Investigating the Effect of Two-point Surface Attachment on Enzyme

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Investigating the Effect of Two-point Surface Attachment on Enzyme Stability and Activity Xingquan Zou, Shuai Wei, Somayesadat Badieyan, Mckenna Schroeder, Joshua Jasensky, Charles L. Brooks, E. Neil G. Marsh, and Zhan Chen J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08138 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 7, 2018

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Journal of the American Chemical Society

Investigating the Effect of Two-point Surface Attachment on Enzyme Stability and Activity

Xingquan Zou,*,1 Shuai Wei,*,1 Somayesadat Badieyan, *,1 McKenna Schroeder, 1 Joshua Jasensky,1 Charles L. Brooks III**,1,2 E. Neil G. Marsh, **,1,3 and Zhan Chen, **,1,2 1

Department of Chemistry, 2Department of Biophysics, 3Department of Biological Chemistry University of Michigan, Ann Arbor, Michigan 48109, United States

1 ACS Paragon Plus Environment

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Abstract Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitro-reductase (NfsB), to demonstrate that engineered variants with two strategically positioned surfacetethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.


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1. Introduction The immobilization of enzymes on supporting surfaces provides various practical advantages for using enzymes in biotechnological applications, which has been widely used in research and in applications. Such advantages include easier reusability, enzyme recovery, generally improved enzyme stability under storage and operating conditions, enhanced activity, optimized selectivity or specificity etc.1-13 Physical adsorption and covalent binding are the two most frequently used methods for surface immobilization of enzymes. Physical adsorption relies on relatively weak and non-specific interaction, such as van der Waals forces, hydrophobic interactions, hydrogen bonding, or charge-charge interactions.14-17 Although simple to perform, leaching of the enzyme from the surface and immobilization in inactive conformations reduce enzyme activity and long-term stability.18 Covalent immobilization of enzymes relies on the attachment of the enzyme to chemically reactive surfaces through protein sidechains bearing amino, carboxyl, hydroxyl, or sulfhydryl groups, etc.19-24 Attachment through residues that are commonly found on the surface of proteins, such as Lys or Glu. Bundy et al.21, 25 applied “click” chemistry together with non-natural amino acids to facilitate their “protein residue-explicit covalent immobilization for stability enhancement” (PRECISE) strategy and demonstrated improved stability of a model protein, T4 lysozyme. In this work, we have immobilized enzymes on maleimide-terminated surfaces through engineered cysteine residues, as we reported previously.26-31 Previously,26,

27

we described studies on nitro-reductase (NfsB, PDB: 1DS7), an

enzyme that is able to reduce a range of organic molecules containing nitro-groups and serves as a convenient model system. By studying NfsB variants that displayed unique reactive



cysteinyl residues on their surfaces, we were able to covalently immobilize them on maleimidefunctionalized surfaces. These studies revealed the important roles that enzyme orientation, surface loading and surface topology play in determining enzyme activity and stability. Some previous studies revealed that with a single tethering site for a protein to bind to a surface, the orientation of the protein might not be fully controlled.32 The orientational flexibility may lead to an unfavorable orientation of the surface immobilized protein to lose activity with the active site interacting with the solid surface or neighboring proteins on the surface. Therefore, more tethering strategies have been investigated for a better protein orientation control. One straightforward way is to employ one more tethering point on the protein surface so that the protein can be immobilized with two sites simultaneously. Despite of many efforts of trying and testing, very few double tethering site trials led to good protein stabilities with the expected orientation control.32 Mansfeld et al.33, 34 studied the stability of several mutants of the thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) immobilized via protein cysteine. Their results showed that enzyme stabilization depends strongly on the position and number of the tethering sites. The greatest stabilization was achieved by tethering the enzyme through its postulated unfolding region. However no direct evidence of the postulated structure of the protein after surface immobilization was reported. Excellent research has also been performed on multiple-site enzyme immobilization on epoxy and agarose substrates to improve the surface immobilized enzyme properties, such as enzyme stability.35-40 Especially with herterofunctional support, the final immobilized enzyme properties can be further improved.38 Such research focused on the enzyme property


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investigation, enzyme structural study could further provide molecular mechanisms to interpret the enzyme property change via multiple site immobilization. Here we report the use of coarse-grained molecular dynamics (MD) simulations to guide the design of NfsB mutants with two neighboring tethering sites on each enzyme that should result in immobilized enzymes with better controlled surface orientation and increased stability. We experimentally characterized three NfsB mutants, each containing two surfacereactive cysteinyl residues, and immobilized them on maleimide-terminated SAM surfaces at the molecular level. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the orientations of these surface immobilized NfsB enzymes. Stability assay results confirmed the simulation predictions that NfsB mutants immobilized via two tethering sites, (423, 247) or (423, 111), have better structural stability compared to other NfsB variants. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

2. Experimental Section 2.1 Materials Right-angle CaF2 prisms were purchased from Altos Photonics (Bozeman, MT). O(propargyl)-N-(triethoxysilylpropyl) carbamate (90%) was purchased from Gelest (New Orleans, LA). Azido-PEG3-Maleimide Kit was purchased from Click Chemistry Tools (Scottsdale, AZ). Potassium phosphate (monobasic and dibasic) solution (1.0 M), Tris(2-



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carboxyethyl) phosphine hydrochloride (TCEP) solution (0.5 M), toluene, copper sulfate, (+)sodium L-ascorbate (≥99.0%), dimethyl sulfoxide (DMSO, ≥ 99.9%), and sodium dodecyl sulfate

(SDS,

98%)

were

purchased

from

Sigma-Aldrich

(St.

Louis,

MO).

Ethylenediaminetetraaceticacid (EDTA) was obtained from Fisher Biotech (Pittsburgh, PA).

2.2 Protein expression, purification and mutagenesis These experiments used a modified ‘single-subunit’ version of NfsB in which the two subunits were genetically fused through a flexible linker sequence and endogenous cysteine residues replaced by serine or alanine, as described previously.26 Cysteine mutations were introduced at the desired positions in the enzyme using standard methods. All the NfsB enzyme variants were expressed and purified using standard methods as described previously.26-28

2.3 Enzyme immobilization for activity studies For enzyme activity studies, self-assembled monolayers terminated with maleimide functional groups were formed at the surface of 75 m glass beads as described previously.28 Briefly azide-functionalized EG3 linkers terminated with either –maleimide or –OH were reacted in molar ratio of 1:10 respectively with the alkyne-functionalized glass bead surfaces by a click reaction. This ratio was previously demonstrated to provide the maximum specific activity of NfsB upon chemical immobilization.28 Enzyme immobilization was conducted by incubating the functionalized beads with the various cysteine-containing variants of NfsB at 5 M concentration in 1X PBS buffer at pH 7.5 for 16 hour at 4°C with gentle shaking. Then the beads were rinsed with PBS buffer and non-ionic detergent to remove non-specifically bound


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enzyme, as described previously.29 The cysteine-free variant of NfsB was used as a control to determine background levels of non-specifically bound enzyme.

2.4 Activity assay and surface coverage The activities of soluble and surface-immobilized NfsB enzymes were measured as previously described.28 In brief, the activity of NfsB with 4-nitrobenzenesulfonamide as substrate was monitored by following the reduction in absorbance at 340 nm due to NADH using plate reader. Assays were performed at 20 °C with three replicates. The activity of the control sample (cysteine-free variant of NfsB) was always negligible compared to that of the chemically immobilized NfsB molecules. This is reasonable because under our experimental condition, the maleimide group is ~1000 times more reactive toward a free sulfhydryl than to an amine. The surface coverages of covalently immobilized NfsB via one or two cysteine sites were determined by BCA assay and found to be similar for various mutants.28

2.5 Thermal stability assay The half-lives of single (with one surface immobilized cysteine) and double (with two neighboring cysteines) mutants of NfsB were measured by incubating enzyme-functionalized beads at 45 °C in a water bath (the approximate melting temperature of this enzyme) in presence of PBS buffer. Four batches of beads were used for each variant studied. Samples were taken at various time points and cooled immediately on ice. The residual activity of each sample at each time point was then measured as described above. The half-life was measured individually and independently for each batch of sample, while overall half-life for each mutant was



measured by including all the measured activities from each batch of that mutant. The activity curves were fitted to a single exponential decay function using Kaleidagraph 4.0 to determine the half-lives of the enzyme variants. Using the exponential decay curve, the half-life is defined as the time point that the immobilized enzyme shows half of the activity it has at time 0 (Supporting Information, Figures S7 and S8).

2.6 SFG spectroscopy SFG vibrational spectroscopy as a surface sensitive technique has been widely used to study chemical and biological molecules on surface or interface.41-51 SFG theory52-55 and experimental details56-62 have been extensively reported and will not be repeated here. In this study, SFG experiments were performed using a commercial SFG system from EKSPLA. The pumping laser delivers picosecond (ps) laser pulses (20 ps pulse width) at a repetition rate of 50 Hz. Such output goes through a nonlinear optical system to generate two kinds of laser pulses for SFG detection. One pulse has a fixed wavelength at 532 nm, and the other pulse has tunable wavelength from 2.3 to 10 μm. The two pulses overlap spatially and temporally at the sample surface to generate sum frequency signal. A near total reflection geometry for SFG signal collection was adopted in order to maximize the collected SFG signal.63, 64 SFG spectra were collected with different polarization combinations of the input visible, input IR, and generated SFG beams, including ssp (s-polarized SFG signal, s-polarized visible input, and ppolarized input IR) and ppp. More details about how to deduce enzyme orientation from SFG data were extensively published previously,61, 65 and some discussions are presented in the supporting information.


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SiO2-coated CaF2 prisms were thoroughly cleaned by detergent, DI water, and oxygen plasma, then immersed in a 10 μM O-(propargyl)-N-(triethoxysilylpropyl) carbamate-toluene solution for more than 24 h to grow an alkyne-terminated self-assembled monolayer (SAM). Azido-PEG3-maleimide linker was prepared by mixing the 10 mg reaction kits in 224 μL of DMSO for 60 min to generate a 120 mM azido-PEG3-maleimide stock solution. The click reaction between the surface terminated alkyne group and the azide group in phosphate buffer (PB, pH = 8.0) was catalyzed by 100 μM copper (I) ions for more than 12 h at room temperature (∼24 °C). The SAM surface was washed by 100 mM TCEP solution and DI to remove the copper (I) ions, then immersed in a 5 μM NfsB enzyme solution to immobilize the NfsB enzyme molecules onto the SAM surface. Before characterization, the substrates were washed with DI water, 1% sodium dodecyl sulfate (SDS) solution, and DI water for a second time, to remove any physically adsorbed NfsB enzymes. More detailed procedures of SAM surface preparation and biomolecules immobilization have been published previously.66 The schematic showing the SAM preparation and enzyme immobilization can be found in the supporting information (Figure S3).

2.7 Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy ATR-FTIR experiments were performed on a commercial Nicolet 6700 FTIR spectrometer. Maleimide terminated SAM surface was prepared on Germanium ATR trapezoid prism (Crystran Ltd., Dorset, United Kingdom) coated with a thin layer of SiO2. To avoid spectral confusion between the water O−H bending mode and NfsB enzyme amide-I mode, D2O instead of H2O was used in the ATR-FTIR measurements. Both s- and p-polarized ATR-



FTIR spectra in the amide I frequency region were collected to determine the enzyme orientation.26, 30, 67 The background ATR-FTIR spectra were collected from maleimide SAM surface without NfsB immobilization. Then, after NfsB was immobilized on the SAM surface, ATR-FTIR spectra were collected under the same condition using the previous spectra as backgrounds. More details about enzyme orientation determination from ATR-FTIR are presented in the supporting information.

2.8 Circular Dichroism (CD) spectroscopy The secondary structures of NfsB mutants immobilized were measured by a J-1500 CD spectrometer (Jasco Inc., Japan) using a continuous scanning mode at room temperature. NfsB enzymes were immobilized on maleimide-terminated SAM surface constructed on high quality z-cut quartz slides. After removing physically adsorbed NfsB by a 1% sodium dodecyl sulfate (SDS) and phosphate buffer (PB) solution wash, the CD spectrum was collected between 240 and 190 nm at a 1 nm resolution and 50 nm/min scan rate, and averaged by five scans. To increase CD signal, six slides with immobilized NfsB enzymes were stacked together in the buffer solution and CD spectra were collected.

2.9 Molecular Dynamics Simulations of Surface Tethered Proteins Molecular dynamics simulations were performed using coarse-grained simulation methods based on a well-studied protein-surface interaction model.68 This model employed the Karanicolas and Brooks Go-like protein model69-72 and a corresponding surface potential that quantitatively describes surface hydrophobicity. Several studies have demonstrated the


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accuracy of predictions from this model for protein conformation and orientation while interacting with various sensor surfaces.31, 32, 63, 64, 66, 73-76 The detailed surface potential and various parameters were developed and published in earlier work68, 77 and are included in the supporting information for completeness. The simulation details are also described in the supporting information.

3. Results and Discussion 3.1 Design of NsfB with two neighboring cysteine sites for surface immobilization 3.1.1 Selection of the second tethering site based on the NfsB H360C Previously we studied the effect of surface orientation on the activity of immobilized NfsB. 26, 27 Because wild type NsfB enzyme is a homodimer, we engineered the enzyme to be a single subunit, pseudo-dimeric enzyme by genetically connecting the two subunits through a short linker sequence, as described previously.26, 27 Additionally, the single, naturally occurring cysteine in NfsB was mutated to alanine, allowing cysteine residues to be placed at selected surface positions, e.g., position 423 or position 360, for surface immobilization.26, 27 As part of that study, two variants, NfsB-H360C and NfsB-V423C were investigated,26 and it was found that NfsB-H360C when immobilized exhibited higher activity. This was attributed to the fact that tethering NfsB through position 360 orients both of the active sites in the pseudo-dimeric enzyme away from the surface. Therefore in this current study, our initial investigations for immobilizing NfsB via two cysteine sites focused on identifying a second tethering site (in addition to site 360) that would potentially stabilize the enzyme further and limit the orientations that the enzyme could adopt. Based on these considerations, position-133 was



identified by visual inspection as a promising second tethering point that could form an additional covalent cross-link to the surface without distorting the protein. We introduced a second mutation into NfsB-H360C to produce the variant NfsBH133C+H360C, which could be over-expressed and purified from E. coli by standard methods.26 After covalently immobilizing NfsB-H133C+H360C on the maleimide-terminated SAM surface, both SFG and ATR-FTIR spectra were successfully collected from the immobilized enzyme (data not shown). These data indicated that, when tethered to the surface at two positions, the enzyme maintained its secondary structure and was specifically oriented on the surface. However, although in free solution this NfsB variant had similar activity to the wild-type NfsB, no enzymatic activity could be detected from the surface immobilized NfsBH133C+H360C. To better understand this unexpected result, coarse grained MD simulations were undertaken to model the interaction of the tethered enzyme with the surface. These simulations demonstrated that when NfsB-H133C+H360C was immobilized via two tethering sites, the two subunits of the NfsB pseudo-dimer became separated due to strong hydrophobic interactions between the enzyme and the surface. Although each monomer largely maintained its tertiary structure, the interface between the two original subunits in the pseudo-dimeric enzyme, which contains the active sites, was extensively disrupted by the strong protein-surface hydrophobic interactions, shown in Figure 1. The simulation results provided a reasonable explanation for the experimentally observed loss of the enzymatic activity coupled with the detection of strong SFG signal.


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360

133

360

133

Figure 1. MD simulation results of snapshots of initial and final structures of NfsB immobilized on maleimide-SAM surface with tethering sites 133C and 360C. The red arrows indicate the active sites.

3.1.2 Rational selection of the second tethering site to achieve stable protein structures based on NfsB V423C simulation results We next considered the possibility of introducing a second tethering site adjacent to position-423 of NfsB, as studies on NsfB-V423C variant had validated this position as a good tethering site. To screen for sites that would allow a second tethering point to the surface without disrupting the enzyme’s structure, we carried out coarse-grained MD simulations for NfsB V423C immobilized on the maleimide-terminated SAM surface at position 423. From our analysis of these simulations, we identified three residues (111, 247, and 290) that consistently maintained physical interactions with the solid surface, and choose each of these as a candidate for the introduction of a second tethering site (P111C, K247C, and S290C, Figure 2).



Figure 2. MD simulation results of NsfB structure after immobilization on a maleimide SAM surface via site 423C, illustrating that positions 111, 247 and 290 are in close proximity to the surface. Based on the modeling, position 423 was chosen as the first immobilization site and each of the three other positions (111, 247, or 290) were then investigated as the second immobilization site.

The simulations indicated that the NfsB structure was maintained when tethered to the surface at position-423, suggesting that a second tethering site introduced at position-111, 247, or 290 should likely preserve the NfsB structure. To explore this hypothesis, we performed further MD simulations in which NfsB was tethered through position-423 and each of the other three positions in turn. Figure 3 shows snapshots from the simulations, which in each case spanned timescales sufficient to elicit the response of the protein to the additional tethered position. Although the modeling revealed some changes in the conformation of each protein due to surface interactions, the region surrounding the active site remained unperturbed in most cases. NfsB immobilized through positions-423+247 or 423+111 exhibited good structural stability throughout the entire simulation. NsfB immobilized through positions-423+290 appeared less stable and denatured towards the end of the simulation. All these immobilized NsfB variants appeared much more stable than NfsB immobilized through positions-133+360 we discussed above in Figure 1, which denatured rapidly during simulation.


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Figure 3. MD simulation results of structures of surface immobilized NfsB 111+423, NfsB 247+423, and NfsB 290+ 423 via two tethering sites.

3.2 Structural Characterization Based on our simulation results, we expressed and purified three NfsB enzyme variants designated NfsB-423+290, NfsB-423+247, and NfsB-423+111 in which Cys was introduced at each of the positions indicated. These variants were all expressed as soluble proteins, could be purified by Ni-NTA affinity chromatography, and were better than 95% homogeneity as determined by SDS-PAGE. In free solution, these variants exhibited similar reductase activity to wild-type NsfB, of 4.2 ± 0.2 mol.min-1.mg-1. These proteins were then immobilized on maleimide-terminated EG3 SAM surfaces and their structures interrogated using a combination of SFG vibrational spectroscopy and ATR-FTIR spectroscopy. Figures 4(a)-4(c) show the SFG spectra collected from surface immobilized NfsB variants NfsB-423+290, NfsB-423+247, and NfsB423+111 in phosphate buffered solution, respectively. Both ppp and ssp spectra show a strong amide I peak centered around 1650 cm-1, 15 ACS Paragon Plus Environment


which arises from α-helical structure in the immobilized NfsB enzymes. These spectra were fitted using  ( 2 )

2

2) ( 2) to obtain quantitative SFG signal strength and the ratio of  (ppp . /  ssp

Polarized ATR-FTIR spectra were also collected from the immobilized enzymes and are shown in Figures 4(d)-4(f). Gaussian functions were used to fit and decompose the spectra into the components contributed by α-helices, β-sheets, and random coil structure within the protein.

Figure 4. SFG polarized ssp (s-polarized SFG signal, s-polarized input visible, and ppolarized IR input) and ppp spectra of immobilized NfsB (a) 423+290, (b) 423+247, and (c) 423+111, and their correspondingly s and p polarized ATR-FTIR spectra (d), (e) and (f). The spectral data was used to deduce the orientations, specified by a combination of tilt (θ) and twist angles (ψ), of the immobilized enzymes with respect to the SAM surface using methods described previously (The zero position was defined as the orientation of the protein in the PDB file 1DS7).26, 28, 30, 67 Figure 5(a), 5(c) and 5(e) show heat maps of the possible orientation angle regions of the three NfsB variants deduced by SFG and ATR-FTIR measurements, with the color indicating the probability that the deduced orientational angles match the data. More details of how to obtain such heat maps were published26, 30, 67 and 16 ACS Paragon Plus Environment

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presented in the supporting information. The results indicate that the three NfsB variants adopt similar, well-defined orientations with respect to the surface normal.

Figure 5. Possible orientation angle regions deduced using the SFG and ATR-FTIR measurements and the most likely orientations of NfsB immobilized on SAM surface. x-y plane denotes the SAM surface. (a) and (b) NfsB 423+290; (c) and (d) NfsB 423+247; (e) and (f) NfsB 423+111. The red arrows indicate the active sites. The color scale in (a), (c), and (e) indicates the percentage matching between the calculated SFG/ATR-FTIR values as a function of enzyme orientation and experimental measured SFG/ATR-FTIR results, which has been extensively used in our previous publications.78

3.3 Effects on Enzyme Activity and Stability 17 ACS Paragon Plus Environment


The activity and stability of both the single-site immobilized and double-site immobilized enzymes was compared. Upon immobilization, all the enzymes exhibited at least a 50% reduction in their activity relative to enzyme in free solution (Figure 6(a)). This decrease in activity, which has been observed in previous studies,28, 29 may be attributed to the reduced accessibility of one active site in the engineered NfsB pseudo-dimer that is positioned close to the surface when the enzyme is immobilized. Other, non-specific surface effects may also contribute to the reduced activity. The NfsB enzymes immobilized through two tethering sites have generally slightly lower activity compared to the variants immobilized through a single site. This might reflect a loss of protein conformational motion that contribute to activity.2 To quantify the effects on enzyme stability of single-site tethering versus two-site tethering, the half-life (t1/2) of each of the immobilized NfsB variants was measured at 45 °C. This temperature was chosen as it represents the melting temperature of wild-type NfsB in solution; therefore differences in half-life due to changes in thermal stability are most readily revealed.28 As illustrated in Figure 6(b), the NfsB-111+423 and NsfB-247+423 variants immobilized through two tethering points showed small but statistically significant (P < 0.05) increases in their half-lives compared to any of the single-site immobilized NfsB variants. The other two-site immobilized enzyme, NfsB-290+423, demonstrated no improvement in its thermal stability in relative to the single-site immobilized enzymes NfsB-290 or NfsB-423. These results are in a good agreement with the predictions of the coarse-grained MD simulations of surface-immobilized NfsB, which indicated that immobilized NfsB-290+423 would be less stable than NfsB-111+423 and NsfB-247+423, which remained folded at the end of the simulation. The higher thermal stability of immobilized NfsB with two cysteines is also


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well correlated to the enzyme structural stability in air as illustrated by SFG studies. While the NfsB immobilized at the surface via a single tethering site lost the secondary structure in the air (in absence of bulk water), the immobilized NfsB with two cysteine tethering sites kept its conformation at least partially upon drying at air. Such SFG results in air are presented in the supporting information.

Figure 6. (a) Activity assay of immobilized NfsB on maleimide-terminated SAM surface. The reduction in NADH absorbance in the presence of 4-nitrobenzenesulfonamide substrate was monitored at 340 nm. (b) Thermal stability assay of immobilized NfsB by measuring the halflife of enzyme from its time-dependent deactivation profile at 45 °C. The values in the graph show the p-values of t-test analyses between the calculated half-life of each immobilized double mutant relative to its corresponding single mutants.

It is interesting to observe that comparing to all other enzymes immobilized via one site, NfsB 111 has the best stability. Site 111 is on a helix, while other sites such as 247, 290, and 423 are on coils. Our previous study on a different enzyme, beta-galactosidase, also showed that the enzyme immobilized with a site on a helix had a better stability compared to an enzyme immobilized via a site on a coil.31 Similarly, here for NfsB, the surface immobilization via a



cysteine site on helix leads to a better activity. With the second immobilization site in addition to the site on a helix, the enzyme’s stability was further enhanced, as demonstrated by NfsB111+423.

4

Conclusions The studies on the effects of protein immobilization site and number on protein activity

and stability have been reported.33, 34, 79 For protein immobilization inside pores of porous materials, it was shown that the protein stability depends on the number of immobilization sites.79 This can be different for proteins immobilized onto a flat surface because multiple immobilization sites may lead to the denaturation of the immobilized protein. Previous research indicated that the immobilization site and number of proteins on flat surfaces influence the protein stability, but no direct evidence on the structure-function relationships of the surface immobilized proteins was reported.33 We have applied a combined approach to study immobilized enzyme on surface with SFG experiments and MD simulations. Our previous studies showed that the SFG experimental and MD simulation results matched with each other very well. For example, with SFG and simulation results, we showed that the immobilization of an enzyme via sites on different secondary structures (coil and helical structure) leads to varied stability,31 and the immobilization of the same enzyme on surfaces with different hydrophobicity have different mobility.80 The development and utilization of coarse grained simulation models of enzymes tethered to SAM surfaces in this research has enabled us to design pairs of attachment sites on


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NfsB through which the enzyme could be immobilized without loss of structure. We experimentally characterized three enzyme variants that were each immobilized through two cysteine attachment points: NfsB 423+247, NfsB 423+111 and NfsB 423+290. It is worth mentioning that for NfsB immobilized via two cysteine sites, after one cysteine site is reacted, reaction of nearby lysine residues with maleimide is possible and cannot be rigorously ruled out. However, we consider that this is unlikely because lysine is intrinsically less reactive than cysteine and at the pH at which the immobilization reaction is conducted, the lysine residues will be protonated and unreactive. Characterization by SFG and ATR-FTIR spectroscopy on NfsB 423+247, NfsB 423+111 and NfsB 423+290 indicated that the orientations of these NfsB variants were consistent with the simulation predictions. Although the two-site immobilized enzymes showed broadly similar activity and stability, two immobilized enzyme variants, NfsB-423+247 and NsfB-423+111 were more stable than any of the single-site immobilized enzymes. However, these enzymes were also less active than the single-site immobilized enzymes, suggesting that there is a trade-off between activity and stability. This observation can be described by the likely effect of two-point immobilization on the enzymes’ dynamics. Increasing the number of tethering sites is expected to reduce the flexibility of the enzyme upon surface immobilization, which could impact the enzyme activity. However, it is reported in many cases81 that rigidifying an enzyme (in solution) is correlated with higher thermal or chemical stability. In addition, after immobilization of the enzyme onto the substrate via one site, even though very likely most of the enzyme molecules would take a specific orientation, they still can move and interact with the support surface in all directions, while such interactions with the support could easily occur. The immobilization of the enzyme via two



sites makes the enzyme only can move backward and forward, which makes the enzyme still can interact with the support, but such interactions are less compared to those of enzyme immobilized via one site. The different degrees of the interactions with the support surface are also responsible for the different behavior of the enzyme immobilized via one or two sites. More generally, we believe that the systematic approach we have developed utilizing coarse-grained MD simulations to guide protein engineering and using advanced experimental tools to protein the molecular structure of immobilized proteins will be valuable for rationally designing immobilized enzymes to maximize both activity and stability in the future. In the future further systematic research will be carried out to optimize the selection of multiple immobilization sites (by computational simulations) that enhance the stability of enzyme while maintaining (or increasing) the enzyme activity.

AUTHOR INFORMATION Corresponding author **E-mail: [email protected], [email protected], [email protected] Tel: (+1) 734-615-6628, (+1) 734-647-6682 Author Contributions *Authors contributed equally to this paper. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests.


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ACKNOWLEDGEMENT This research was supported by the Defense Threat Reduction Agency (HDTRA1-16-1-0004 and HDTRA1-11-1-0019), Army Research Office (W911NF-11-1-0251) and National Science Foundation (CHE-1505385). Supporting Information Available: SFG data analysis, additional figures, CD data, MD simulation parameters, SFG and ATR-FTIR spectral fitting parameters, additional activity measurement data, and additional discussion on choosing enzyme immobilization sites. This material is available free of charge via the Internet at http://pus.acs.org.

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Investigating the Effect of Two-point Surface Attachment on Enzyme

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