Stability of Proteins on Hydrophilic Surfaces - Langmuir (ACS

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Stability of Proteins on Hydrophilic Surfaces Georges Belfort Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503865b • Publication Date (Web): 23 Dec 2014 Downloaded from http://pubs.acs.org on December 27, 2014

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Stability of Proteins on Hydrophilic Surfaces

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Langmuir la-2014-03865b.R3 Article 16-Dec-2014 Grimaldi, Joseph; Rensselaer Polytechnic Institute, ; Rensselaer Polytechnic Insittute, Chemical and Biological Engineering Radhakrishna, Mithun; Columbia University, Department of Chemical Engineering Kumar, Sanat K.; Columbia University, Department of Chemical Engineering Belfort, Georges; Rensselaer Polytechnic Insittute, Chemical and Biological Engineering

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Stability of Proteins on Hydrophilic Surfaces Joseph Grimaldi1, Mithun Radhakrishna2, Sanat K Kumar2*, Georges Belfort1* 1

Howard P. Isermann Department of Chemical and Biological Engineering and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY- 12180-3590 2 Department of Chemical Engineering, Columbia University, New York NY 10027 Key words: Enzyme activity, concave and convex pores, surface polarity, 64mer model peptide, activity measurements and molecular simulations Abstract The physical and chemical properties of solid substrates or surfaces critically influence the stability and activity of immobilized proteins such as enzymes. Reports of increased stability and activity of enzymes near/on surfaces as compared with those in solution abound, however, a mechanistic understanding is wanting. Simulations and experiments are used here to provide details towards such a mechanistic understanding. Experiments demonstrate increased activity of alcohol dehydrogenase (ADH) inside moderate hydrophilic mesopourous silica (SBA-15) pores but drastically decreased activity inside very hydrophilic NH2-SBA-15 surfaces as compared with that in solution. Also, the temperature stability of ADH was increased over that in solution when immobilized in a cavity with a mildly hydrophilic surface. Simulations confirm these experimental findings. Simulations calculated in the framework of a hydrophobic-polar (H-P) lattice model, show increased thermal stability of a model 64-mer peptide on positive and zero curvature surfaces over that in solution. Peptides immobilized inside negative curvature cavities (concave) with hydrophilic surfaces, exhibits increased stability only inside pores that are only 3 to 4 nm larger than the hydrodynamic radius of the peptide. Peptides are destabilized however, when the surface hydrophilic character inside very small cavities/pores becomes large. *Corresponding authors: Georges Belfort and Sanat K. Kumar < [email protected]>.

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Introduction Understanding the effect of surface chemistry and curvature on the stability and activity of a protein or peptide is crucial when developing immobilization strategies for enzymatic conversion. Immobilized protein systems have gained importance in the past decade in the fields of drug delivery1, biofuel cells2, biosensing1, 3 and biofuels4. The activity and stability of several immobilized proteins were investigated previously using a variety of support materials including silica nanoparticles,5, 6 mesoporous silica,7, 8 self-assembled monolayers,9 acid treated single wall carbon nanotubes10, 11, agarose particles12 and diatoms13. Recently, immobilization on mesoporous materials like silica has been extensively studied due to their large pore volume and tunable pore size7, 8. Katchalski et al.14, in the 1970s, determined that enzymes immobilized on surfaces while confined in cavities, displayed higher activity than those in free solution. Since this seminal discovery, many researches have confirmed that adsorbing15 and covalently tethering10, 11 proteins to suitable solid substrates will protect16, 17 and also allow the enzymes to remain stable18 and highly active19, 20. Confinement of enzymes by exploiting surface curvature21 and surface passivation with chemistry22, 23 have been used to minimize losses in stability and activity of immobilized enzymes. The properties of the surface play a major role in proteins stability24; hydrophobic surfaces can cause proteins to lose their αhelical structure and gain β-sheet structure25, 26. Sang et al. reported increased activity of the enzyme myoglobin and lysozyme when immobilized inside hydrophilic SBA-15 pores and dramatically decreased activity when immobilized on hydrophobic SBA-15 pores8. Zhou et al.27 also have reported increased activity and stability of lipases immobilized inside hydrophobic mesoporous organosilicas. These results are a direct consequence of the accessibility of the active site of the enzymes and increased stability of the enzyme, both due to enzyme-surface and enzyme–enzyme interactions. Despite the vast amount of research reported in the literature on enzyme activity and stability at solid interfaces, there is no commonly agreed mechanism of how surface interactions affect the activity/stability of surface-associated

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enzymes. Previous work from the Belfort and Kumar groups has shown how proteins immobilized inside highly hydrophobic pores lose stability, while those immobilized inside weakly hydrophobic materials retain stability.28 To build upon these findings, the research reported here focused on the effect of protein adsorption on hydrophilic surfaces with positive, zero (flat surface) and negative curvatures. Our results combine simulations, which calculate thermal stability of model peptides in the framework of hydrophobic-polar (H-P) lattice models on different curvature surfaces, with experimental findings of enzymes confined inside hydrophilic pores. We also provide a rationale to explain previous findings of increased stability of the enzymes immobilized onto mildly hydrophilic surfaces. Material and Methods Experiments Materials: All materials and reagents were used as received. All buffers are at physiological pH and room temperature unless specified otherwise. Alcohol dehydrogenase from Saccharomyces cerevisiae [A3263] (ADH, 146.8 kDa, pI 5.429), β-nicotinamide adenine dinucleotide (NADH), isobutyraldehyde, thiamine pyrophosphate (ThDP), (3-Aminopropyl) triethoxysilane (APTES), and buffer salts were purchased from Sigma-Aldrich Chemicals (Milwaukee, WI). SBA-15 was synthesized according to published protocols. SBA-15 is a mesoporous silica with an isoelectric point of 37 and ADH has an isoelectric point of 5.429. 96 well plate ADH assay conditions: The reaction mixture contains: 105 µL buffer A (50 mM potassium phosphate buffer, pH 6.8, 2.5 mM MgSO4, 0.1 mM ThDP), 20 µL NADH 2.5 mM in buffer A (final concentration 0.25 mM) and 25 µL 25 mg particles/mL buffer A. The assay was started by addition of 50 µL isobutaldehyde 120 mM in buffer A (final concentration 30 mM) to each well. ADH converts isobutaldehyde to isobutanol using NADH as a cofactor; NADH absorbs light at 340 nm and NAD+ does not.

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Kinetic activity was followed by observing the decrease in absorbance over time (∆ABS). SBA-15 functionalization with NH2 groups: SBA-15 (100 mg) was reacted with 2% APTES in toluene (3 mL) overnight30. Toluene was removed using a rotovap and solid SBA-15 was rinsed 3 times with ethanol and 3 times with DiH2O. NH2 functionalization was confirmed by FTIR. (Fig. S1). SBA-15/ NH2-SBA-15 ADH immobilization: SBA-15 or NH2-SBA-15 (25 mg) was mixed on an end-over-end mixer with 1 mL (1 mg ADH/mL buffer A) solution for 4 hours. The particles were centrifuged and the supernatant assayed at 280 nm on a nanodrop UV-Vis spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) to determine protein concentration. SBA-15 particles were washed with fresh buffer A, then the SBA-15 particles were assayed according to the 96 well plate assay conditions described above. Kinetics of immobilized enzyme was compared to those of equivalent enzyme free in solution. ADH in solution and immobilized ADH on SBA-15 Heat Stability: Solutions (2 mL) of either ADH (50 µg/mL) or ADH immobilized on SBA-15 (25 mg of particles/mL), were heated to various temperatures (below) for 10 minutes in glass vials using a Fisher Scientific Isotemp 125D heating block. The vials were allowed to cool to room temperature in ambient conditions. Selected temperatures were room temperature, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C. Once cooled, 200 µL aliquots of the enzyme solutions were each pipetted into 8 wells of a 96-well plate. 50 µl of isobutaldehyde 120 mM in buffer A (50 mM potassium phosphate buffer, pH 6.8, 2.5 mM MgSO4, 0.1 mM ThDP), (final concentration 30 mM) was added to each well to start the reaction. ADH converts isobutaldehyde to isobutanol using NADH as a cofactor; NADH absorbs light at 340 nm and NAD+ does not. Kinetic activity is tracked by observing the decrease in absorbance over time.

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Simulations Model: In the current paper, we have used a Hydrophobic– Polar (H-P) lattice model introduced by Dill to model a generic protein or peptide31. The peptide chain is grown as a self-avoiding walk in a three dimensional cubic lattice with periodic boundary conditions imposed in all the directions. The bond length between the adjacent amino residues is held constant equal to the unit space. Solvent molecules (water) are modeled implicitly. To account for the presence of an implicit solvent, an attractive potential is introduced between the non-bonded hydrophobic resides to mimic the hydrophobic effect. Two ‘H’ groups interact with an energy, - ε H − H (ε H − H = 1) , when they are the nearest non-bonded neighbors. All other interactions ε P− P and ε H − P are set to zero. To simulate surface adsorption on a flat surface, a plane at z=0 is modeled as a hydrophilic surface. λ is a measure of the surface hydrophilicity when the peptides occupy the z=1 plane. λ=0 represents an athermal (neutral) surface. An athermal surface is placed at z=zmax to confine the peptide to a finite volume. Only the ‘P’ (polar) groups of the peptide interact with the surface. Strong interactions with the surface result in lower energies; therefore, the energy of interaction for a polar group is -λ. Simulations on the inside (negative curvature) and on the outside (positive curvature) were performed to study the effect of curvature on protein adsorption. A detailed description on the validation of this model is presented in our previous works28. Following our previous convention, we fix the length scale for peptide-surface interaction to be √3 lattice unit. In the current study, we have used a 64 mer HP model designed by Yue et.al32. The model peptide forms a four-helix coil in its folded state exhibiting typical thermal transitions similar to that of proteins. The size of the simulation box ( 65 × 65 × 65 ) is chosen so as to avoid any interactions between the periodic images of the peptide. The peptide folding is modeled as a two state process. The peptide with ground state energy of -56 ε is treated as the folded state and all the other energy states are treated as unfolded state. Monte Carlo simulations33 were carried out at different temperatures

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(T * =

ε k BT

) and surface hydrophilicity (λ). In the reduced temperature range we

are at ε~4kT (0.25 22°C. However, ADH immobilized on SBA-15 at room temperature exhibited a kinetic activity of (slope) 0.1884 ± 0.031 ∆ABS(-)/∆time (s). This is just less than a 3fold increase in activity. In order to compare ADH activity in solution versus adsorbed to the inner surface of concaved weakly hydrophilic SBA-15, we normalized the enzymatic activity values (slopes) by the initial room temperature activity in Fig. 2 and show the effect of temperature. After heating and cooling ADH kinetic activity in solution declined with increased temperature. This was

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not the case for ADH immobilized on SBA-15 which retained over 90% of it’s activity up to 55°C. Molecular modeling is used below to qualitatively explain these results. Simulation Results Neutral surfaces: The effect of surface hydrophilicity (λ) on the folding temperature of a 64-mer model peptide under different geometries is shown in Fig. 3. For a neutral surface (λ=0), the model peptide inside a spherical concave cavity (radius, R=4 and R=6) has a higher folding temperature indicative of higher thermal stability when compared with that on a flat surface and on a positive curvature surface. This is purely an entropic effect as explained in our previous paper28. Confining model peptides helps in eliminating many open chain conformations of the unfolded state of the peptide due to physical constraints. Consequently this loss in entropy of the unfolded state drives the equilibrium towards the folded state. This is further reinforced whereby the folding temperature inside a cavity of radius 4 is higher than that of inside a cavity of radius 6. The 64-mer model peptide, when placed onto a flat surface and onto the outside of a convex surface with radius 10, behaves as if it were in bulk solution since at a value of λ=0, it has no propensity to adsorb onto the surface. Also, due to the large open volume available above the surface, it does not sense confinement due to the presence of the surface. The folding temperature of the 64-mer model peptide for an athermal surface with different geometries is listed in Table 1. Hydrophobic surfaces: As the value of surface hydrophilicity (λ) is gradually increased, we see that it has a substantial effect on the folding temperature (Tm ) of the 64-mer model peptide depending on the geometry (Table 1 for λmax at Tm,max). a) Adsorption on the inside (negative curvature): We observe contrasting behavior of the peptide depending on the size of the cavity. The model peptide inside the smaller cavity of radius 4 is dramatically destabilized by

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the increase in surface hydrophilicity (λ). This is explained by the fact that, inside smaller cavities surface energy dominates entropy. As a result, the model peptide denatures (unfolds) to gain peptide-surface contacts indicated by a decrease in the folding temperature. On the other hand, the folding temperature of the model peptide increased inside the larger cavity of radius 6 with an increase in the value of λ up to a critical value of λ=0.3 from Tm=0.295 to Tm=0.32 and then decreased. For the values of λ > 0.52, the peptide is destabilized even relative to its bulk behavior. This behavior can be explained as follows. For low values of λ, the model peptide gains stability due to two independent factors. One from the entropic stability from the confinement and second from the energetic stability due to interactions between the ‘P’ (polar) groups of the model peptide and the surface. The surface energetics are strong enough to stabilize the folded state but not strong enough to denature the model peptide, i.e. the energetic gain by unfolding is countered by the energetic gain upon adsorption in the folded state and the entropic stabilization. For values of λ > 0.52 the surface energetics become strong enough to rupture the model peptide and hence it is destabilized. b) Adsorption on flat surface: The folding temperature of the model peptide increased with increase in value of surface hydrophilicity from T=0.29 for λ=0 to T=0.317 for λ=0.5 and then decreased with the further increase in λ. The percentage increase in the folding temperature (~6%) is less than that for adsorption on the inside of a cavity of radius 6 (~9%) possibly due to absence of entropic stabilization of confinement. c) Adsorption on the outside of a (convex) surface: A maximum percentage increase in the folding temperature (~13%) is observed for adsorption of the 64-mer model peptide on the outside of a hydrophilic surface with positive curvature (convex). This increased stabilization comes from the two independent factors – weak peptide-surface interactions and availability of greater local open volume above the surfaces. Due to weaker peptide surface interactions on a convex surface, the 64-mer model

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peptide is stabilized at higher values of λ compared with that on a flat surface and on the inside of a negative curved surface (concave). Further, due to more local volume available above the surface due to its positive curvature, the 64-mer model peptide in its folded state is exposed to relatively more surface area (more ‘P-S’ contacts) resulting in higher stability. For very high values of λ though, the peptide denatures due to strong peptide-surface interactions.

The difference in free energy differences between the folded and unfolded state (∆∆G / k BT = (∆G folded − ∆Gunfolded ) / k BT ) of the 64-mer model peptide placed on various geometric surfaces as a function of λ measured at their respective melting temperatures is shown in Fig. 4. A negative value of the free energy difference indicates that the 64-mer peptide is stabilixed in the folded state relative to that in bulk solution. The data in Fig. 4 confirm the findings shown in Fig. 3. For the pore with radius 4 (inside), the free energy difference is always positive i.e ∆G unfolded < ∆G folded indicative of the fact that the unfolded state is the minimum free energy state over the entire range of surface hydrophilicity (λ), whereas in all other cases the folded state is stabilized up to a critical value of λ (λcrit) depending on the geometry studied i.e. the protein prefers to adsorb in its folded state and not unfold between λ=0 and λcrit. Discussion Qualitative agreement between the simulations and the experiments are shown here for the enzyme, ADH, and the model 64-mer peptide. The 64-mer model peptide serves as a good model for a solvent stable protein. ADH is a wellstudied protein with 31% α-helical structure, 30% beta sheets, 21% turns and 18% random (PDB: 2HCY) and is stable in solution. Even though ADH has 347 residues in each of its subunits, the 64-mer model peptide which forms a four helix bundle in its folded state is known to be a common structural motif among natural proteins and has previously been shown to exhibit structural behavior similar to proteins (i.e. repressor of primer, ROP, 63 residues; cytochrome b562, 10


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106 residues; T4 lysozyme, 164 residues) 32, 37, 38. The Belfort group has shown that proteins lose their α-helical structure and gain β-sheet structure25, 26 when exposed to surfaces, especially hydrophobic surfaces35. By modeling a 64-mer α-helical peptide on various geometric and hydrophobic surfaces, insight into the stability of a full-length protein was established. Immobilizing ADH on an intermediate hydrophilic surface (SBA-15) was most active and more so than in solution or on a very hydrophilic surface (Fig 1), and more stable than in solution when exposed to heating (Fig 2). These results are consistent with the simulation results, which clearly indicate a critical intermediate surface hydrophilicity for maximum stability The simulation results should be analyzed with some caveats as the HP protein model is an implicit solvent model and as a result does not capture the effects of solvent, pH, ionic strength and also coulombic interactions. The driving force for adsorption is not due to electrostatic interaction since the pH of the solution is 7.4; both SBA-15 and ADH are negatively charged. Note however the major driving forces for protein adsorption in the current study are hydrophobic and van der Waals interactions between the polar (P) residues of the protein and the surface. Further, our simulations results are also in agreement with previous experimental work which shows that enzymes adsorbed onto hydrophilic surfaces retain higher activity in organic solvents compared with that in bulk solution39. The 64-mer protein forms a 4 helix bundle in its native state, whereas ADH is known to contain 31% helical content, 30% beta sheets, 21% turns and 18% random in its native state. However, most experimental works attribute the loss in activity to the loss of helical content. Therefore in this context the 64mer protein can serve a model to associate enzymatic activity and helical content of the protein. Conclusions Concave geometries, which confine ADH, offer higher enzymatic kinetics than free equivalent protein in solution. This finding only holds true for surfaces that have weakly hydrophilic pore surfaces. When the surface chemistry is 11


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changed to a strongly hydrohopbic28 or strongly hydrophilic cavity, ADH is destabilized. This suggests that for a hydrophilic cavity, destabilizing interprotein interactions are dominant and the protein-surface interactions are not sufficiently intense to stabilize the protein. These stabilizing effects are confirmed during heat denaturing studies. ADH immobilized on weakly hydrophilic SBA-15 retains over 90% of it’s activity up to 55°C while ADH free in solution continuously loses activity for T > 22°C. Simulations in the framework of hydrophobic-polar lattice model provide us a rationale to explain and understand the experimental results. Increased thermal stability of the 64-mer model peptide on both surfaces of positive and zero curvature was observed. For immobilization inside cavities with hydrophilic surfaces with negative curvature, we observe increased stability only inside hydrophilic pores of optimal sizes. The 64-mer model peptide was destabilized with increasing hydrophilicity inside very small pores. For low values of λ, inside negative curvature pores, the 64-mer model peptide gains stability. Surface energetics are sufficiently strong to stabilize the folded state but not strong enough to denature the 64-mer peptide. This is due to two independent factors: (1) the entropic stability from the confinement and (2) the energetic stability due to interactions between the polar groups of the model peptide and the surface. The simulations are consistent with the experimental findings and provide a qualitative explanation. Increased activity was observed for ADH inside weak to moderate hydrophilic SBA-15 pores; however, a drastic decrease in the activity was detected for very hydrophilic NH2-SBA-15 surfaces.

Acknowledgement J G and G B thank Dr. Marc-Olivier Coppens for providing SBA 15. The authors also thank the Department of Energy (Grant No DE-FG02-11ER46811) & (Grant No. DE/SC0006520) for funding the project.

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Table 1: Folding temperature for an athermal surface and at its maximum value for a 64-mer HP peptide adsored onto different geometries Geometry

Folding temperature (Tm) at λ=0*

Hydrophobicity λmax

Radius 4 (negative curvature) Radius 6 (negative curvature) Flat surface Radius 10 (positive curvature) * Athermal surface: λ=0

0.302 0.295 0.290 0.290

0.0 0.3 0.5 0.70

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Maximum folding temperature Tm, max 0.300 0.320 0.317 0.330

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Figure Legend Figure 1: Normalized ADH kinetic activity in solution [] (previously published28) and immobilized passively (adsorbed) onto porous SBA-15 [] (previously published28) and NH2-SBA-15 particles [▲]. Figure 2: Percent of activity retained vs. temperature. Activity of ADH in solution (solid) bars and immobilized passively onto porous SBA-15 particles (open). Figure 3: Folding (melting) temperature (Tm) of the 64-mer HP peptide as a function of surface hydrophilicity (λ) for different types and degrees of confinement. Radius 4 (inside) [], Radius 6 (inside) [], Flat surface [▲], Radius 10 (outside) [] Figure 4: Free energy difference between the folded and unfolded state of the 64mer HP peptide with varying surface hydrophobicity (λ) at different degrees of confinement calculated at the melting temperature of the protein at λ=0, of the confining surface. (T*=0.29 for flat surface, T*=0.295 for a cavity of radius 6, T*=0.302 for a cavity of radius 4,T*=0.29 for adsorption outside of the cavity). Radius 4 (inside) [], Radius 6 (inside) [], Flat surface [▲], Radius 10 (outside) []

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Figures and captions

1.05 Absorbance, ABS340 (-)

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1 ~0 0.95

∆ABS (-) ∆time (s)

0.9 0.19 ± 0.03

0.066 ± 0.003

0.85 0.8 0.75 0

50

100 150 Time, t (s)

Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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References: 1. Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Carbon nanotubes in biology and medicine: in vitro and in vivo detection, imaging and drug delivery. Nano research 2009, 2 (2), 85-120. 2. Kim, J.; Jia, H.; Wang, P. Challenges in Biocatalysis for Enzyme-Based Biofuel Cells. Biotechnology Advances 2006, 24 (3), 296-308. 3. Boozer, C.; Ladd, J.; Chen, S.; Jiang, S. DNA-directed protein immobilization for simultaneous detection of multiple analytes by surface plasmon resonance biosensor. Analytical Chemistry 2006, 78 (5), 1515-1519. 4. Grimaldi, J.; Collins, C.; Belfort, G. Toward cell-free biofuel production: Stable immobilization of oligomeric enzymes. Biotechnology progress 2014. 5. Shang, W.; Nuffer, J. H.; Muñiz-Papandrea, V. A.; Colón, W.; Siegel, R. W.; Dordick, J. S. Cytochrome C on Silica Nanoparticles: Influence of Nanoparticle Size on Protein Structure, Stability, and Activity. Small 2009, 5 (4), 470-476. 6. Vertegel, A. A.; Siegel, R. W.; Dordick, J. S. Silica Nanoparticle Size Influences the Structure and Enzymatic Activity of Adsorbed Lysozyme. Langmuir 2004, 20 (16), 6800-6807. 7. Wang, Y.; Caruso, F. Mesoporous Silica Spheres as Supports for Enzyme Immobilization and Encapsulation. Chem. Mater. 2005, 17 (5), 953-961. 8. Sang, L. C.; Coppens, M. O. Effects of Surface Curvature and Surface Chemistry on the Structure and Activity of Proteins Adsorbed in Nanopores. Phys. Chem. Chem. Phys. 2011. 9. Asuri, P.; Bale, S. S.; Pangule, R. C.; Shah, D. A.; Kane, R. S.; Dordick, J. S. Structure, Function, and Stability of Enzymes Covalently Attached to Single-Walled Carbon Nanotubes. Langmuir 2007, 23 (24), 12318-12321. 10. Amador, S. M.; Pachence, J. M.; Fischetti, R.; McCauley, J. P.; Smith, A. B.; Blasie, J. K. Use of Self-Assembled Monolayers to Covalently Tether Protein Monolayers to the Surface of Solid Substrates. Langmuir 1993, 9 (3), 812-817. 11. Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Structure and Function of Enzymes Adsorbed onto Single-Walled Carbon Nanotubes. Langmuir 2004, 20 (26), 1159411599. 12. Bolivar, J. M.; Wilson, L.; Ferrarotti, S. A.; Guis√°n, J. M.; Fern√°ndez-Lafuente, R.; Mateo, C. Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxylagarose. Journal of Biotechnology 2006, 125 (1), 85-94. 13. Poulsen, N.; Berne, C.; Spain, J.; Kröger, N. Silica Immobilization of an Enzyme through Genetic Engineering of the Diatom Thalassiosira Pseudonana. Angewandte Chemie 2007, 119 (11), 1875-1878. 14. Katchalski, E.; Silman, I.; Goldman, R. Effect of the Microenvironment on the Mode of Action of Immobilized Enzymes. Adv. Enzymol. Relat. Areas Mol. Biol. 1971, 34, 445-536. 15. Klibanov, A. M. Enzyme Stabilization by Immobilization. Anal. Biochem. 1979, 93 (1), 1. 16. Kim, J.; Grate, J. W.; Wang, P. Nanostructures for Enzyme Stabilization. Chem. Eng. Sci. 2006, 61 (3), 1017-1026. 17. Kim, J.; Grate, J. W.; Wang, P. Nanobiocatalysis and its Potential Applications. Trends Biotechnol. 2008, 26 (11), 639-646. 18. Zoungrana, T.; Findenegg, G. H.; Norde, W. Structure, Stability, and Activity of Adsorbed Enzymes. J. Colloid Interface Sci. 1997, 190 (2), 437-448. 19. Martinek, K.; Klibanov, A.; Goldmacher, V.; Berezin, I. The Principles of Enzyme Stabilization I. Increase in Thermostability of Enzymes Covalently Bound to a Complementary

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39. Khmelnitsky, Y. L.; Belova, A. B.; Levashov, A. V.; Mozhaev, V. V. RELATIONSHIP BETWEEN SURFACE HYDROPHILICITY OF A PROTEIN AND ITS STABILITY AGAINST DENATURATION BY ORGANIC-SOLVENTS. Febs Letters 1991, 284 (2), 267-269.

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Table of Contents Only

Native Structure

Unfolded Structure

SBA-15 λ=0.5 (Mildly Hydrophilic)

NH2-SBA-15 λ=1 (Very Hydrophilic)

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Stability of Proteins on Hydrophilic Surfaces - Langmuir (ACS

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