Probing the Effects of Cysteine Residues on ... - ACS Publications

Aug 8, 2013 - Kumudu Siriwardana , Ailin Wang , Manuel Gadogbe , Willard E. Collier , Nicholas C. Fitzkee , and Dongmao Zhang. The Journal of Physical...
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Probing the Effects of Cysteine Residues on Protein Adsorption onto Gold Nanoparticles Using Wild-Type and Mutated GB3 Proteins Kumudu Siriwardana,† Ailin Wang,† Karthikeshwar Vangala,‡ Nicholas Fitzkee,† and Dongmao Zhang†,* †

Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762, United States Research and Development Center, Southern Ionics Incorporated, Columbus, Mississippi 39701, United States

‡

S Supporting Information *

ABSTRACT: The role of cysteine residues in the protein binding kinetics and stability on gold nanoparticles (AuNP) was studied using AuNP localized surface plasmon resonance (LSPR) in combination with an organothiol (OT) displacement method. GB3, the third IgG-binding domain of protein G, was used to model protein-AuNP adsorption. While wildtype GB3 (GB30) contains no cysteine residues, bioengineered GB3 variants containing one (GB31) and two (GB32) cysteine residues were also tested. The cysteine content has no significant effect on GB3 binding kinetics with AuNPs, and most protein adsorption occurs within the first few seconds upon protein/AuNP mixing. However, the stability of GB3 on the AuNP surface against OT displacement depends strongly on the cysteine content and the age of the AuNP/GB3 mixture. The GB30 covered AuNPs can be completely destabilized and aggregated by OTs, regardless of the age of the GB30/AuNP mixtures. Long-time incubation of GB31 or GB32 with AuNPs can stabilize AuNPs against the OT adsorption inducted aggregation. This study indicates that multiple forces involved in the GB3/ AuNP interaction, and covalent binding between cysteine and AuNP is essential for a stable protein/AuNP complex.

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INTRODUCTION Proteins are known to have high binding affinity to gold nanoparticles (AuNPs), and thus they are used extensively for AuNP surface modification to improve AuNP functionality, stability, target specificity, and biocompatibility.1,2 When AuNPs are exposed to the biological medium, protein selfassembles onto the AuNP surface, forming a protein-coated layer that is commonly referred to as the protein corona.2,3 Importantly the protein corona evolves from the soft corona in which protein can be desorbed via centrifugation, to hard corona in which the protein-coated layer is stable against centrifugation desorption.4 Despite the extensive literature on protein interaction with gold, including planar gold film and AuNPs,2,5 the fundamental mechanism regarding the protein/ AuNP interaction and the structural details of proteins on AuNPs are not clear.6 Several groups report that the dominant binding forces between serum protein albumin and gold is electrostatic interactions between the positively charged lysine groups and the negatively charged citrate-stabilized AuNPs,4,7 while some researchers believe that the albumin/AuNP binding is mainly through the formation of a S−Au covalent bond between the cysteine residue and AuNP.8−10 We recently studied the interaction of bovine serum albumin (BSA) with AuNPs using a series of organothiols (OTs) including mercapobenzimidazole (MBI), cysteine, and glutathione as molecular probes.11 One key finding is that a substantial amount of OTs is adsorbed onto the AuNPs without inducing significant displacement of BSA preadsorbed onto the AuNPs. Another important observation is that, while © 2013 American Chemical Society

BSA association with AuNP is an exceedingly rapid process, and over 90% of the protein is adsorbed onto AuNP within the first a few seconds of the sample preparation, the fraction of the AuNP surface area passivated by each BSA molecules against OT adsorption keeps increasing during the entire 3 days of the experimental period.11 These results have several important implications: First, the binding affinity of BSA to AuNP is stronger than that of the OTs we tested. This strongly suggested that the BSA is covalently bonded to AuNPs through one or more of its 35 cysteine residues. Second, the AuNP surface areas passivated by BSA molecules increase as a function of aging time of the BSA/AuNP mixture, indicating that BSA cannot reach static-state adsorption even with 3 days of sample incubation. This calls to question the reliability and usefulness of “equilibrium” BSA−AuNP binding constants reported in literature. The goal of this present study is to probe the significance of cysteine on the kinetics and stability of the protein/AuNPs. Proteins are structurally complex, and therefore protein/AuNP interactions are expected to be highly dynamic in nature. Multiple intermolecular forces including covalent bonding and nonspecific forces such as electrostatic, hydrophobic (entropydriven), and van der Waals interactions can simultaneously contribute to the protein/AuNP binding, and the dominant force between protein/AuNP likely varies over time. Our Received: June 14, 2013 Revised: August 6, 2013 Published: August 8, 2013 10990

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Figure 1. (Top) Cartoon representation of GB3 (from PDB 2OED)14 highlighting residues 11 (top) and 19 (bottom) in yellow CPK spheres. GB32 contains both the T11C and K19C mutations, while GB31 only contains T11C.14 Image created using PyMOL software.17 (Bottom) The amino acid sequence of wild type GB30 and the variants used in this study.

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hypothesis is that the initial protein−AuNP binding mainly involves long-range or nonspecific intermolecular forces such as electrostatic, hydrophobic (entropy-driven), and van der Waals interactions that bring protein into contact with AuNPs, while covalent interactions between AuNP and protein cysteine residue occur mostly after the protein deforms on the AuNPs. This hypothesis is derived from the consideration that cysteine accounts for only 2% of protein amino-acid residues in mammalian proteins,12 and cysteine residues exist in buried disulfide bonds, shielded from the protein surface. Consequently, the covalent Au−S bonding for these proteins may be possible only after the protein is adsorbed and deformed onto the AuNPs. Direct experimental observation of the Au−S bond in a protein/AuNP complex is challenging; therefore, we have probed cysteine/AuNP binding indirectly using OT displacement experiments. In this study, we use the third IgG-binding domain of Protein G (GB3) and its variants as model proteins (Figure 1). The wild-type GB3 has a molecular weight of 6208 g/mol. It consists of 56 amino acid residues with no cysteines13−15 (thus denoted as GB30). Wild type GB3 has six lysine residues, and an isoelectric point (pI) of ∼4.9.14 Two variants of GB30 were designed to contain either one or two cysteine residues on the protein surface (K19C, termed GB31; and T11C K19C, termed GB32). The AuNP binding kinetics of the GB3 variants and the GB3 stability on AuNPs against OT displacement were studied using time-resolved UV−vis spectroscopy. The model OTs used in this study include MBI and homocysteine (Hcy). These were chosen because both MBI and Hcy adsorption induce AuNP aggregation, which can be readily monitored using UV−vis spectroscopy.16 We also compared the GB3 adsorption onto AuNPs with polyethylene glycol with one terminal thiol (PEG1) binding with AuNPs. One key learning from these comparisons is that the nonspecific interactions between protein and AuNPs delay formation of covalent bonding between the protein cysteine residue and AuNPs. In this paper, we adopt a convention where (AuNP/GB3)/OT refers to a sample of AuNP/GB3 that has been incubated before subsequently adding OT. Similarly, AuNP/(GB3/OT) refers to a sample where AuNPs are added to a premade GB3 and OT mixture.

EXPERIMENTAL SECTION

Materials. All chemicals used were purchased from Sigma−Aldrich unless otherwise notified. The nominal molecular weight of the thiolated PEG (729159-1G) is 5000 g/mol, and the samples are used as received. Nanopure water (ThermoScientific) was used in all our measurements. GB3 and GB3 Variants. A pET-11b plasmid encoding for GB3 was provided as a generous gift from Ad Bax (National Institutes of Health). After heat-shock transformation, E. coli Bl21*DE3 cells (Invitrogen) were incubated in 1 L of LB media at 37 °C. When the culture reached an OD600 of 0.5−0.7, expression was induced with 1 mM IPTG, and the cells continued to grow at 25 °C overnight. After they reached an OD600 of 2, the cells were harvested and then resuspended in lysis buffer. The resuspended cells were sonicated (Branson Sonifier 250) on ice at power level 6. Processed lysate was heated to 85 °C for 15 min and was swirled every 3−4 min. After the mixture was cooled on ice, DNA was precipitated by adding 0.5% streptomycin sulfate and swirling an additional 10 min. The lysate was centrifuged (Beckman Coulter) at 18000g for 45 min, with GB3 remaining in the soluble fraction. Further purification was performed by gel filtration chromatography on a HiLoad 26/600 Superdex 75pg column (Amerisham biosciences/GE healthcare). Pooled protein fractions (A280 > A260) were dialyzed in nanopure water overnight and frozen at −80 °C. The protein was then lyophilized, and purity was estimated at >98% by SDS-PAGE (Biorad) analysis. The K19C and T11C/K19C variants were designed based on prior work which introduced mutations in GB3 at these locations.18 Variants used in this study were created by site-directed mutagenesis using QuickChange II kits (Agilent). Ampicillin-resistant colonies were isolated, and mutations were confirmed by sequencing plasmid DNA. The structure of variants was analyzed by examining 15N transverse relaxation optimized spectroscopy-heteronuclear single quantum coherent spectroscopy (TROSY-HSQC) NMR spectrum.19 AuNP synthesis. AuNPs were synthesized using the citrate reduction method.20,21 Gold(III) chloride trihydrate (0.0415 g, 0.105 mmol) was dissolved in 100 mL of distilled water and refluxed with vigorous stirring. Then, 10 mL of aqueous 1.14% (w/v) sodium citrate dihydrate was added to the solution after solution was boiling and refluxing was continued for 20 min. The average AuNP size was ∼13 nm in diameter with peak UV−vis absorption centered at 519 nm (Figure S1, Supporting Information). The concentration of AuNPs was calculated as 11.1 nM by assuming that all gold(III) ions were reduced to atomic gold, which is also consistent with the AuNP concentration estimated using the UV−vis absorbance of the assynthesized AuNPs. 10991

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AuNP Ligand-Binding Experiments. Lyophilized GB3 was dissolved in nanopure water. The GB3 concentration was determined with UV−vis method using the GB3 molar absorptivity of GB3 of 9970 M−1 cm−1 at 280 nm. All the ligand binding experiments in this work were conducted under ambient conditions and with OT and GB3 dissolved in nanopure water. The solution pH of the AuNP/ GB3/OT mixtures was ∼6.5. This small acidity is because the assynthesized AuNP colloidal solution is slightly acidic (pH ≈ 6.3). Time-Resolved UV−vis Measurements. Time-resolved UV−vis measurements were taken to monitor the protein adsorption kinetics onto AuNP as well as the stability of protein−AuNP complexes. To probe the effect of cysteine on adsorption kinetics of protein onto AuNP, the time-resolved UV−vis spectra were taken immediately after the addition of GB3 into the AuNP solution that is under constant stirring. The time interval between consecutive spectral acquisitions was 0.5 s. The dead (lead) time of the instrument was about 4 s, and the integration time of each spectrum was 1.1 s. The protein− nanoparticle stability of AuNPs in (AuNP/GB3)/OT and AuNP/ (GB3/OT) were studied by acquiring LSPR spectra at various intervals after the addition of the third component. In the case of (AuNP/GB3)/OT samples, AuNP/GB3 was incubated for either 5 min or 1 day prior to the addition of OT.

Adsorption Kinetics of GB3 on AuNP. We noticed that GB3 dissolved in 1X PBS buffer (phosphate buffer saline) induced immediate AuNP aggregation regardless of the cysteine content in the GB3 variants. To eliminate the electrolyte interference with the GB3 binding with AuNPs, all GB3 adsorption experiments described in this work were conducted with GB3 dissolved in nanopure water. UV−vis is one of the very few techniques suitable for in situ study of AuNP interfacial interactions in solution, and it has been studied extensively for protein and AuNP binding.1,4,22,23 The protein adsorption changes the dielectric constant of the medium surrounding AuNPs, inducing modifications in the AuNP peak UV−vis absorbance and/or wavelength. The time-resolved UV−vis spectra in all AuNP/GB3 solutions are remarkably similar (Figure 3), indicating that cysteine content has no significant effect on the GB3 and AuNP binding kinetics. Immediately after the addition of GB3 to AuNP (∼4 s of the instrumental dead time), the peak UV−vis absorbance increases ∼0.16 absorbance units, which is more than 3 times higher than the total change in the AuNP UV−vis absorbance induced by the subsequent 800 min of sample incubation. This spectral change is very similar to what is observed when BSA is mixed with AuNPs,11 and it indicates that GB3 adsorption is an exceedingly rapid process and that most of the protein adsorption occurs within the first few seconds of the initial mixing. It should be noted that it is not currently possible to correlate the change of the AuNP UV−vis absorbance with the protein packing density on the AuNPs surfaces. Imaginably however, the AuNP UV−vis absorption, that is due to the AuNP localized surface plasmonic resonance (LSPR), can be changed by both protein adsorption and by any subsequent protein conformational changes on the AuNP surface. Presumably the degree of spectral change induced by the protein conformational change is significantly smaller than that induced by protein adsorption itself. Recent work with BSA binding with AuNPs showed that the amount of protein adsorbed onto AuNPs remains unchanged once the BSA/ AuNP is mixed for more than ∼5 min, even though the AuNP UV−vis peak increases very slowly during the entire 24 h experimental period.11 The similarity in the protein adsorption kinetics onto AuNP among GB3 variants is noteworthy. The effect of cysteine on protein adsorption kinetics on AuNPs has not been investigated prior to this. Our data strongly suggests that cysteine residues are unlikely to play any significant role in the protein adsorption per se. Otherwise, the rates of GB32 and GB31 adsorption onto AuNPs should be much higher than that of GB30. Evidently, other factors such as electrostatic, van der Waals, and entropy-driven interactions are needed to explain the similar binding kinetics of GB3 variants with AuNPs. Importantly, even though both GB3 (pI ≈ 4.9) and the AuNPs are negatively charged under our experimental conditions (pH ≈ 7), electrostatic interactions may still play a role in protein− AuNP binding through the positively charged domain. To demonstrate this, we performed electrostatic calculations of the surface charge distribution of folded GB3 using the linearized Poisson−Boltzmann equation.24 GB3 contains both negatively (red) and positively (blue) charged regions at pH 7 solution (Figure 4).24 It is possible that the initial GB3−AuNP attraction is mediated by the positively charged patch formed by lysines 4, 19, 28, and 50. Besides the electrostatics and the universal van der Waals interactions, entropy maximization may also contribute to the

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RESULTS AND DISCUSSION Characterization of GB3 Variants. Cysteine mutants of wild-type GB3 were created by site-directed mutagenesis. To maximize surface exposure and flexibility, cysteine residues were introduced at loops on opposite ends of the protein. Previous studies have determined that residues 11 and 19 are tolerant of structural changes, so these sites were selected for introduction of Cys residues.18 Size-exclusion chromatography indicated that GB3 Cys variants were folded and monomeric (data not shown). To determine whether the native structure was retained, 15N-labeled GB31 and GB32 were expressed as described previously.18 2D 15N HSQC spectra were obtained for both wild-type and Cys variants. Only small changes in chemical shifts were observed, localized to residues surrounding sites T11 and K19 where mutation occurs. (Figure 2; complete data were shown in Figure S2, Supporting Information). This strongly suggests that these mutations have not induced significant conformational changes in GB3.

Figure 2. Comparison of 15N TROSY-HSQC spectra for wild-type GB30 (red) and GB32 (cyan). With few exceptions, chemical shift differences between the variants are small, and the exceptions are typically located close to the mutation sites in the folded structure. Spectra were recorded on a 600 MHz Avance III NMR spectrometer, using an acquisition time of 60 ms in the F1 dimension. 10992

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Figure 3. Time-resolved UV−vis LSPR spectra of AuNP/GB31. The arrow indicates spectra taken at increasing incubation time. The spectrum in black was taken with AuNP control without GB3. (B) Time-course of the peak UV−vis absorbance of LSPR spectra at 520 nm obtained with (black) AuNP/GB30, (red) AuNP/GB31, and (blue) AuNP/GB32 mixtures where the concentrations of AuNP and GB3 proteins were 5.6 nM and 5 μM, respectively. The first data point (0 min) in the time-course is from the AuNP control where the AuNP concentration is also 5.6 nM.

Hcy were used as the model OTs. In each series, the aging time of the AuNP/GB3 mixtures varies from 0, 5 min, to 24 h before the OT addition. OT induces AuNP aggregation in all the GB30 samples, regardless how long the GB30/AuNP mixtures were aged before the OT addition. The aggregated AuNPs were completely settled to the bottom of the sample container after prolonged sample incubation. The AuNP settlement is evident from the absence of the AuNP surface plasmonic resonance peak in the time-resolved UV−vis spectra (Figure 5). Since the GB30 adsorption onto the AuNPs is known from the LSPR measurement (Figure 3), the only sensible explanation to the AuNP aggregation in the (AuNP/GB30)/OT samples is that GB30 on the AuNPs are not stable enough to resist OT displacement. The scenario is much more complicated for the GB31 and GB32 adsorbed AuNP. The stability of the AuNPs against the OT adsorption induced aggregation depends on both the incubation time of the (AuNP/GB31) and (AuNP/GB32) mixture before the OT addition, as well as the structure of the OT molecules. AuNP aggregation and sedimentation was rapid when OT and AuNPs were simultaneously mixed with either GB31 or GB32. More importantly, the rate of AuNP aggregation and sedimentation in the AuNP/(GB31/OT) or AuNP/ (GB32/OT) are very similar to that of the GB30 samples (Figure 5). This result indicates that there is no substantial covalent bond formation between AuNP and GB1 or GB2 in the AuNP/(GB31/OT) and AuNP/(GB32/OT) samples. Otherwise, one would expect that the AuNP aggregation and settlement speed will be significantly lower in AuNP/(GB31/ OT) and AuNP/(GB32/OT) samples than in AuNP/(GB30/ OT). When OT is added 5 min after the preparation of AuNP/ GB31 and AuNP/GB32 mixtures, AuNPs in (AuNP/GB31) 5min/Hcy, (AuNP/GB31)5min/MBI, and (AuNP/GB32)5min/ MBI aggregated and eventually settled to the bottom of the cuvettes. However, AuNPs in (AuNP/GB32)5min/Hcy remains entirely stable. This result, in combination with the experimental observation that the degree of AuNP aggregation in (AuNP/GB32)24hr/MBI is significantly less than that in (AuNP/GB31)24hr/MBI, unambiguously indicates that GB32 is more effective than GB31 and GB30 in protecting AuNPs against OT-induced AuNP aggregations. The difference in the AuNP aggregation characteristics between the (AuNP/GB32)5min/Hcy and (AuNP/GB32) 5min/MBI is most likely due to the fact that compared to

Figure 4. (Left) Front and (right) back view of the surface charge distribution of GB30. The blue and red represents the positively and negatively charged regions at neutral pH. Calculations were performed using the software package APBS using a solvent dielectric constant of 78 and a protein dielectric constant of 2.24

AuNP/protein binding. Upon the protein binding with AuNPs, the total number of the solvent molecules organized surrounding the proteins and AuNPs are reduced, increasing the entropy of the system. Such an entropy-driven mechanism was previously invoked for explaining the nanoparticle aggregation,25−27 and our data, which precludes the possibility of cysteine-driven association, are compatible with this possibility. Stability of AuNP-GB3 Complexes. The role of cysteine on stabilization of GB3 on AuNPs was studied by monitoring the stability of AuNPs against aggregation induced by OT adsorption. A recent study of BSA/AuNP binding demonstrated that OT can adsorb onto the AuNPs precoated with BSA.11 However the subsequent OT adsorption did not induce detectable BSA desorption or AuNP destabilization (aggregation), suggesting that the BSA/AuNP binding is stronger than that of OT/AuNP, and/or the ligand exchange between BSA and OT on AuNPs is too slow to be detected. The GB3 variants constitute an ideal set of model proteins for probing the correlation between protein stability on AuNPs and its cysteine content. In addition, the study of GB3/AuNP aging dependence of the GB3 stability on AuNP allows us to estimate the time scale for the protein to form a stable binding that is resistant to OT displacement. Figure 5 shows the time-resolved UV−vis spectra obtained with two series of AuNP/GB3/OT mixtures where MBI and 10993

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Figure 5. Representative time-resolved UV−vis spectra of (A1 to C1) AuNP/(GB3/MBI), (A2 to C2) AuNP/(GB3/HCy), (D1 to I1) (AuNP/ GB3)/MBI, and (D2 to I2) (AuNP/GB3)/HCy samples. The GB3 variants in (AuNP/GB3)/MBI samples were incubated with AuNPs for (D1 to F1) 5 min and (G1 to I1) 1 day prior to the addition of MBI. The GB3 variants in (AuNP/GB3)/HCy samples were incubated with AuNPs for (D2 to F2) 5 min and (G2 to I2) 1 day prior to the addition of HCy. The GB3 proteins used in the plots are (A1, A2, D1, D2, G1, G2) GB30, (B1, B2, E1, E2, H1, H2) GB31, and (C1, C2, F1, F2, I1, I2) GB32, respectively. The concentration of AuNP, OT (MBI/HCy), and GB3 were 3.7 nM, 10 μM, and 6.6 μM, respectively.

The fact that AuNP is mostly stable in (AuNP/GB32)5min/ Hcy, but not in (AuNP/GB31)5min/Hcy indicates that a larger fraction of the AuNP surfaces in (AuNP/GB32)5min/Hcy is passivated by GB32 against the Hcy adsorption than that in (AuNP/GB31)5min/Hcy. With two cysteine residues, GB32 will likely form a covalent S−Au bond faster than GB31. As a result, a higher fraction of GB32 may be covalently attached to AuNPs than GB31 before the Hcy addition. This hypothesis is consistent with our recent observation that BSA can rapidly stabilize AuNPs against Hcy- and MBI-adsorption-induced aggregation. As a protein that contains 35 cysteine residues, BSA is likely more efficient than GB32 in forming covalent S− Au binding with AuNPs. Importantly, even though it contains only one thiol group, PEG1 is much more effective than GB31 and GB32 in protecting

Hcy, MBI is much more effective at inducing AuNP aggregation. Figure 6 showed AuNP aggregation kinetics as a function of OT concentration in the AuNP/MBI and AuNP/ Hcy solutions. While 5 μM MBI induces immediate AuNP aggregation and complete sedimentation, AuNP remains highly stable in the AuNP/Hcy mixture even when the Hcy concentration is 10 μM Hcy. This result indicates that Hcy has to reach a near saturation packing on the AuNPs before it induces appreciable AuNP aggregations. This hypothesis also explains why 10 μM Hcy induced AuNP aggregation in Figure 5, but not in Figure 6 where the AuNP concentrations are 3.7 nM and 5.7 nM, respectively. In the case of both MBI and Hcy inducing AuNP aggregations, the AuNP aggregation kinetics in AuNP/MBI samples is much faster than in AuNP/Hcy mixtures. 10994

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residue with AuNPs and subsequently the S−Au bond formation.

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CONCLUSIONS The protein−gold nanoparticle interaction is an exceedingly complicated phenomenon. It is highly dynamic in nature and involves multiple intermolecular interactions. The present work probes the role of cysteine residues on protein adsorption kinetics and stability on AuNPs using the GB3 protein and its bioengineered variants. One key finding is that cysteine does not have significant contribution to the initial GB3 adsorption onto AuNPs, but is critical in maintaining protein stability on AuNP against OT displacement. The aging GB3/AuNP mixture promotes the formation of a covalent S−Au bond between protein and AuNPs, and the rate or the extent of the S−Au bond formation increases with the number of the cysteine residues in the GB3 variants. Besides providing new insight into the fundamental mechanism of protein/AuNP interactions, this work is also important for designing stable protein-functionalized AuNPs for biological/biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

Complete 15N TROSY-HSQC comparison between GB30 and GB32; UV−vis spectrum and TEM images of AuNPs; stability of PEG-SH covered AuNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (662) 3256752.

Figure 6. The time-resolved UV−vis spectra of AuNP/MBI and AuNP/Hcy samples. The concentrations of MBI used were 1 μM, 2 μM, 5 μM, and 10 μM and those of Hcy were 2 μM, 5 μM, 10 μM, and 25 μM. The spectrum in red is taken after incubating samples for overnight. The AuNP concentration in these samples was 5.7 nM, which was higher than the 3.7 nM AuNP used in Figure 5.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an NSF CAREER Award (CHE 1151057), NSF Fund (EPS-0903787), and a seed grant provided to D.Z. from Agricultural Research Service, U.S. Department of Agriculture, under Project No. 5864022729.

AuNPs from subsequent OT adsorption-induced aggregation. AuNPs are entirely stable in the (AuNP/PEG1)5min/OT) samples regardless that the OT is MBI or Hcy (Figure S3, Supporting Information). This result is in stark contrast with the (AuNP/GB31)5min/MBI) and (AuNP/GB32)5min/MBI) in which MBI induces complete AuNP aggregation and settlement (Figure 5). The difference in the AuNP stability against MBI adsorption induced aggregation strongly suggests that the reaction speed between GB3 cysteine with AuNP is significantly slower than PEG-SH. This result is not surprising given the drastic difference in their structural characteristics. Unlike PEG1 where the terminal thiol is presumably the only functional group that has high affinity to AuNPs, GB31 and GB32 have multiple amino acids residues that are known to have high affinity to AuNPs. Besides cysteine residues, amino acid groups such as lysines, methionine, aromatic amino acids tryptophan, phenylalanine, and tyrosine can also bind to AuNPs. These nonspecific interactions on one hand may enhance the rate of GB3 adsorption onto the AuNPs. On the other hand, it likely hinders the covalent bond formation between protein cysteine and AuNP. Imaginably, once GB3 is attached onto AuNP through a nonspecific interaction, the rotational/translational motion of GB3 will be constrained, which will inevitably reduce the collision rate of cysteine

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REFERENCES

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dx.doi.org/10.1021/la402239h | Langmuir 2013, 29, 10990−10996