows that many possible combinations of tilt and twist angles can satisfy the measured ATR-FTIR data. As we discussed above, SFG and ATR-FTIR measure different orientation parameters. The deduced NfsB orientations should satisfy both SFG and ATR-FTIR measurements. That is to say, the possible NfsB V424C orientation regions should be the overlapped regions in Figures 4 and 5. Such overlapped regions are displayed in Figure 6, left panel, showing the four possible orientation regions with (tilt, twist) angles at (25, 30), (65, 120), (115, 300), and (160, 210). Clearly, we can exclude tilt angles larger than 90° because such orientations orient the enzyme in a way in which the surface cysteine is too far from the surface, making it impossible to form chemical bonds with surface maleimide groups. Another possible orientation region with a tilt angle of 65° also orients the cysteine group too far from the maleimide surface; therefore, we believe that the most likely orientation is the region around (25, 30), which is plotted in Figure 6, right panel. Indeed, using SFG we can measure the absolute orientation of chemically immobilized NfsB to confirm the above conclusion that the most likely orientation is the orientation angle region around (25, 30). The absolute orientation of covalently attached NfsB (V424C) was determined based on a spectroscopic interference method. In the past we have studied the orientation of an α-helical peptide, cecropin P1 with a cysteine-modified c-terminus (CP1c), immobilized on a SAM terminated with maleimide groups. The c-terminus cysteine reacts with the SAM surface, and the n-terminus is pointing toward the solution. According to our analysis, if a surface has both immobilized CP1c and NfsB (V424C) with an orientation around (25, 30), then SFG signals from CP1c and NfsB will be enhanced by each other. We first immobilized NfsB (V424C) on the SAM surface. Before the NfsB SFG signal reaches equilibrium, we replaced the NfsB solution with CP1c solution to contact the SAM surface. The time-dependent SFG signal

SFG was then used to study the interfacial orientation of NfsB with one cysteine group on the surface at different locations. After the addition of NfsB solution to the PBS buffer solution, the SFG signal at ∼1650 cm−1 can be detected from the maleimide SAM/solution interface, where the intensity gradually increased and reached a plateau after 3 h. SFG ssp and ppp spectra were then collected, and strong SFG signals were detected (Figure 2, middle panel). We replaced the enzyme solution initially in contact with the SAM with a PBS solution and then replaced this PBS solution with new PBS solutions twice to wash off the physically adsorbed NfsB on the maleimide SAM. SFG ssp and ppp spectra were collected again from the SAM/PBS solution interface (Figure 2, left panel), which are the same as those detected before washing, indicating that cysteine-modified NfsB enzymes were chemically immobilized onto the SAM surface through the maleimide−thiol coupling chemistry. Both ssp and ppp spectra were fitted using the standard spectral fitting method28,53 (shown in the Supporting Information). The spectral fitting results demonstrated that one strong peak centered at 1655 cm−1 can fit the spectra very well, showing that the SFG spectra were mainly contributed by the α-helical component. The fitted signal strength ratio (ppp/ ssp) for the V424C construct is 1.74, from which χzzz/χyyz (due to the surface isotropy, χyyz = χxxz) is calculated to be 1.79 after considering the Fresnel factor.53 This ratio can be used to determine the possible orientation range of the immobilized NfsB, which will be presented in more detail below. The same method was applied to determine the orientation of the NfsB H360C as well. The SFG spectra collected from the immobilized NfsB H360C are shown in Figure 2, bottom panel. The fitting results indicate that for NfsB H360C, χzzz/χyyz is 1.45. 3.2.2. ATR-FTIR Studies on NfsB on Maleimide SAM. Polarized ATR-FTIR measures different (and independent) orientation parameters than does the SFG experiment, providing more measured parameters to more accurately deduce the enzyme orientation. Here, ATR-FTIR spectra were collected from cysteine-modified NfsB after immobilization on the maleimide SAM surface using both the s and p polarizations (Figure 3). Our above SFG studies showed that SFG spectra collected from cysteine-modified NfsB at the SAM/solution interface are contributed mainly by the α-helices. SFG spectra are more sensitive to the ordered secondary structure. Here, ATR-FTIR spectra cannot be fitted using a single peak centered at 1655 cm−1. ATR-FTIR signals are sensitive to different secondary structures. In this study, the ATR-FTIR spectra were fitted using the following peaks centered at 1631 cm−1 (contributed by β-sheets), 1644 cm−1 (contributed by random coil/ disordered structure), 1655 cm−1 (contributed by α-helices), and 1671 cm−1 (contributed by turn/β-sheet). Figure 3 shows that the p-polarized ATR-FTIR spectrum has a stronger signal. The spectral fitting result (Supporting Information) showed the p/s signal strength to be 1.75 for the V424C construct and 2.05 for the H360C construct. This ratio will be used to deduce the orientation of immobilized NfsB, which will be discussed in more detail below. 3.2.3. Orientation Determination and Comparison of Chemically Immobilized NfsB. We combined polarized SFG and ATR-FTIR measurements to determine the immobilized NfsB orientation. Our group has recently developed a software program written in Python that allows us to calculate the SFG 5935

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monitored at the amide I signal frequency indicated that the SFG signal intensity increased continuously (Figure 7), showing that the SFG signals from immobilized CP1c and NfsB were enhanced by each other. This confirms that the orientation of NfsB is around (25, 30). Similar methodology can be applied to determine the orientation of surface-immobilized NfsB H360C using a combined SFG and ATR-FTIR approach. According to the SFG measurements, the possible orientation angle region is shown in Figure 8, top left panel. The possible orientation angle region of NfsB H360 C determined by ATR-FTIR is shown in Figure 8, top right panel. As we discussed above, SFG and ATR-FTIR provide independent measurements; therefore, the final orientation should satisfy both SFG and ATR-FTIR measurements, which is shown in Figure 8, middle panel. The schematics of the NfsB H360C with two possible orientation regions are plotted in Figure 8, bottom panel. Apparently, the angle region (145, 280) positioned the cysteine near the surface to enable the immobilization, while the angle region (35, 100) positioned the cysteine far from the surface. We therefore believe that the angle (145, 280) describes the orientation of NfsB H360C after immobilization. To further compare the orientation of NfsB immobilized via different cysteine sites, we plotted the deduced orientation angle regions of NfsB V424C and NfsB V360C in the same figure and labeled both positions 424 and 360 in the NfsB structure for each orientation region (Figure 9). It is clearly shown that the orientation of NfsB is different while immobilized via different cysteine sites. For each case, the cysteine is near the SAM surface, enabling surface coupling. At the same time, the other position is far from the surface. This study demonstrates the feasibility of controlling the orientation of enzyme molecules on surfaces through enzyme engineering. In this study, we assumed that immobilized NfsB adopts the same structure as the crystal structure. We believe that such an assumption is valid. Our recent MD simulation using a coarsegrained model indicates that the conformational change of immobilized NfsB compared to the crystal structure is negligible. The MD simulation results will be published in the future.

Article

ASSOCIATED CONTENT

S Supporting Information *

Detailed experiments on NfsB thiol characterization, surface immobilization, and surface concentration determination. SFG and ATR-FTIR data analysis and spectral fitting parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Defense Threat Reduction Agency (HDTRA1-11-1-0019) and the Army Research Office (W911NF-11-1-0251). We thank Dr. Xiaofeng Han and Yuwei Liu for assistance with the experiments.

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REFERENCES

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4. CONCLUSIONS Nitro-reductase (NfsB) has been engineered with one cysteine residue (V424C) close to one of its active sites and one farther away from the active sites (H360C). This genetic engineering allows the surface attachment of NfsB through the thiolmaleimide coupling chemistry with site-specific immobilization on the abiotic self-assembled monolayers. This immobilization strategy along with the position-selective mutation technique facilitates the investigation of the enzyme orientation upon immobilization. We have successfully applied SFG and ATRFTIR spectroscopy simultaneously to deduce the most likely orientation of nitro-reductase (NfsB) covalently immobilized on the self-assembled monolayers (SAMs). As we designed, after surface immobilization, the H360C NfsB mutant has two active sites available while the V424C mutant has only one active site available. This interprets the specific activities of the immobilized enzymes, which were found to be 14 ± 1 nM/ min/ng enzyme for the H360C NfsB mutant and 7 ± 2 μM/ min/μg enzyme for the V424C NfsB mutant. Our results demonstrated that it is feasible to control the orientation of enzymes chemically immobilized on surfaces. 5936

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