Engendering Materials Directing Peptides with Non-Native

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C: Physical Processes in Nanomaterials and Nanostructures

Engendering Materials Directing Peptides with Non-Native Functionalities Through Synthetic Sequence Modifications Catherine J. Munro, and Marc R. Knecht J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b07198 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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The Journal of Physical Chemistry

Engendering Materials Directing Peptides with Non-Native Functionalities Through Synthetic Sequence Modifications Catherine J. Munro and Marc R. Knecht* Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida, 33146, United States. ABSTRACT: Recent efforts in bio-inspired Au nanomaterial synthesis have identified that Trp residues of Au binding peptides AuBP1 (WAGAKRLVLRRE) and AuBP2 (WALRRSIRRQSY) have the capacity to drive metal ion reduction. Such a capability could be intrinsically valuable for material production under sustainable conditions that limits the number of reagents required for nanoparticle generation. Additionally, it could also allow for precise localization of inorganic materials based upon peptide positioning. These advances in material peptide design could prove to be significant for applications in catalysis, sensing, plasmonic, etc. Herein we examine this reduction capability of tryptophan modified peptides to identify strategies to incorporate such reactivity into non-reactive peptides to enhance their individual functionality for material production. This is examined using peptide mutation studies that incorporate Au3+ reductive Trp residues into non-reactive materials binding peptides. The results demonstrate that reactivity can be incorporated into non-functional biomolecules where the location of the Trp, the neighboring residues in direct contact with the Trp, and the complete sequence all can be tuned to greatly modulated Au3+ reduction reactivity. Additionally, the binding strength of the peptide to the free metal ions in solution is shown to alter the reactivity where stronger affinity between the biomolecules and metal ions leads to diminished reduction. Taken together, these results present pathways toward selective biological modifications of material directing peptides to increase their inherent capabilities for the design, production, and stabilization of functional inorganic nanomaterials.

Introduction Nature has used millennia of evolution to develop biomineralization pathways to generate inorganic materials,1-3 including nanoparticles composed of noble metals such as Au and Ag.4 In many cases, biological pathways are responsible for the reduction of metal ions, as well as the nucleation, growth, and stabilization of the resultant zerovalent nanoparticles.5 Unfortunately, translation from nature to the single pot approach for noble metal nanoparticle fabrication remains elusive, but would be an important advancement in material synthesis for a variety of applications, including optics,6-7 catalysis,8-9 and plasmonics.10-11 Biomolecules such as DNA,12-14 proteins,15-16 viruses,17-18 and single cell organisms19 are becoming routinely used for the synthesis of inorganic nanomaterials due to their unique molecular-level structures.20-21 For instance, DNA is a popular ligand for nanoparticle design and synthesis,12-14 where recent efforts have exploited DNA to generate crosslinked hydrogels with embedded Au nanoparticles for use as hydrogenation catalysts.22 There have been additional efforts to exploit DNA to passivate low23 and high24 energy facets of Pd nanoparticles prepared using seed mediated methods resulting in uniform catalysts with precise control over the resultant material structure and properties. Like DNA, proteins such as the cage protein ferritin, which is used in biological systems for Fe storage, have been widely adapted as templates for material synthesis. Ferritin derivatives have been engineered to template Ag15-16 and Au16 nanoparticles where an exogenous reductant was required for metal ion reduction inside the cage protein. Beyond DNA and ferritin, other biomacromolecules have been used to generate inorganic nanomaterials including materials binding peptides,25 hyper branched amino acids,26 phycocyanin,27 Matricaria chamomilla extract,28 Rice protein,29 etc.

While exquisite property control has been achieved using biomolecules for noble metal material production, metal ion reduction more commonly occurs through the use of exogenous reagents. To more closely mimic biomineralization processes, biomimetic ligands that control the synthesis process from metal ion reduction to particle stabilization are required. Peptides represent intriguing biological molecules to achieve full synthetic control of nanoparticle production based upon their simple molecular structure, numerous functional groups, and ability to recognize and bind inorganic nanomaterials. For Au materials specifically, peptides have been identified from biocombinatorial selection techniques with the ability to bind the zerovalent surface and stabilize the growing materials;30-33 however, identification of sequences that fully mimic the biomineralization processes (i.e. to include metal ion reduction capabilities) remains problematic. While the ability to reduce Au3+ ions has not been used as a peptide selection criteria, previous studies have identified Au0 binding sequences including the AuBP1 (WAGAKRLVLRRE) and AuBP2 (WALRRSIRRQSY).34 These peptides were solely selected based upon their affinity for Au0 with binding abilities that rival thiols on the metal surface. Our recent work has demonstrated that these two sequences also possess the ability to reduce Au3+ ions to Au0, resulting in nanoparticle formation and eventual stabilization.34-35 These studies identified that the Trp residue is crucial for reduction; oxidation of Trp to several potential products has been suggested to occur,36-37 driving metal ion reduction and nanoparticle formation. This was supported by control studies using the Pd4 peptide (TSNAVHPTLRHL), which also has the ability to bind Au surfaces.38 Pd4, a Trp free peptide, was not able to reduce Au3+ to Au0.34 These studies suggested that simple incorporation of a Trp residue into a materials binding peptide may be sufficient to generate a multifunctional biomolecule with the ability to better mimic

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biomineralization approaches for materials production; however, it is likely that the total amino acid composition of the peptide and its sequence play an important role in controlling the reduction. In this contribution, we demonstrate the ability to selectively mutate materials binding peptide sequences to engender the biomolecules with non-native capabilities for materials synthesis. We show that Au3+ reduction capabilities can be integrated into the biomolecules through the direct addition of a Trp residue. As a result, unreactive peptides can be modified to incorporate Au3+ reductive capabilities, leading to the single pot production of Au nanoparticles without the need for exogenous reagents. Remarkably, these studies demonstrate that the complete peptide sequence plays a substantial role in controlling the reduction capabilities of the modified peptides. Furthermore, the binding between the Au3+ ions and the peptides in solution is shown to play a critical part in controlling the reduction reactivity of the biomolecules. These results provide new information on the ability to manipulate peptide sequences for controlled materials production, which could be important for the a priori design of multifunctional biomolecules for the generation of inorganic materials with tailored property control. Experimental section Materials. HAuCl 4 was purchased from Acros Organics, while trifluoroacetic acid (TFA) and tri-isopropyl silane (TIS) were purchased from Alfa Aesar. Acetonitrile, methanol, and N,N-dimethylformamide (DMF) were purchased from BDH. FMOC-protected amino acids, Wang resins, and coupling reagents were acquired from Advanced Chemtech. All reagents were used as received and ultrapure water (18.2 MΩ·cm) was employed in all experiments. Peptide Synthesis. Solid phase peptide synthesis was used following standard protocols39 on a TETRAS synthesizer (CreoSalus). Peptides were cleaved from the Wang resins using a cocktail of TIS:H 2 O:TFA (25 μL: 25 μL: 950 μL) and purified using reversephase HPLC (Waters). The purified peptides were confirmed using ESI and/or MALDI-TOF mass spectrometry. Peptide-Induced Au3+ Reduction Reaction. For each system, the peptide-driven Au3+ reduction reaction was processed in water. The reactions were performed using various peptide:Au3+ ratios that ranged from 1-5. A description of the reaction for a peptide:Au3+ ratio of 1 is described; however, the volumes of peptide solution, Au3+ solution, and/or the water added were changed to maintain a constant reaction volume of 200 µL. In this regard, to a reaction well in a clear 96-well plate, 20.0 μL of an aqueous 1.0 mM peptide solution was diluted in 160.0 μL of water. To this mixture, 20.0 μL of 1.0 mM aqueous HAuCl 4 was added and the mixture was slightly agitated. The well plate was subsequently inserted into the plate reader (Synergy|Mx) to monitor the absorbance of the reaction at 540 nm for 1500 – 21700 min, depending upon the specific peptide sequence being studied to ensure that the reaction reached completion. The nominal temperature of the reaction analysis in the plate reader was ~29 °C. All reactions were run in triplicate and the analyses represent the average plus or minus one standard deviation. Photoluminescent Quenching Analysis. For every peptide where a Trp residue was present, a photoluminescent quenching analysis was performed in water. For this, 3.0 mL of 1.0 μM peptide was added to a cuvette to which 1.0 μL aliquots the Au3+ solution was added and mixed prior to each fluorescence reading. The concentration of the aqueous Au3+ was 100.0 mM for the studies using the AuBP1, AuBP2, WPd4, WAPd4, Pd4W, and WAgBP2 peptides; however, a lower Au3+ concentration of 1.0 mM was employed for the quenching studies using WAgBP1, WAAgBP1, and AgBP1W. No more than 1000 μL of the Au3+ solution was added to any quenching experiment and no experiment took more that 10 min for complete quenching. Quenching experiments were only run to meet the concentration of metal used in the reduction reaction (0.1 mM). All fluorescence studies were performed on a Perkin-

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Elmer LS 55 Fluorescence Spectrometer where the excitation was set to 277.0 nm and the emission was read from 287.0 to 520.0 nm. The nominal temperature of these reactions was ~26 °C and all reactions were run in triplicate. Characterization. TEM analysis was performed using a JEOL JEM-2010 microscope operating at 80 kV. The samples were prepared by drop-casting 5 μL of the nanoparticle solution onto a carbon-coated 200 mesh Cu grid (EM Sciences) and allowed to dry overnight. Sizing of the generated materials was performed on all samples over at least ten images for each grid, including at least 400 particles at their widest sections. Results and Discussion The ability to predictively manipulate materials binding peptides to engender them with additional capabilities represents new avenues in the biomimetic production of inorganic nanomaterials. Bio selection techniques can identify sequences with affinity for a zerovalent inorganic surface;40 however, the ability to isolate peptides that can reduce metal ions in situ remains challenging. Previous work has shown that Au-specific peptides with a Trp residue have the capacity to reduce Au3+ to Au0.34 The rate of the reduction reaction is dependent upon solution conditions35 and peptide sequence context effects34 where the amino acids that neighbor the Trp residue alter the reaction kinetics. To increase the capabilities of non-reactive sequences, we identified peptides with affinity for Au0 that were unreactive for Au3+ reduction. Through simple Trp addition to the sequence, we anticipated modifying the capabilities of the biomolecules, making them reactive for metal ion reduction and nanoparticle formation. For these studies, a library of peptides with known affinity for Au was identified,38 including those with the ability to reduce Au3+ (AuBP1 and AuBP2)31 and those that are not reactive for Au3+ reduction (AgBP2 – EQLGVRKELRGV,41-42 AgBP1 – TGIFKSARAMRN,42-43 and Pd4 – TSNAVHPTLRHL).32, 44 To identify if a new functionality could be incorporated into a peptide sequence, Trp residues were appended to the N-terminus of the AgBP2, and both the N- and C-terminus for AgBP1 and Pd4. All of the peptide sequences used in this study are presented for comparison in Table 1 where their chemical structures are shown in Table S1 of the Supporting Information. Trp modifications were done to facilitate Au3+ reduction from the non-reactive peptides where the sequences should also stabilize the final Au nanoparticles. Peptide synthesis was used to prepare the biomolecules and the sequences were confirmed using mass spectrometry. To monitor their reactivity for Au3+ reduction, UV-vis analysis was employed where the peptide was mixed with HAuCl 4 in a 96 well plate at selected peptide:Au3+ ratios. The formation of a plasmon band, indicative of nanoparticle production and thus Au3+ reduction, was tracked at 540 nm for at least 1500 min for all reactions. From this analysis, the growth of the plasmon absorbance can be fit using kinetic parameters, where the reaction rate constants (k) can then be extracted to compare the reactivity of the different biomolecules.

Table 1. A list of all the peptides used in the experiments where modifications are identified by color.

Figure 1 presents the reactivity analysis of the parent AgBP2 peptide and its modified analog, WAgBP2. The AgBP2 peptide was originally isolated with affinity for Ag surfaces;41-42 however,


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Figure 1. Analysis of peptide-driven Au3+ reduction via Au nanoparticle plasmon growth. Parts (a and c) present the UV-vis spectra of plasmon growth over time at a peptide:Au3+ ratio of 1 for the parent AgBP2 and mutant WAgBP2 peptides, respectively. Parts (b and d) display images of the reactions after 30 h at the indicated peptide:Au3+ ratios for the (b) AgBP2 and (d) WAgBP2 systems. Part (e) shows the pseudo first order kinetic analysis of the WAgBP2-driven Au3+ reduction reaction, while part (f) compares k values for the reactions. it was later demonstrated with the ability to bind Au0 and stabilize Au nanoparticles in the presence of an exogenous reducing agent.43 The WAgBP2 peptide incorporates a Trp residue at the N-terminus to induce Au3+ reduction capabilities in the peptide. Figure 1a presents the Au3+ reduction analysis for the parent AgBP2 peptide studied at an AgBP2:Au3+ ratio of 1. Should Au3+ reduction occur over time leading to zerovalent Au nanoparticle formation, a plasmon band would be observed at ~520 nm in the UV-vis spectrum. This band would grow in intensity over time, reflective of the metal ion reduction, nucleation, and growth process. As anticipated, no substantial change in the absorbance of the reaction was observed throughout the duration of the study (1500 min), indicating that the AgBP2 peptide was unable to reduce Au3+ to Au0. This was confirmed through the image of Figure 1b, which displays the reaction wells after 1500 min at AgBP2:Au3+ ratios of 0-5 from left to right. For all the reactions, the solutions remained clear and colorless and not a shade of red that would be anticipated should Au nanoparticles have been prepared. Figure 1c presents the plasmon evolution analysis for the reaction using the WAgBP2 peptide. Upon integration of the reactive Trp residue, plasmon formation was clear, confirming the role that Trp plays in the reduction process. The figure shows the UV-vis analysis using a WAgBP2:Au3+ ratio of 1 over 1500 min. In this system, a plasmon band is evident at 60 min, which is centered at 550 nm. Over the reaction timeframe, the intensity of the band increases concomitant with a blue shift. After the reaction, the peak is centered at 530 nm. Such plasmon formation was confirmed by the image of the reaction wells (Figure 1d), where the solution was pink in color without any observable precipitate for all peptide:Au3+ ratios studied > 0. This vibrant solution color change strongly suggests nanoparticle production and colloidal stabilization. Using the spectral data of Figure 1c, the absorbance at 540 nm can be plotted as a function of time (Figure 1e), from which reaction rate constants (k) can be calculated. Note that this analysis con-

flates all aspects of nanoparticle formation, including Au3+ reduction, Au0 atom nucleation, and Au nanoparticle growth; however, it is the most convenient approach for reaction comparison. For the WAgBP2 system at a peptide:Au3+ ratio of 1, a k value of (4.33 ± 0.21) × 10−3 min-1 was determined. As the WAgBP2:Au3+ ratio was increased, the rate of plasmon band formation also increased (Figure 1f). In this regard, at the highest peptide:Au3+ ratio studied, 5, a k value of (14.6 ± 0.10) × 10−3 min-1 was determined. Increased reactivity as a function of higher peptide:Au3+ ratios was anticipated. Interestingly, the reactions with the WAgBP2 peptide are substantially faster than those driven with the Trp-containing AuBP1 and AuBP2 peptides previously reported.34 To this end, when using the AuBP1 and AuBP2 peptides at a peptide:Au3+ ratio of 5, k values of (5.0 ± 0.06) × 10−3 min-1 and (5.5 ± 0.46) ×10−3 min-1 were observed, which are notably smaller than the rate constant for the same reaction driven with the WAgBP2. This demonstrates that while the presence of Trp enhances Au3+ reduction, the effects of the sequence and amino acid composition of the complete peptide are important in controlling the reaction process. TEM analysis of the reactions driven with the WAgBP2 peptide were completed to confirm nanoparticle production and to identify their overall structure. Figure 2 presents TEM images of the nanoparticles that were prepared after 1500 min for reactions processed at WAgBP2:Au3+ ratios from 1-5. TEM was performed for reactions of WAgBP2 where a plasmon was generated. For all the WAgBP2-based syntheses, spherical or oblong materials were prepared, regardless of the peptide:Au3+ ratio. The sizes were similar across all the reaction conditions, which ranged from 10.1 to 16.7 nm. While differences in average sizes were present, somewhat large standard deviations in these sizes were evident, suggesting

Figure 2. TEM analysis of the materials generated after 1500 min at a WAgBP2:Au3+ ratio of (a) 1, (b) 2, (c) 3, (d) 4, and (e) 5.



that all the particles were similar in dimension irrelevant of peptide concentration. For each sample, sizing of at least 400 nanoparticles over 20 different images of the grid was used to generate the average size plus or minus one standard deviation. The integration of a Trp residue into the AgBP2 sequence introduced reductive capabilities into the biomolecule, but it remains unclear if this is a universal capability. As such, Trp integration into the Pd4 peptide was processed to identify sequence effects on Trpactivated reduction. The Pd4 peptide was originally identified with affinity for Pd0 surfaces,32, 44 but has been shown to bind Au surfaces and Au nanoparticles in the presence of exogenous reductants.38, 43 To confirm that the parent Pd4 peptide was inactive for

Figure 3. Analysis of Au3+ reduction via modified Pd4 peptides. Parts (a-d) present the UV-vis spectral analysis of particle growth over a 1500 min reaction time at a peptide:Au3+ ratio of 1 driven by the (a) Pd4, (b) WPd4, (c) Pd4W, and (d) WAPd4 peptides. Parts (e-h) display the same analysis as in parts (a-d); however, the reaction time was extended to 21,700 min. Part (i) compares the k values for the Au3+ reduction reaction calculated after 21,700 min of reaction for the peptides at the indicated peptide:Au3+ ratios.

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Au3+ reduction, the standard reaction was processed at peptide:Au3+ ratios of 1 through 5, where no changes in the UV-vis absorbance of the solution were observed after 1500 min (Figure 3a). This confirms that the Pd4 peptide, which does not possess a Trp residue, is non-reactive toward Au3+ reduction within the 1500 min reaction window. To incorporate reductive capabilities into the Pd4, a Trp residue was first appended to the N-terminus, generating the WPd4 peptide. When this newly formed peptide was employed to drive the reaction at a WPd4:Au3+ ratio of 1, only negligible degrees of reactivity were evident (Figure 3b) with a very small increase in absorbance at ~500 nm. No plasmon band was noted over the 1500 min reaction timeframe; however, a general increase in the overall absorbance of the system was noted, differing from the analysis using the parent Pd4. Similar effects were noted when the Trp was appended onto the C-terminus of the Pd4 peptide (Pd4W) (Figure 3c). For this sample, there was the formation of a small absorbance at 540 nm over 1500 min, but the intensity of the peak was very minor. Since the WPd4 and Pd4W demonstrated negligible degrees of reactivity over the 1500 min reaction timeframe, two reaction modifications were processed to identify the basis of the unexpectedly low reactivity. First, the WPd4 variant of the peptide sequence was further changed to swap out the Thr1 residue of the peptide for an Ala. Such an exchange was used to better reflect the N-terminus of the AuBP1 peptide that presents an Ala residue immediately adjacent to the terminal reactive Trp.34 When this peptide (termed WAPd4) was employed in the reaction analysis, almost no change in the UV-vis absorbance of the reaction over the 1500 min timeframe was noted (Figure 3d). This suggests that while local residue effects are likely to manipulate Trp-based reduction, the complete peptide sequence appears to present substantial influence over the reductive capabilities of the Trp residue. In the second reaction modification, the reaction time was increased to 21,700 min (~15 days) as the shorter timeframe may not be long enough to observe the reduction process. As anticipated for the parent Pd4 peptide at a peptide:Au3+ ratio of 1, no change in the UV-vis spectra of the reaction throughout the extended study was observed (Figure 3e), again confirming that the Trp-free peptide was unable to reduce Au3+ ions. Remarkably, when the WPd4 peptide was employed as the reductant over the longer reaction time (Figure 3f), clear plasmon formation was evident at times > 2800 min. This absorbance grew in intensity and slightly blue shifted over the extended reaction time. This trend was also apparent in Pd4W- (Figure 3g) and WAPd4- (Figure 3h) driven reactions, confirming that incorporation of the Trp residue does indeed engender the peptide with reductive capabilities; however, the rates of the reactions are strongly influenced by the overall sequence. Using the UV-vis analysis from the longer reaction times with the Trp-containing Pd4 peptides, the k values were calculated for each system at peptide:Au3+ ratios of 1-5 (Figure 3i). When considering the WPd4-driven system at a ratio of 1, a k value of (0.07 ± 0.01) × 10−3 min-1 was determined. Interestingly, when the ratio was increased to 2, the k value decreased slightly to (0.04 ± 0.003) × 10−3 min-1, which remained relatively stable for the k values determined at higher WPd4:Au3+ ratios. Similar effects were observed for the Pd4W- and WAPd4-driven systems where maximum reactivity was noted at a peptide:Au3+ ratio of 1 that diminished at higher values. Interestingly, while the WPd4 and WAPd4 peptides demonstrated roughly equivalent reactivity, the Pd4W system presented notably higher reduction rates; however, these values are substantially lower than the reaction rates observed for AuBP1-, AuBP2-, and WAgBP2-driven reductions. For the Pd4-derived reduction system, two notable differences in reactivity trends were observed as compared to the WAgBP2 system. First, the reaction process was substantially slower, requiring a 14.5-fold increase in reaction time to reach completion. Second,


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an increase in the peptide concentration resulted in decreased reductive capacity. Additionally, notable differences in k values were observed based upon the position of the Trp residue in the Pd4 peptide (either N- or C-terminus). Taken together, this strongly suggests that the amino acid composition of the complete peptide sequence, as well as the identity of the neighboring residues of the reactive Trp species (e.g. context effects) play an important role in the reductive capacity of the amino acid. Since the AgBP2 and Pd4 peptides provided different results concerning Trp incorporation for reactivity, a third sequence, AgBP1 was also examined (Figure 4). For this, two mutations of AgBP1 were processed to incorporate Trp at the N-terminus (WAgBP1) and at the C-terminus (AgBP1W). Like the Pd4 peptide, AgBP1 also presents a Thr at the first residue position, thus the WAAgBP1 sequence was analyzed that incorporates the Trp at the N-terminus and replaces the Thr1 for an Ala. Figure 4a shows the UV-vis reduction analysis for the parent AgBP1 at a peptide:Au3+ ratio of 1 over 1500 min. As anticipated, no reactivity was evident with no plasmonic changes over the reaction timeframe due to the lack of a Trp residue. For the other three mutated peptides, clear reactivity within 1500 min was evident with plasmon formation for all the systems at a peptide:Au3+ ratio of 1. To this end, k values of (0.7 ± 0.04) × 10−3 min-1, (0.8 ± 0.16) × 10−3 min-1, and (0.4 ± 0.18) × 10−3 min-1 were observed for the reactions driven with the WAgBP1, AgBP1W, and WAAgBP1 peptides, respectively.

Figure 4. Analysis of Au3+ reduction via modified AgBP1 peptides. Parts (a-d) present the UV-vis spectral analysis of particle growth over a 1500 min reaction time at a peptide:Au3+ ratio of 1 driven by the (a) AgBP1, (b) WAgBP1, (c) AgBP1W, and (d) WAAgBP1 peptides. Parts (e-h) display images of the reactions after reaction completion at the indicated peptide:Au3+ ratios for the (e) AgBP1, (f) WAgBP1, (g) AgBP1W, and (h) WAAgBP1 systems. Finally, part (i) compares the calculated k values at a peptide:Au3+ ratio of 1 for the indicated sequences. While the three AgBP1 modified peptides demonstrated reactivity at peptide:Au3+ ratios of 1 over the shorter 1500 min timeframe, a surprising event was observed as the peptide:Au3+ ratio was in-

creased. When increased amounts of peptide were studied to increase the ratio, the reduction process was completely inhibited. This is evident in the images of Figures 4e-h which show the reaction wells for each peptide-driven reduction system after 1500 min. For Figure 4e, no color change in any reaction was noted using the inactive AgBP1 parent; however, for the three mutated species, a reaction color change was only observed at a peptide:Au3+ ratio of 1. For all the reactions at higher ratios, the wells remained clear and colorless with no evidence of bulk Au precipitation, indicative of no reaction occurring. This complete lack of reduction at higher peptide:Au3+ ratios for the WAgBP1, AgBP1W, and WAAgBP1 peptides is unique to this sequence. It provides a third reactivity trend to demonstrate unique effects of Trp addition to incorporate metal ion reduction capabilities to non-reactive biomolecules. From the different peptides studied, great variances in reactivity were observed. In our previous work, we have shown that solution conditions, notably the reaction pH, can significantly alter the reactivity.35 In the present study, the different systems were not pH controlled where all reactions initially had an acidic pH that ranged from 3.2 to 5.7. Such a value is likely determined via the peptide sequence and HAuCl 4 in the system where most of the reactions were between a small pH range of 3.5 to 4.0. As expected, the solutions remained acidic over the course of the 1500 min reaction period where the final pH values were between 3.2 and 4.2. No substantial differences were observed, suggesting that the pH of the reaction was not responsible for the reactivity differences. To directly compare the reactivity for peptide-driven Au3+ reduction, Figure 5 presents a plot of the k values for each peptide at the indicated peptide:Au3+ ratio. This comparison figure only presents peptide-driven systems that demonstrated some degree of reduction capabilities; those sequences with no reactivity were not included for clarity. In general, the k values ranged from (0.025 ± 0.00095) × 10−3 min-1for the slowest reaction (WPd4) to (14.6 ± 0.1) × 10 −3 min-1 for the fastest system (WAgBP2). It is important to reemphasize here that these k values conflate Au3+ reduction with nanoparticle nucleation and growth; however, they are the most direct method to compare the reactivity in the different systems. Previous studies from our group have exploited this approach to compare reduction rates for the fabrication of peptide-capped Au nanoparticles using the AuBP1, AuBP2, and Pd4 peptides where three different exogenous reductants were employed: hydrazine, ascorbic acid, and NaBH 4 .49 When these well-known reductants drove Au3+ reduction and eventual nanoparticle formation, k values that ranged from (636.0 ± 72.0) × 10−3 min-1 to (4890.0 ± 720.0) × 10−3 min-1 were observed. As anticipated, the reactions using NaBH 4 , an extremely powerful reductant, were unable to be monitored due to the exceedingly fast reaction, thus no rate constants could be determined. Such values are ≈ 44 to 335 times greater than the fastest k value of the present system (WAgBP2 at a peptide:Au ratio of 5). Additionally, differences in the reaction rates were observed using the exogenous reductants based upon the peptides used to in the reaction, suggesting differences in Au3+ reduction based upon the biomolecule passivant. When comparing the reactions with and without exogenous reductants, this demonstrates that the slow rate of plasmon formation observed with the peptide-only systems is due to Au3+ reduction and not with Au nanoparticle nucleation and growth. Should the latter steps of nanoparticle production dominate the system, identical k values would have been observed. Since significant differences are evident, this is anticipated to arise from the rate of Au3+ reduction. Consistent with the previous studies49 using exogenous reductants, notable differences in Au3+ reduction were evident based upon the peptide in the reaction. As shown in Figure 5, the three specific regimes of Trp-driven peptide reduction of Au3+ ions. In regime one (AuBP1, AuBP2, and WAgBP2), substantial reductive capabilities can be achieved using the peptides. This gives rise to the



Figure 5. Comparison of the calculated k values for Au3+ reduction reaction driven by the indicated peptides at the selected peptide:Au3+ ratios. Values were calculated at 21,700 min for the Pd4derived sequences and 1500 min for all other biomolecules. Note that only those that demonstrated reactivity were included. largest rate constants that increase as the peptide:Au3+ ratio is increased. It is interesting to note that the peptide with the most reactivity is the WAgBP2, which was mutated with the Trp residue to incorporate reactivity. In the second regime (Pd4 derivatives), Au3+ reduction is observed; however, it is significantly slower and required substantially longer reaction times to reach completion as compared to the biomolecules of regime one. This is evidenced by k values that are ~30 to ~60 times slower as compared to the values noted WAgBP2 at a peptide:Au3+ of 1. Beyond being notably slower to react, increasing the peptide:Au3+ ratio resulted in diminished reactivity, suggesting that higher biomolecule concentrations alter the reactivity. Finally, the third regime (AgBP1 variants) presents the most unique reactivity. In this regard, incorporation of a Trp residue to the inactive AgBP1 peptide does give rise to reduction capabilities at a peptide:Au3+ ratio of 1, albeit with diminished rate constants as compared to regime one. Interestingly, the peptides of regime three demonstrated complete inactivity for Au3+ reduction as the peptide:Au3+ ratio increased above 1 under the standard reaction conditions. Taken together, these results do indicate that incorporation of a Trp residue can engender a peptide sequence with the ability to reduce Au3+ ions; however, the position of the residue, the immediate amino acids in direct contact with the Trp, and the complete peptide sequence appear to play notable roles in modulating and controlling the overall reduction reactivity of the biomolecules. It is also important to point out that for all the reactive peptides, stable nanoparticle suspensions were prepared, thus demonstrating that the inherent affinity of the mutated peptides for the metal materials remained sufficient to prevent material aggregation to bulk precipitates. To support the spectroscopic observations, TEM imaging of the solution of a reaction driven using the non-reductive AgBP1peptide was processed. For this system, it is important to note that no plasmon band was observed spectroscopically, the solution remained clear and colorless, and no bulk material precipitation was observed in the reaction well. Together this would support the complete lack of Au3+ reduction from this system. Remarkably, TEM analysis of this reaction did indeed observe nanoparticles in the system (Supporting Information, Figures S16 and S17). As an additional control, the exact same reaction was processed; however, no peptide was added into the system. In this case, only HAuCl 4 was dissolved in water and was spectroscopically monitored for 1500 min. This system demonstrated no plasmon formation, solution color change, or bulk material precipitation, as anticipated. TEM imaging of the

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reaction solution again demonstrated Au nanoparticle formation. This latter result was even more remarkable considering that no reductant or capping agent was present in the system. These two key control studies suggest that negligible degrees of reduction may be occurring for nanoparticle formation as a result of the drying process in the TEM preparation. In this regard, no plasmon band was evident and no bulk material precipitation was indicated, which would be required should Au3+ reduction was occurring from these samples during the reaction. As a first step to identify the basis for the differences in reactivity regimes, comparison of the amino acid composition of the peptide sequences was conducted (Figure 6). For all the reactive biomolecules, a Trp residue must be present to induce Au3+ reduction. When comparing the sequences with the greatest reactivity (regime one), which includes AuBP1, AuBP2, and WAgBP2, it is evident that these sequences are rich in positively charged residues with terminal amines where at least 23% of the residues are either Arg or Lys. Beyond this similarity, these sequences are noticeably different with varying compositions of hydrophobic, hydroxyl, and acid containing residues. When comparing these peptides to those sequences in the slow reactivity regime (Pd4 derivatives), the main difference arises from the Pd4 sequences containing a high percentage of His and Pro residues. His is known to strongly bind to many metals16, 45-49 where the imidazole ring acts as a Lewis base facilitating this interaction.50-51 Because of this strong binding, it is possible that the His residues are sequestering the Au3+ ion in such a way to prevent reduction at the Trp residue. In addition, the Pd4 peptide possesses a Pro at the seven position. Due to the Pro52-53 structure, this residue establishes a prominent kink within the biomolecule, limiting the degrees of rotation. This limited range of motion, coupled with binding of the metal ions at the His residues may limit the interaction of the Au3+ ions with the reducing Trp residues, resulting in diminished reductive capabilities from the Pd4-based sequences.

Figure 6. Amino acid composition comparison of the peptides reactive for Au3+ reduction. Interestingly, the peptides in the third regime (AgBP1 variants) also possess a high composition of Arg and Lys residues. As such, they look somewhat like the peptides in regime one; however, their reactivity is substantially different with reduction occurring only at a peptide:Au3+ ratio of 1. Noticeably, the AgBP1 derived sequences possess a Met residue, which is known to bind exceedingly strong to metal ions because of the thioether side chain; the lone pair of electrons on the sulfur atom act as a soft ligand to strongly interact with Au3+.54-55, 56-58 These sequences also possess a Phe residue that has known interactions with Au3+ that could potentially participate in the reaction process.59-60 Such capabilities are not present in the other peptides of the study, thus the affinity of the biomolecules for


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the metal ions during the reaction may have an effect on the overall rate of the reduction process. To quantify the interaction between the peptides and Au3+ ions in solution, photoluminescent quenching studies61-62 were performed for each biomolecule with the ability to drive Au3+ reduction. A Stern-Volmer61-62 analysis was then applied to calculate the change in the free energy (∆G) based upon the binding event. Figure 7a presents the photoluminescence intensity analysis using the AuBP1 peptide. This analysis monitors the emission from the Trp residue of the sequence, which upon Au3+ binding results in a quenching of the photoluminescence of the residue. The intensity of the peak diminishes as the concentration of Au3+ in the analysis increases. The Stern-Volmer analysis of this quenching was performed at the Trp emission maximum of 365 nm, as seen in Figure 7b. In this analysis, the intensity ratio is plotted against the concentration of the quencher (Au3+ in this case). Using I o /I = K a [Q] + 1, where I 0 is the initial fluorescence intensity, I is the fluorescence intensity at time t, and K a is the association between the photoluminescent peptide (I o /I) and the Au3+ quencher (Q). The slope of this linear fit is defined as K a . This K a value can then be used to calculate ΔG for the peptide/Au3+ binding event, which can then be applied to each peptide-Au3+ pair. Figure 7c presents the calculated ΔG values for each peptide:Au3+ pair. In general, the peptides from the first (AuBP1, AuBP2, and WAgBP2) and second regimes (Pd4 derivatives) all presented similar free energy values, which ranged from –20.2 kJ/mol at the lowest for Pd4W to –25.4 kJ/mol at the highest for AuBP1. Interestingly, the peptides in the third regime (AgBP1 derivatives) presented notably greater binding affinity, which ranged between –30.3 and –32.8 kJ/mol. These higher ΔG values likely arise from the Met residues of the AgBP1-based peptides, which can strongly bind with Au3+ ions. This binding event likely sequesters the metal ions away from the Trp residue, thus inhibiting reactivity at peptide:Au3+ ratios > 1. The reaction would be dependent upon peptide concentration where more peptide in the system would likely enhance the Met/Au3+ binding and diminish metal ion reduction, consistent with the observed reactivity. Based upon the observed reactivity differences and binding affinities of the peptides, it is possible that the Au3+ ions are bound to the peptide in such a manner that it is unable to encounter the Trp residues. For instance, in the WAgBP1 sequence, the metal ions could be complexed to the Met residues, preventing their reduction at the Trp at higher peptide:Au ratios due to the structure of the complexed system. This could also play a role in the enhanced reactivity for peptide-based systems where exogenous reductants are used that could more readily react at the Met-bound Au3+ ions, negating the proposed steric effects.49 This effect was examined using the parent AgBP1 sequence without the Trp residue, which was allowed to complex with Au3+ ions in solution. To this mixture, free Trp amino acid molecules were added at a peptide:Trp ratio of 1:1 and the reaction was monitored. In this system, Au3+ reduction was evident via plasmon formation, giving rise to k values that ranged from (0.86 ± 0.21) × 10−3 min-1 to (0.63 ± 0.06) × 10−3 min-1 for AgBP1:Au ratios of 1 and 5. Such values are comparable in magnitude to the WAgBP1 system that reacts only at a peptide:Au3+ ratio of 1. Remarkably, Trp itself was also able to reduce Au3+ ions and passivate the generated Au nanoparticles, generating k values of (13.3 ± 0.6) × 10−3 min-1 for a Trp:Au ratio of 1 and (44.7 ± 3.6) × 10−3 min-1 at a ratio of 5. These values are ~3 times higher than rate constants for the WAgBP2 peptide under the same conditions. Such differences in reactivity may arise from shifts in the reduction potential of the Au3+ once complexed with the moieties of the peptides, which has been observed previously for other metal ions.63-64 Nevertheless, these results suggest that the peptide coordination of the Au3+ ions appears to be critical in controlling the

Figure 7. Photoluminescent quenching analysis of Au3+/peptide binding for reactive peptides. Part (a) presents spectra of photoluminescent quenching of AuBP1 with increasing amounts of Au3+ in the system, while part (b) displays the Stern-Volmer analysis of the data in part (a) to calculate the ∆G of Au3+/AuBP1 binding. Part (c) compares the ∆G values for Au3+/peptide binding for all reactive peptides. reaction rates, potentially from both steric and electronic considerations. Taken together, it is likely that the peptide sequence composition, binding affinity, and peptide/Au3+ complex structure all play important roles in controlling the overall reactivity. This gives rise to the three different reactivity regimes of the studied biomolecules. Integration of reductive reaction capabilities were achieved through addition of the Trp residue to the peptides; however, the full sequence of the biomolecule played a substantial role in controlling the overall reactivity. Intriguing new directions to probe the fundamental basis of this reactivity have been presented, including additional mutation studies for specific new sequences, which are presently under study. Conclusions The main goals of this study were to demonstrate the ability to integrate non-native functionality into materials binding peptides. This was achieved through the addition of terminal Trp residues



into peptides that lacked Au3+ reduction capabilities. Interestingly, while Trp incorporation did give rise to reductive capabilities for nanoparticle formation, the other residues of the biomolecules have been suggested to play important and compounding roles in controlling the overall reactivity and reaction rates. This is anticipated to arise from the three-dimensional structures of the peptides in solution and their overall binding affinity for the Au3+ ions. New peptides that mutate these characteristics are presently under consideration to access a fundamental understanding of these effects.

ASSOCIATED CONTENT Supporting Information Pseudo first order kinetic and Stern-Volmer analyses of peptides with Au3+ reduction reactivity, peptide purification analysis, and reaction pH studies.

AUTHOR INFORMATION Corresponding Author *MRK: Phone: (305) 284-9351. E-mail: [email protected].

ACKNOWLEDGMENT We greatly acknowledge the University of Miami for support. C.J.M. would also like to thank the University of Miami Dean’s Summer Fellowship and the Dean’s Dissertation Year Fellowship for support. We also thank Dr. James N. Wilson for assisting in the photoluminescent quenching studies and the TEM Core at the University of Miami for microscopy analysis of our samples.

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Engendering Materials Directing Peptides with Non-Native

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