Langmuir 2007, 23, 11347-11350
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Conformational Control of Inorganic Adhesion in a Designer Protein Engineered for Cuprous Oxide Binding Woo-Seok Choe,† M. S. R. Sastry,‡ Corrine K. Thai, Haixia Dai,§ Daniel T. Schwartz, and Franc¸ ois Baneyx* Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195-1750 ReceiVed February 21, 2007. In Final Form: September 19, 2007 Combinatorial selection of peptides that bind technological materials has emerged as a valuable tool for roomtemperature nucleation and assembly of complex nanostructured materials. At present, the parameters that control peptide-solid binding are poorly understood, but such knowledge is needed to build the next generation of hybrid materials. Here, we use a derivative of the DNA binding protein TraI engineered with a disulfide-bonded cuprous oxide binding sequence called CN225 to probe the influence of sequence composition and conformation on Cu2O binding affinity. We previously reported a statistically significant enrichment in paired arginines (RR) among a family of cuprous oxide binding peptides and hypothesized that this is a key motif for binding. However, systematic alanine (A) substitutions in the CN225 RR motif (creating RA, AR, and AA pairs) do not support the hypothesis that RR is critical for Cu2O binding by CN225. Instead, we find that the presentation of the peptide in a disulfide-constrained loop (i.e., the conformation present during combinatorial selection) is crucial for binding to the metal oxide. Our results suggest that caution should be exerted when extrapolating from statistical data and that, in some cases, conformation is more important than composition in determining peptide-inorganic adhesion.
Introduction Recent years have seen a surge of interest in using polypeptides selected for their ability to bind inorganic or synthetic compounds to fabricate advanced nanostructured materials for applications ranging from biomaterials and engineered tissues to electrooptical, magnetic, and photonic devices.1-6 Traditionally, short polypeptides displayed on the surface of a cell or bacteriophage in a linear or disulfide-constrained conformation are isolated from large, random populations of nonbinders or weak binders based on their affinity for a technological material.7 The resulting peptides have been used in a synthetic form,8-15 in the context of the displaying organism,16-23 or following genetic engineering * To whom correspondence should be addressed. E-mail: baneyx@ u.washington.edu. † Current address: Department of Chemical Engineering, Sungkyunkwan University, Suwon, Korea 440-746. ‡ Current address: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri Kansas City, Missouri 64110. § Current address: Cambrios Technologies Corp., 2450 Bayshore Parkway, Mountain View, California 94043. (1) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042. (2) Merzlyak, A.; Lee, S.-W. Curr. Opin. Chem. Biol. 2006, 10, 246. (3) Sarikaya, M.; Tamerler, C.; Jen, A. K.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577. (4) Sarikaya, M.; Tamerler, C.; Schwartz, D. T.; Baneyx, F. Annu. ReV. Mater. Res. 2004, 34, 373. (5) Seeman, N. C.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6451. (6) Zhang, S. Nat. Biotechnol. 2003, 21, 1171. (7) Baneyx, F.; Schwartz, D. T. Curr. Opin. Biotechnol. 2007, 18, 312. (8) Ahmad, G.; Dickerson, M. B.; Church, B. C.; Cai, Y.; Jones, S. E.; Naik, R. R.; King, J. S.; Summers, C. J.; Kro¨ger, N.; Sandhage, K. H. AdV. Mater. 2006, 18, 1759. (9) Naik, R. R.; Brott, L. L.; Clarson, S. J.; Stone, M. O. J. Nanosci. Nanotechnol. 2002, 2, 95. (10) Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Nat. Mater. 2002, 1, 169. (11) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Acta Biomater. 2005, 1, 145. (12) Sano, K.; Sasaki, H.; Shiba, K. Langmuir 2005, 21, 3090. (13) Umetsu, M.; Mizuta, M.; Tsumoto, K.; Ohara, S.; Takami, S.; Watanabe, H.; Kumagai, I.; Adschiri, T. AdV. Mater. 2005, 17, 2571. (14) Slocik, J. M.; Stone, M. O.; Naik, R. R. Small 2005, 1, 1048. (15) Slocik, J. M.; Naik, R. R. AdV. Mater. 2006, 18, 1988.
within a host protein scaffold24-28 to nucleate, grow, assemble, and organize target compounds in a (somewhat) controlled fashion. Understanding how a particular inorganic-binding peptide interacts with its cognate solid is desirable both from a fundamental standpoint and as a guiding principle to tailor the specificity and selectivity of nucleation and control the growth and spatial positioning of inorganic nanostructures. In theory, amino acid sequences that have been selected via phage or cell surface display should specify all the information that is necessary to mediate solid interaction. This includes the chemistry, order, and spacing of amino acid side chains. Several recent studies have revealed that charged residues (R, K, H, D, and E) play an important role in mediating peptide-solid interactions.29-37 However, how a peptide is presented to the material and how (16) Huang, Y.; Chiang, C. Y.; Lee, S.-K.; Gao, L.; Hu, E. L.; De Yoreo, J.; Belcher, A. M. Nano Lett. 2005, 5, 1429. (17) Lee, S.-K.; Yun, D. S.; Belcher, A. M. Biomacromolecules 2006, 7, 14. (18) Lee, S. W.; Mao, C.; Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892. (19) Li, C.; Botsaris, G. D.; Kaplan, D. L. Cryst. Growth Des. 2002, 2, 387. (20) Mao, C.; Flynn, C. E.; Hayhurst, A.; Sweeney, R.; Qi, J.; Georgiou, G.; Iverson, B.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 6946. (21) Nam, K. T.; Kim, D. W.; Yoo, P. J.; Chiang, C. Y.; Meethong, N.; Hammond, P. T.; Chiang, Y.-M.; Belcher, A. M. Science 2006, 312, 885. (22) Reiss, B. D.; Mao, C.; Solis, D. J.; Ryan, K. S.; Thornson, T.; Belcher, A. M. Nano Lett. 2004, 4, 1127. (23) Yoo, P. J.; Nam, K. T.; Qi, J.; Lee, S.-K.; Park, J.; Belcher, A. M.; Hammond, P. T. Nat. Mater. 2006, 5, 234. (24) Brown, S.; Sarikaya, M.; Johnson, E. J. Mol. Biol. 2000, 299, 725. (25) Brown, S. Nano Lett. 2001, 1, 391. (26) Dai, H.; Choe, W. S.; Thai, C. K.; Sarikaya, M.; Traxler, B. A.; Baneyx, F.; Schwartz, D. T. J. Am. Chem. Soc. 2005, 127, 15637. (27) Kramer, R. M.; Li, C.; Carter, D. C.; Stone, M. O.; Naik, R. R. J. Am. Chem. Soc. 2004, 126, 13282. (28) Sano, K.-I.; Sasaki, H.; Shiba, K. J. Am. Chem. Soc. 2006, 128, 1717. (29) Chen, H.; Su, X.; Neoh, K.-G.; Choe, W. S. Anal. Chem. 2006, 78, 4872. (30) Hayashi, T.; Sano, K.; Shiba, K.; Kumashiro, Y.; Iwahori, K.; Yamashita, I.; Hara, M. Nano Lett. 2006, 6, 515. (31) Kumada, Y.; Tokunaga, Y.; Imanaka, H.; Imamura, K.; Sakiyama, T.; Katoh, S.; Nakanishi, K. Biotechnol. Prog. 2006, 22, 401. (32) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Langmuir 2005, 21, 6929. (33) Sanghvi, A. B.; Miller, K. P.; Belcher, A. M.; Schmidt, C. E. Nat. Mater. 2005, 4, 496. (34) Sano, K.; Shiba, K. J. Am. Chem. Soc. 2003, 125, 14234. (35) Serizawa, T.; Sawada, T.; Matsuno, H.; Matsubara, T.; Sato, T. J. Am. Chem. Soc. 2005, 127, 13780.
10.1021/la702414m CCC: $37.00 © 2007 American Chemical Society Published on Web 10/06/2007
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its structure deforms upon surface binding are likely to be equally important in determining binding affinity.11,38-43 Previously, we used the multivalent FliTrx cell surface display to identify disulfide-bonded dodecapeptides binding to the metal oxides Cu2O and ZnO.44,45 A subclass of binders was enriched in hydrophilic and basic residues and contained a statistically significant number of paired arginines, leading us to postulate that the RR motif may be important in metal oxide recognition.45 One such sequence termed CN225 was engineered as a CGP/ GPC-flanked, disulfide-bonded loop, within a permissive site of the DNA-binding protein TraI, to mimic FliTrx peptide presentation. The resulting designer protein (TraIi1753::CN225) bound Cu2O with an equilibrium dissociation constant (Kd) of 12 nM and could be used for the mineralization and organization of cuprous oxide nanoparticles on DNA.26 Because it otherwise lacks cysteines, contains a single inorganic binding motif, exhibits high affinity for Cu2O, and can be readily modified by genetic engineering, TraIi1753::CN225 provided us with a suitable platform to determine how conformation and the signature RR motif modulate the affinity of the CN225 dodecapeptide for Cu2O. Here, we show by quartz crystal microbalance (QCM) measurements that whereas the twin arginines have modest influence on Cu2O affinity, presentation in a disulfide-bonded loop is critical for the binding of both TraIi1753::CN225 and a synthetic version of the peptide to cuprous oxide. Experimental Methods All mutations were introduced in plasmid p99I::i1753::CN22526 which carries the gene encoding TraIi1753::CN225 using the QuickChange site-directed mutagenesis kit (Stratagene). Primer pairs 5′-ACCGATGGTCTGGCGCGTATTGCGGCGCGT-3′ and 5′ACGCGCCGCAATACGCGCCAGACCATCGGT-3′, 5′-GATGGTCTGCGTGCGATTG CGGCGCGT-3′ and 5′-ACGCGCCGCAATCGCACGCAGACCATC-3′, and 5′-ACCGATGGTCTGGCGGCGATTGCGGCGCGT-3′ and 5′-ACGCGCCGCAATCGCCGCCAGACCATCGGT-3′ were used to introduce the R10A, R11A, and R11A mutations, respectively. The presence of the correct mutations was verified by sequencing using primer 5′-GACTCTTATACACAAGTAGCGTTC-3′. Proteins were expressed and purified as before.26 To carboxymethylate TraIi1753::CN225, the disulfide bond was first reduced by incubating 500 µL of protein (218 mg/L) with 25 µL of 100 mM dithiothreitol (DTT) in phosphate buffer (90 mM Na2HPO4, 10 mM NaH2PO4, pH 8.0) for 1 h at 37 °C. The mixture was supplemented with 50 mM iodoacetate, incubated at 37 °C for 3 h, and dialyzed twice against 1 L of phosphate buffer. Protein concentration was determined with the Coomassie dye-binding protein assay kit (Sigma). QCM experiments and calculation of equilibrium dissociation constants were performed as described previously.26 For the experiments of Figure 1, purified proteins were injected at a final (36) Serizawa, T.; Sawuda, T.; Kitayama, T. Angew. Chem., Int. Ed. 2007, 46, 723. (37) Willett, R. L.; Baldwin, K. W.; West, K. W.; Pfeiffer, L. N. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7817. (38) Gray, J. J. Curr. Opin. Struct. Biol. 2004, 14, 110. (39) Kulp, J. L., III; Shiba, K.; Evans, J. S. Langmuir 2005, 21, 11907. (40) Wang, S.; Humphreys, E. S.; Chung, S.-Y.; Delduco, D. F.; Lustig, S. R.; Wang, H.; Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y.-M.; Jagota, A. Nat. Mater. 2003, 2, 196. (41) Oren, E. E.; Tamerler, C.; Sarikaya, M. Nano Lett. 2005, 5, 415. (42) Kantarci, N.; Tamerler, C.; Sarikaya, M.; Haliloglu, T.; Doruker, P. Polymer 2005, 46, 4307. (43) Schnirman, A. A.; Zahavi, E.; Yeger, H.; Rosenfeld, R.; Benhar, I.; Reiter, Y.; Sivan, U. Nano Lett. 2006, 6, 1870. (44) Dai, H.; Thai, C. K.; Sarikaya, M.; Baneyx, F.; Schwartz, D. T. Langmuir 2004, 20, 3483. (45) Thai, C. K.; Dai, H.; Sastry, M. S.; Sarikaya, M.; Schwartz, D. T.; Baneyx, F. Biotechnol. Bioeng. 2004, 87, 129.
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Figure 1. Reductive carboxymethylation of TraIi1753::CN225 abolishes Cu2O binding. QCM resonant frequencies were measured in the presence of 14 nM (a) TraIi1753, (b) TraIi1753::CN225, and (c) reduced and carboxymethylated TraIi1753::CN225. Proteins were injected at the times indicated by vertical arrows. Schematic structures showing the TraIi1753 backbone in green, the CN225 dodecapetide in orange, and the flanking tripeptides in gray are shown on top of each panel. Experiments were conducted in phosphate buffer which causes a baseline drift due to slow dissolution of the 20 nm Cu2O film (∼2 Å/h).26 concentration of 2.8 µg/mL (∼14 nM). The CN225 (RHTDGLRRIAAR), cyclic CN225 (CGPRHTDGLRRIAARGPC), and CN97 (LRRRRGWSNLVW) peptides were commercially synthesized and resuspended in phosphate buffer at 1 mg/mL. Cyclic CN225 was incubated at 4 °C with shaking for 7 days to promote formation of the disulfide bridge. The QCM data of Figure 2were collected following injection of ∼60 µM of each peptide. For the experiments of Figure 3, purified TraIi1753::CN225 and its R10A, R11A, and R10A-R11A variants were injected at the indicated concentrations. Data shown are averages obtained with at least three different batches of purified proteins and different quartz crystals. For molecular dynamics simulations, cyclic CN225 and its alanine substitution mutants were manually constrained with a disulfide bridge. Allatom structures were built using the CHARMm27b4 package, optimized by the steepest gradient and conjugate gradient method, solvated in a water box described by the TIP3 model, and the energy of the solvated peptides was minimized as described in detail elesewhere.45 Molecular surfaces and electrostatic potentials were calculated using the SwissPdb viewer package.46
Results and Discussion The Ph.D. M13 phage display system (New England Biolabs) and the FliTrx cell surface display system (Invitrogen) have been used to identify most solid-binding peptides described to date. In the Ph.D.-7 and Ph.D.-12 libraries, linear heptapeptides (or dodecapeptides) are fused to the N-terminus of the minor M13 phage coat protein pIII via a flexible GGGS spacer and should not be subjected to conformational constraints other than those imparted by the short-range interactions that dictate how the random domain will fold. Indeed, linear peptides synthesized on the basis of sequences identified through the use of the Ph.D.(46) Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714.
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Figure 2. A cyclic conformation is required for the binding of synthetic CN225 peptides to Cu2O. QCM resonant frequencies were measured in the presence 60 µM (a) cyclic CN225, (b) linear CN225, and (c) linear CN97. Peptides were injected at the times indicated by vertical arrows, and their respective structures are shown on top of each panel.
12 display retain solid-binding activity and have proven useful for the mineralization of a variety of materials.8-15,47 By contrast, the M13 Ph.D.-C7C and FliTrx systems display heptapeptides and dodecapeptides (respectively) that are constrained in a loop when a disulfide bridge forms between the thiol groups of flanking cysteine residues. How such a restricted conformation affects solid binding affinity is currently unknown. To gain insights on this issue, we made use of TraIi1753:: CN225, a derivative of TraIi175348 engineered to contain a single copy of the CN225 Cu2O-binding loop at the protein C-terminus.26 Control QCM measurements conducted at a protein concentration of 14 nM (∼1 × Kd) showed that whereas adsorption of TraIi1753::CN225 to the Cu2O-coated crystal caused an ∼60 Hz decrease in crystal resonant frequency, TraIi1753 did not adhere to the metal oxide, as evidenced by the absence of a frequency decrease upon addition of the parent protein (Figure 1a and b). To investigate the role of the CN225 conformational context in binding, we reduced the disulfide bridge of TraIi1753::CN225 with DTT and carboxymethylated the free thiols with iodoacetate. The modified protein was next dialyzed against phosphate buffer to eliminate unreacted DTT whose presence causes dissolution of the Cu2O film on the quartz crystal. Figure 1c shows that the reduced carboxymethylated (RCM) form of TraIi1753::CN225 exhibited no affinity for Cu2O, suggesting that CN225 must be presented to the surface within the context of a constrained loop to mediate cuprous oxide binding. To confirm this result, a linear and a cyclic version of the CN225 dodecapeptide were chemically synthesized and tested for Cu2O binding as above. Surprisingly, the concentration of (47) Reiss, B. D.; Rai, G.-R.; Auciello, O.; Ocola, L. E.; Firestone, M. A. Appl. Phys. Lett. 2006, 88, 083903. (48) Haft, R. J. F.; Palacios, G.; Nguyen, T.; Mally, M.; Gachelet, E. G.; Zechner, E. L.; Traxler, B. J. Bacteriol. 2006, 188, 6346.
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Figure 3. Amino acid sequences and Langmuir adsorption isotherms of the TraIi1753 variants. (a) Wild type and mutant CN225 amino acid sequences of the four TraIi1753 derivatives. Invariant tripeptides flanking the CN225 dodecapeptide are shaded in gray, and the N-terminal cysteine is assigned the number 1. Basic residues are shown in blue, acidic residues are shown in red, and hydroxylbearing residues are shown in orange. Substituted alanines are in green. (b) Langmuir adsorption isotherms were constructed for each of the TraIi1753 variants shown in panel (a). Data shown are averages obtained for three independent experiments. Typical standard deviations were 10% or less.
cyclic CN225 required to cause a 70 Hz decrease in crystal resonant frequency was 60 µM, more than 4,000-fold that of TraIi1753::CN225 needed to achieve a comparable frequency reduction (Figure 2a). While the reasons underpinning the poor adhesive properties of the cyclic peptide remain obscure, it is clear that insertion of the CN225 loop within a protein scaffold that does not possess any particular affinity for Cu2O can significantly enhance its binding to the metal oxide (Figure 1b). In agreement with the results of Figure 1c, addition of 60 µM of the linear CN225 peptide to the QCM chamber did not cause a frequency decrease indicative of binding. Rather, we observed a rise in the crystal resonant frequency, which is characteristic of a loss in mass in the ∼20 nm thick Cu2O film (Figure 2b). The dissolution rate calculated from the Sauerbrey equation was ∼215 ng/h, almost 10-fold that measured in buffer alone (25 ng/h). The increase in film dissolution upon addition of linear CN225 suggests that it chelates surface coppers (cuprous or cupric ions, since oxygen is present), making them soluble, either because the CN225 sequence is a specific chelator or because of nonspecific chelation due to the large concentration of free amine termini in solution. To distinguish between the two possibilities, we repeated the experiment with a different synthetic dodecapeptide (CN97) that contains a double RR motif but shows very little affinity for cuprous oxide in the context of the FliTrx display (unpublished data). At a concentration of 60 µM, the rate of CN97-mediated film dissolution was virtually identical to that observed with linear CN225 (Figure 2c). Thus, both linear peptides exhibit a nonspecific surface activity against Cu2O that is unrelated to their intrinsic affinity for the material when displayed as disulfide-bonded loops. Taken together, the above results indicate that the primary structure of CN225 alone is insufficient to confer Cu2O binding affinity and that the
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Figure 4. Simulated molecular surfaces and electrostatic potentials of the disulfide-bonded CN225 peptide (a) and of the R10A (b), R11A (c), and R10A-R11A (d) mutants. Structures on the left correspond to a “front” view of the disulfide-constrained peptides, while structures on the right correspond to a “top” view rotated by 45°. In the front view, disulfide bridges are at the “bottom” of the structure. Electrostatic surfaces are colored between -3kT (red) and +3kT (blue).
conformation of the peptide plays a more important role than its composition in determining cuprous oxide binding. Several mechanistic studies conducted with synthetic or phageborne linear polypeptides have revealed that electrostatic interactions play an important role in mediating adhesion to a wide range of materials.12,29,30,32-34,37,49 A similar mechanism may be at play in the case of Cu2O binders identified via the FliTrx display because they are enriched in positively charged arginine residues.45 In fact, a statistically significant number of paired arginines in randomly sequenced clones led us to suggest that the basic RR motif contributes to cuprous oxide binding.45 To test this hypothesis, R10 (using the numbering system of Figure 3a) and R11 of the CN225 sequence were converted individually or in combination to alanine, an amino acid containing an uncharged methyl side chain. QCM experiments conducted at increasing protein concentrations were used to generate the Langmuir isotherms of Figure 3b. Compared to the complete loss of binding affinity observed upon reduction of the disulfide bond, conversion of either or both arginine residues to alanine only had a minor impact on the affinity of TraIi1753::CN225 for Cu2O (Figure 2b). In all cases, monolayer coverage was achieved at a protein concentration of ∼50 nM (∼10 µg/mL) and fitting of the isotherms provided comparable equilibrium dissociation constants (Kd) of 14 ( 1, 22 ( 2, 19 ( 2, and 34 ( 4 nM for TraiI1753::CN225 and its R10A, R11A, and R10A-R11A derivatives, respectively. Thus, while it exerts a minor modulating influence, the RR motif of CN225 is not essential for cuprous oxide binding. Exchange of the large, positively charged side chain of arginine for that of alanine should affect both the electrostatic potential and conformation of the CN225 Cu2O-binding loop. To gain insights on these effects, we predicted the structure, molecular surface, and charge of the four disulfide-bonded peptides depicted in Figure 3a through molecular dynamics simulations. Figure 4 shows that the wild type CN225 cyclic peptide, which contains four arginines, is predicted to exhibit a rather uniformly distributed (49) Goede, K.; Busch, P.; Grundmann, M. Nano Lett. 2004, 4, 2115.
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positive charge. Moreover, the side chains of R10 and R11 of the RR motif project away from each other and toward the “front” and “back” sides of the molecular surface, suggesting that the pairing of these residues has a structural role rather than contributing to the creation of a positive hot spot. Interestingly, in both the wild type and the R10A mutant, residues R11 and R15 form a “rabbit ears” structure that we previously proposed to participate in cuprous oxide recognition.45 Obviously, the R11A and R10A-R11A variants do not contain this feature; they also exhibit a significantly less basic character than either the wild type or the R10A mutant. Because the affinity of all three mutants for cuprous oxide is comparable to that of the wild type (Figure 3b), neither the rabbit ears motif nor a highly positive character appear to be necessary for Cu2O binding. On the other hand, the topology of the top (and presumably inorganic-contacting) surfaces of the R11A and R10A-R11A mutants is similar to that of the wild type. Furthermore, for all variants, R4 and R15 are located at the ends of the molecule. Thus, an extended conformation tipped with positively charged residues, rather than a strong overall or even local positive charge, may be sufficient for cuprous oxide binding.
Conclusions Recent studies aiming at understanding how inorganic binders interact with their cognate materials have mostly focused on amino acid composition and have been conducted either with synthetic linear peptides or within the multivalent context of the displaying organism.12,29,30,32-34,37,49 However, amino acid composition is not all that matters; sequence order can also modulate binding and surface specificity, even for linear polypeptides with identical overall composition.33,49 Thus, conformation should be an important contributor to peptide-solid recognition, particularly for those sequences mined from disulfide-constrained libraries. Indeed, in the case of CN225, we found that compositional changes to the twin arginine motif, though predicted to be significant in cuprous oxide binding, had little impact on metal oxide affinity. However, presentation of the sequence in a disulfide-bonded loop analogous to that found in the screening library was essential to mediate the adhesion of TraIi1753:: CN225 to Cu2O. Molecular dynamics simulations support the idea that a highly positive character is not critical for adhesion and that a significant amount of conformational sampling is possible without a drastic effect on Cu2O binding affinity provided that it occurs within the context of the loop environment. Whether this phenomenon is general and indicative of loop deformation so that it can “fit” the cuprous oxide surface is being investigated. Our observation that the binding of TraIi1753::CN225 to Cu2O is completely abolished upon reduction of the disulfide bond opens the door to the design of reconfigurable hybrid materials in which solid binding activity can be addressed (electro)chemically or enzymatically. Considering that the average protein contains multiple permissive sites which can each be engineered with a different solid binding sequence, this additional level of control may prove useful in part-to-part assembly and to alter the functional properties of bio-nanoinorganic assemblies. Acknowledgment. This work was supported by the Army Research Office through the DURINT program (DAAD19-011-04999) and by the National Science Foundation through the MRSEC program (DMR-0520567). We are grateful to Jian Zhou and Shaoyi Jiang for help with molecular dynamics simulations. LA702414M
