Photochemical Identification of Molecular Binding Sites on the Surface of Amyloid-β Fibrillar Aggregates
Aliyan et al. have studied binding sites on amyloid-b fibrils by using a rhenium dipyridophenazine complex. The binding interactions were characterized by spectroscopic and computational methods, which indicated a hydrophobic binding site between Val18 and Phe20 of the amyloid-b fibril. Photoirradiation of the rhenium complex produced oxidation on Met35, leaving a footprint on the fibril, which provides empirical confirmation for the binding site. The identification of molecular binding sites in amyloid-b can guide therapies and drugs design for Alzheimer's disease.
INTRODUCTION<!>RESULTS<!>DISCUSSION<!>Conclusions<!>EXPERIMENTAL PROCEDURES<!>Ab Purification<!>Dilution of Basic Stock Ab to Biological pH<!>Binding Assay
<p>Photochemical processes play important biological roles ranging from the photosynthetic generation of carbohydrates in plants and algae 1 to photoreceptors in a variety of organisms (e.g., cryptochromes 2 ). In humans, photochemical reactions are important in the biosynthesis of vitamin D 3 and in the photoisomerization of retinal in the eyes' rod cells. 4 Light has also been used for the treatment of certain human diseases such as cancer 5 and tuberculosis. 6 The fact that nature has been harvesting light as a reactant for chemical reactions for millions of years has served as an inspiration to our group in the use of photochemical transformations for studying biomolecules that are hard to characterize by standard techniques, i.e., X-ray crystallography and nuclear magnetic resonance (NMR).</p><p>Protein misfolding and aggregation plays a causal role in the onset of several human diseases including Alzheimer's disease (AD) 7 and Parkinson's disease. 8 In the case of AD, development of the disease is linked to the deposition of insoluble amyloidbeta (Ab) plaques in the patient's gray matter. 7,[9][10][11] Because of the link between AD and Ab aggregation, numerous studies on the process of Ab aggregation have been performed. 12 For the last few years, we have studied photoluminescent metal complexes as a new family of probes for monitoring the fibrillation of Ab. [13][14][15] Metal complexes have remarkable applications in diverse fields of chemistry such as solar energy conversion, 16 sensing, 13,17,18 and medical treatments. 5 Attractive characteristics of metal complexes include red emission, large Stokes shifts, and long lifetime,</p><p>The Bigger Picture Alzheimer's disease is a form of dementia affecting over 44 million people worldwide, and its symptoms include agitation, confusion, and memory loss. This disease is characterized by aggregates of the amyloid-b (Ab) peptide in the brain. The transition of Ab from the soluble to the aggregated form is linked to the onset of Alzheimer's disease. Molecules that inhibit Ab aggregation or quench its harmful effect are highly sought after. However, how molecules bind to Ab is still uncertain. Ab aggregates are disordered in nature, preventing the use of common methods for studying structure and binding. To address this, we used a rhenium complex that binds to Ab. Upon light exposure, this complex produces oxidation on Ab, leaving a mark at the place of binding. Spectroscopic and computational studies allowed elucidation of locations and binding modes of these molecules on Ab. This information will guide the production of potent drugs with better binding affinities to Ab for the treatment of Alzheimer's disease.</p><p>which are key features that distinguish this family of probes from organic dyes, and open new windows to address current challenges in materials, medicine, and energy.</p><p>Recently, we reported an unprecedented light-switching behavior of [Re(CO) 3 (dppz)(Py)] + with Ab fibrils upon exposure to UV light (362 nm). 15 Binding of [Re(CO) 3 (dppz)(Py)] + to Ab aggregates produced an instantaneous 18-fold increase in photoluminescence (compared with the photoluminescence of the complex in buffer), which was enhanced to 105-fold increase after irradiation with 362 nm light from the fluorometer lamp. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of oxidized forms of Ab after exposure to UV light. This is a remarkable observation given that the oxidative stress hypothesis [19][20][21] has been postulated as a possible molecular root for AD, where the formation of reactive oxygen species (ROS) is mediated by the ions attached to Ab fibrils. Although the production of ROS in the presence of Ab can be deleterious for the human brain, in vitro, we show that the controllable reaction of photogenerated ROS with Ab can be used to study molecular binding sites.</p><p>Experimental characterization of the binding sites of small molecules to proteins is often done by X-ray crystallography, 22,23 where the protein is co-crystalized with the small molecule. In the case of Ab fibrils, the diverse morphology of the fibrils, which includes regions with a high degree of disorder, makes the characterization of binding sites a challenging task, and quite often X-ray structures are obtained for fibrils that are grown from short segments of Ab. [24][25][26][27] Fibrils that are prepared from shorter segments of Ab are not necessarily similar in structure to those made from full-length Ab. For instance, Ab 16-22 , 28 Ab 17-21 , 29 Ab 11-25 , 29 and Ab 34-42 30 are reported to form antiparallel b-sheet packing, whereas parallel b-sheet packing is expected for full-length Ab. 31 Thus, the determination of binding sites on Ab fibrils produced by the full-length peptide is not trivial. In this work, we use a combination of spectroscopic and computational techniques to propose a binding site for [Re(CO) 3 (dppz)(Py)] + to Ab, that is further probed by a novel photochemical footprinting technique. The photoexcitation of [Re(CO) 3 (dppz)(Py)] + photooxidizes Ab near its binding site, leaving a mark (footprint) on the peptide. The peptide can then be interrogated by tandem mass spectrometry (MS-MS) for identification of the place of oxidation and thus the binding site. The use of orthogonal techniques such as spectroscopy, molecular dynamics (MD) simulations, and photochemical footprinting with MS-MS provide an unprecedented level of detail on identifying molecular binding sites in protein aggregates, which can be extrapolated to other systems.</p><!><p>The study of the binding of [Re(CO) 3 (dppz)(Py)] + to fibrillar Ab was performed according to different research strategies. Characterization of the binding equilibrium was done by performing a saturation titration experiment. Here, a series of solutions with a constant Ab fibril concentration of 4 mM was prepared with varying concentrations of [Re(CO) 3 (dppz)(Py)] + ranging from 1 to 60 mM. The photoluminescence intensity was recorded for each of these solutions at 560 nm instead of the whole spectra to minimize the irradiation time. We have previously demonstrated that the photoluminescence of [Re(CO) 3 (dppz)(Py)] + increases upon binding to Ab fibrils and further strengthens with light irradiation. However, we also showed that if irradiation is carefully controlled and kept to a minimum, the photoluminescence can be measured without an appreciable change in intensity over time. 15 The data on photoluminescence intensity as a function of [Re(CO) 3 (dppz)(Py)] + concentration can be seen in Figure 1A. The data were fit initially to a simple equilibrium model that assumes a 1:1 binding ratio, resulting in a dissociation constant (K d ) of 2.8 G 0.6 mM (Equation 1, with n = 1; the binding constant (K b ) is the reciprocal of the K d , and is equal to 3.6 3 10 5 M À1 ). This K d value is slightly higher than that previously reported for [Ru(bpy) 2 (dppz)] 2+ (2.1 G 0.5 mM) 14 with Ab in the same model. A larger value for the dissociation constant implies a less favorable binding of [Re(CO) 3 (dppz)(Py)] + to Ab. The slightly higher dissociation constant observed for [Re(CO) 3 (dppz)(Py)] + is possibly due to a weaker electrostatic interaction of [Re(CO) 3 (dppz)(Py)] + with the slight negative charge of the Ab fibrils (pI = 5.31) in contrast to the dicationic [Ru(bpy) 2 (dppz)] 2+ , which has one more positive charge. 32 Nevertheless, the similarity between the dissociation constants suggests that the binding site is similar and most likely driven by hydrophobic effects instead of electrostatic interactions. In contrast, the reported dissociation constant of [Re(CO) 3 (dppz)(Py)] + with calf thymus DNA (2.4 3 10 1 mM 33 ) is around two orders of magnitude larger than that reported for [Ru(bpy) 2 (dppz)] 2+ (<1 mM 18 ), suggesting a higher contribution of electrostatic interactions when binding to DNA. A more appropriate binding model based on the assumption that a fixed concentration of binding sites is available at a given concentration of Ab (represented as [Ab]/n, where n is the number of Ab monomers in the fibril necessary to form a binding site) according to the Bard model 34 yields a dissociation constant of 4.2 G 0.6 mM (Equation 1).</p><p>To better understand the structure of the site where [Re(CO) 3 (dppz)(Py)] + binds Ab, the binding stoichiometry was assessed by the continuous variation method (Job plot) (Figure 1B). The Job plot was performed at a total [Ab + [Re(CO) 3 (dppz)(Py)] + ] concentration of 50 mM. The plot maximum lies around 0.26, which corresponds to an Ab:[Re(CO) 3 (dppz)(Py)] + binding stoichiometry of 2.8:1. This value is close to what has previously been reported for [Ru(bpy) 2 (dppz)] 2+ (2.6), 14 suggesting [Re(CO) 3 (dppz)(Py)] + ] will likely bind to the same pocket in the fibril. The maximum is rather broad and irregular in shape, which could indicate more than one binding site or a distribution of binding sites. Given that an Ab fibril is not a rigid rod but rather a flexible rope, it is reasonable to assume that bends, twists, and turns of the fibers can somehow affect the population of binding sites. Thus, we take the value of 2.8 Ab peptides per [Re(CO) 3 (dppz)(Py)] + as an estimated midpoint value. Further details on the binding site of [Re(CO) 3 (dppz)(Py)] + to Ab fibrils can be obtained from docking experiments on the dominant morphology of Ab fibrils. 35 Docking on the fibrils with C2 symmetry presents binding sites within separate domains, termed X and X 0 (Figure 2). These sites are associated with the same amino acids; however, they represent different binding conformations to the Ab fibrils. Docking of [Re(CO) 3 (dppz)(Py)] + onto the two-fold Ab structure produced ten top-ranked sites (Figure 2). In eight of them, the Re complex binds in the same position within separate domains of the fibril structure. The first site, termed A and A 0 , shows interaction with residues Lys16, Val18, and Phe20 and was found 24% of the time (Figures 2A and 2B). Sites B and B 0 were found to interact with residues Gly9, Tyr10, Asn27, Val39, and Val40 and, occurring 34% of the time, are more dominant than the previously obtained docked poses (Figures 2A and  2B). Sites C and C 0 occurred in 20% of the poses analyzed and exist where the Re complex binds to the ends of the fibril, interacting with polar residue Lys28 and non-polar residues Phe19, Ala21, and Asn27 (Figures 2B and 2C). Sites C and C 0 are slightly less dominating than the A and A 0 site because of a much higher surface area in the latter. Site C 0 occurs with slightly higher frequency than site C and interacts with the same residues. The docking results also showed the existence of site D and D 0 , which are similar to site B, and bridge the gap between the two domains of the Ab structure (Figures 2B and 2C). This site occupies 17% of the total poses analyzed and interacts with non-polar residues Gly9, Tyr10, and Asn27 (Figure 2C). Sites E and F show only marginal occupancy, which indicates they might not have a significant and stable interaction with [Re(CO) 3 (dppz)(Py)] + (Figures 2B and 2D).</p><p>Our previous observation that [Re(CO) 3 (dppz)(Py)] + can perform photooxidation on the Ab backbone 15 provides a unique opportunity to use footprinting analysis to identify the binding site. Fibrillary Ab was placed in contact with [Re(CO) 3 (dppz)(Py)] + and irradiated with 365 nm light. Then, the Ab was disaggregated with 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP) 36 and analyzed by LC-MS (Figure S2). Table S1 shows the chromatogram-integrated areas under controlled irradiation conditions. The pristine and oxidized peaks were subject to MS-MS analysis. Analysis of the spectra showing y and b fragmentations are shown in Figures 3A  and 3C. Fragments of the pristine and oxidized peaks showed similar mass until reaching Met35; however, when Met35 was present, the fragments exhibited a mass increase of +16 (Figure 3). MS-MS analyses of y and b ions are shown in Figures 3B and 3D, respectively. Addition of 16 mass units to methionine was consistent with the conversion of the thioether to a sulfoxide. To further confirm this conclusion, fragmentation peaks were simulated with oxidation at different residues and matched to the experimental data. The maximum number of simulated peaks that could be matched with the experimental results are indicative of Met35 oxidation (Figure S3 and Table S2). Control experiments showed that Ab does not get oxidized if irradiated without the rhenium complex (Table S3). Furthermore, if monomeric Ab  S12). This implies that the binding modes for rhenium complexes are different for monomeric and fibrillar Ab.</p><!><p>As a result of the undeniable relationship of Ab and AD, understanding how molecular entities bind to Ab structures, including monomer, oligomers, and fibrils, is of central importance for the design of better drugs and aggregation inhibitory molecules. Still, it is well known that fibrillary aggregates are difficult to study because of their insolubility and high degree of disorder, which makes analysis by conventional techniques (such as X-ray and NMR) difficult. Our studies have shown that binding of [Re(CO) 3 (dppz</p><p>) is stronger than the binding of this complex to DNA by an order of magnitude. On the other hand, the binding is weaker than amyloid-binding molecules such as Congo red 37 and thioflavin T. 38 Nonetheless, [Re(CO) 3 (dppz)(Py)] + binds with similar affinity as [Ru(bpy) 2 dppz] 2+ to Ab. The concordance of the binding constants for these two complexes is consistent with a similar binding site. Furthermore, the fact that the binding constants for the rhenium and ruthenium complexes are so similar, even with the charge disparity between them, implies that electrostatic interactions are not the main driving force for binding, which rather relies on hydrophobic interactions.</p><p>The relatively weak binding constant calculated for this system implies that a surface binding site is responsible for the binding of the metal complex. This was supported by our MD simulations, which showed four distinct binding sites on the surface of the Ab fibril. Interestingly, some of these sites must contribute minimally to binding in full-length Ab fibrils. For example, site C (and C 0 ) was located right at the ends of the fibril where only a maximum of four of these will be accessible, regardless of the length of the fibril. Sites B and D, located on the side of the fibril, were at the interface between Ab domains. This Ab fibril model lacks the first 8 amino acids, which have mostly a random coil conformation. These 8 amino acids would likely occupy most of the space where sites B and D are located. Although this fact alone does not necessarily preclude binding, it is very likely that binding occurs at a less obstructed site, such as site A. Site A is at the surface of the fibril, and it is found between residues Val18 and Phe20. This site is comprised of hydrophobic amino acids that will interact well with the hydrophobic dipyridophenazine ligand. The computational binding model was in agreement with the Job plot values (2.8:1 for Ab: [Re(CO) 3 (dppz)(Py)] + ). The MD model suggests that every three peptides in the fibril can create a binding site A for [Re(CO) 3 (dppz) (Py)] + , from the projection of [Re(CO) 3 (dppz)(Py)] + on the fibrillar axis.</p><p>More than 20 MD simulations were performed to ensure accurate binding of the [Re(CO) 3 (dppz)(Py)] + to the Ab fibril. The stability of sites A and A 0 were investigated utilizing 200 ns MD simulations in explicit solvent. These simulations were performed in GROMACS 39 software with AMBER03 40,41 force field parameters. The interaction persisted between the [Re(CO) 3 (dppz)(Py)] + complex and the Ab fibril throughout the simulation. However, changes in the orientation of the Re complex were observed in many of the simulations (more than 80%). Two distinct binding modes were observed within the same binding pocket, termed primary and secondary (Figure 4). During the simulations, the [Re(CO) 3 (dppz)(Py)] + complex shifted between two conformations; the first was embedded within the fibril's hydrophobic pocket between residues Val18 and Phe20, and the second was raised out of the hydrophobic pocket exposing the dppz ligand to a more hydrophilic environment. The primary binding mode (Figure 4A) was observed to occur more than 75% of the time within the simulations, whereas the secondary mode (Figure 4B) was observed to occur less frequently. This is mostly due to the increased interaction between hydrophobic residues within the pocket, which stabilizes the primary site more often than the secondary binding mode where only two CH-P interactions hold the complex.</p><p>Thermodynamically, the primary mode should dominate and was observed in most of the simulations. The [Re(CO) 3 (dppz)(Py)] + complex interacts strongly through P-P interactions with the fibril surface at distances less than 4 A ˚. CH-P interactions were also observed between residue Val18 and the dppz ligand. However, the pyridine ring shows no interactions with the Ab fibril in both modes.</p><p>The hydrophobic cleft between Val18 and Phe20 has been proposed by our group and others on the basis of MD simulations for the Ab fibril binding site of molecules such as [Ru(bpy) 2 dppz] 2+ , 14 Congo red, 42 N-[2-(3,4-dimethoxyphenyl)ethyl]-3phenyl-acrylamide, 43 and 5-((2E,4E)-5-(4-(dimethylamino)phenyl)penta-2,4-dien-1ylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione. 44 The Eisenberg group was able to co-crystallize Orange G with the 6 amino acid peptide KLVFFA, which contains the Val18-Phe20 region. 25 The X-ray structure shows that Orange G interacts strongly with this peptide wedging the hydrophobic zipper of the amyloid structure.</p><p>Although interaction between full-length Ab fibrils and binding molecules is unlikely to wedge the hydrophobic backbone of the fibril, the reported interaction by the Eisenberg group supports the importance of the Val18-Phe20 region in molecular binding. Our results present the first chemical evidence that the Val18 and Phe20 hydrophobic cleft on the surface of full-length Ab is an important binding site for hydrophobic molecules. On the basis of these results, we propose that the hydrophobic cleft formed between Val18 and Phe20 is a general site for the binding of molecules to Ab, particularly molecules with large hydrophobic and aromatic moieties.</p><p>The information obtained from the binding site can be used to explain the lightswitching behavior observed for the probe in the presence of Ab. The excited state of [Re(CO) 3 (dppz)(Py)] + possesses two closely spaced triplet energy levels, an intraligand state ( 3 IL, emissive) and metal-ligand charge transfer state ( 3 MLCT, nonemissive). 45,46 Polar environments stabilize the 3 MLCT, rendering the complex not photoluminescent. However, when the polarity of the medium decreases, the 3 MLCT is destabilized allowing the population of the 3 IL and the emission of light. When [Re(CO) 3 (dppz)(Py)] + binds to the hydrophobic cleft, between Val18 and Phe20, the microenvironment around the dppz ligand becomes less polar, displaying photoluminescence by the preferential population of the emissive 3 IL state.</p><p>The binding constant, Job plot, and computational models support the occurrence of a non-polar domain on the surface of the Ab fibril between Val18 and Phe20, where molecules with hydrophobic groups can bind. [Re(CO) 3 (dppz)(Py)] + , with a large aromatic and hydrophobic dipyridophenazine ligand, can bind to this hydrophobic cleft on the surface of Ab fibrils. In the previous section, we presented evidence that photoexcitation of [Re(CO) 3 (dppz)(Py)] + leads to Met35 oxidation. Control experiments of [Re(CO) 3 (dppz)(Py)] + with fibrillary Ab purged with nitrogen does not produce oxidation in the peptide. ROS, such as singlet oxygen species, are short lived reactive species and will only modify amino acids that are nearby the place where they are created. The formation of ROS by [Re(CO) 3 (dppz)(Py)] + and their reaction with the Ab fibril creates a mark (or footprint) on the place where they were created, which incidentally is near the binding site. The side chain of Met35 is about 1.5 nm from site A; however, Met35 is buried in the hydrophobic zipper of the protein. We have observed that in the absence of molecular oxygen, Met35 does not get oxidized, which strongly suggests that oxygen molecules can populate the inner hydrophobic core of Ab. Our MD simulations in the presence of 19 oxygen molecules showed that molecular oxygen can be trapped inside the Ab fibrils. Out of 19 molecules, four oxygen molecules (O 1 -O 4 ) were located inside the hydrophobic core of fibrils after 200 ns simulations (Figure 5A). The first (O 1 ) and second oxygen (O 2 ) were embedded deep inside the hydrophobic pocket of the two domains and were in proximity of five Met35 residues (Figure 5B).  5C). However, the fourth oxygen molecule (O 4 ) was considerably distant from Met35 at a distance of 8.55 A ˚(Figure 5D). Oxygen is a small, hydrophobic molecule that can interact with the hydrophobic interior of the fibril. Although these simulations show that oxygen is stable in this hydrophobic environment, they cannot completely explain how oxygen gets there. In the simulations, C a atoms of the Ab 40 fibrils were frozen in place to maintain the stability of the fibril structure, which prevented exploration of the dynamic fluctuations that were likely to contribute to the diffusion of oxygen to these regions in the structure. On the other hand, studies have identified that the maturation and even photobleaching of fluorescent proteins such as mCherry were associated with the penetration of molecular oxygen into the beta barrel through gaps in the structure. 47 Kinks, twists, and bends in the fibril structure could introduce these gaps, allowing oxygen to access the hydrophobic interior of the fibril.</p><p>A reasonable hypothesis for the photooxidation of Met35 is that trapped oxygen molecules in the hydrophobic core of Ab were photosensitized into ROS, which reacted with Met35, converting the thioether into sulfoxide. Experiments using singlet oxygen sensor green (SOSG) show that [Re(CO) 3 (dppz)(Py)] + produces singlet oxygen in MeCN/water mixtures with an efficiency that increases with the decrease in polarity of the medium (MeCN/water 25:75, F D = 0.43; MeCN/water 75:25, F D = 0.74; Figures S13 and S14). On the other hand, experiments with [6-(4 0 -amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), which reacts with hydroxyl radicals but not with superoxide, hydrogen peroxide, or singlet oxygen, 48 show significant fluorescence upon illumination, which becomes stronger with [Re(CO) 3 (dppz)(Py)] + concentration, decreasing the medium polarity and increasing the irradiation time (Figures S15 and S16). This indicates that this rhenium complex could produce a variety of ROS species. Nonetheless, further experiments will still be required to define whether a type 1 or type 2 reaction is occurring, as well as the details of the reaction, which is a topic currently under investigation.</p><p>In contrast to Ab fibrils, LC-MS using the rhenium complex for the analysis of photooxidized Ab in the monomer form showed a significantly different pattern where ions with +14, +16, and +32 masses were present in significant quantity (Figures S4-S8). The Ab peptide contains other amino acids that are prone to oxidation by singlet oxygen, such as His6, His13, His14, and Tyr10. 49 Furthermore, fragmentation of the +32 oxidized species showed that fragment 1-34, which do not contain methionine but rather contain histidine and tyrosine residues, presents oxidation (see Supplemental Experimental Procedures and Figures S9-S12). Tyrosine and histidine oxidation is consistent with reaction with singlet oxygen. 50 Histidine oxidation by singlet oxygen has been reported to produce +14 and +32 mass increases, 51 and other oxidations can occur as well. Although the photooxidation mechanism is still under investigation, the fact remains that photooxidation of monomeric Ab occurs at regions not containing Met35. This observation is crucial because it demonstrates that photooxidation at other regions of the peptide could occur but only when the peptide is in a different conformation (monomeric form). This observation further supports the hypothesis that sites B and D are not available for binding because photooxidation in those regions occurs for monomeric Ab but is not observed for fibrillar Ab, suggesting that amino acids 1-8 occupy this region (Figure 6), and thus prevent binding of the rhenium complex in the fibrillar form.</p><!><p>In this study, we have comprehensively characterized the binding between [Re(CO) 3 (dppz)(Py)] + and Ab and offered chemical proof of a binding site on the surface of Ab. When [Re(CO) 3 (dppz)(Py)] + binds to fibrillar Ab, it presents a remarkable increase in photoluminescence (light switching), as well as oxidation of the peptide backbone. A dissociation constant of 4.2 G 0.6 mM and an Ab:[Re(CO) 3 (dppz)(Py)] + stoichiometry ratio of 2.8 were determined. MD simulations indicated a binding site for [Re(CO) 3 (dppz)(Py)] + on Ab in the hydrophobic cleft formed between Val18 and Phe20. The suggested model predicted a hydrophobic interaction of the aromatic dipyridophenazine ligands with the hydrophobic cleft with a 3:1 stoichiometry ratio for Ab:[Re(CO) 3 (dppz)(Py)] + , which was in agreement with the empirical ratio obtained from the Job plot assay. MS-MS analysis of the irradiated [Re(CO) 3 (dppz)(Py)] + /Ab system showed that the photooxidation occurred at Met35. When the structure of Ab fibrils and the predicted binding model were analyzed, it became evident that Met35 was in close proximity to the binding site. Moreover, MD simulations showed that molecular oxygen can be trapped very close to Met35 in the Ab hydrophobic zipper. The oxidation of this amino acid is considered as strong experimental confirmation for the proposed model. The oxidation of Met35 also discarded other potential binding sites, showing that the hydrophobic cleft between Val18 and Phe20 is the main site where metal dipyridophenazine complexes bind. This study confirms the photochemical footprinting of a binding site in Ab. The confirmation of this hydrophobic cleft as an important site for molecular binding has repercussions in the design of amyloid-binding molecules with potential therapeutic effects.</p><!><p>A detailed procedure for the synthesis of the metal complex, purification, and preparation of basic Ab stock solution is reported elsewhere. 15 The procedures are summarized below.  (dppz)(Py)]PF 6 is provided in Figure S17. The complex was ion exchanged to the chloride salt to improve water solubility.</p><!><p>Bulk Ab 1-40 (from 21 st Century Biochemicals, lot no. 9596) was dissolved in HFIP, and after dilution with 0.1% trifluoroacetic acid (TFA) in water, was desalted by dialysis. A dry film of the peptide was achieved by complete removal of the solvent via lyophilization. The peptide was purified by reverse-phase high-performance liquid chromatography (HPLC) with a gradient of water/ACN containing 0.1% TFA.</p><p>Preparation of Basic Stock of Ab HPLC-purified peptide was suspended in 25 mM aqueous NaOH. After sonication for 2 min, the solution was filtered with 0.2 mm centrifuge tubes, and the concentration was measured in basic media by UV-visible spectroscopy ( 3 292 nm z 2,132 M À1 cm À1 ).</p><!><p>To prepare Ab 1-40 aggregates, the stock basic solution was diluted to 150 mM with water and 100 mM phosphate buffer (PB) containing 0.02% NaN 3 at pH 7.4 to allow a final buffer concentration of 25 mM (see our previous report for more detail 15 ). The solution was incubated at 37 C and 700 rpm for 24 hr.</p><!><p>Ab fibrils (150 mM) were diluted to a 10 mM concentration and was used to prepare series of solutions with a constant Ab and PB concentration of 4 mM and 5 mM, respectively, with a varying concentration of [Re(CO) 3 (dppz)(Py)] + : 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, 30, 40, and 60 mM. Absorbance and photoluminescence of each solution were measured. To minimize the exposure of the samples to UV light, singlepoint photoluminescence measurements (excitation = 362 nm, emission = 560 nm, excitation and emission slit widths = 10 nm) were recorded three times with an integration time of 1 s, 10 s wait time between the measurements, and the average of the three photoluminescence intensities was used. Instrument-related effects and the inner filter effect were corrected according to previous reports. 14,52 The data were fit to the following equation based on Bard's model: 34 Absorbance and photoluminescence of each solution were obtained (as explained above in the binding assay section). Instrument-related effects and the inner filter effect were corrected according to previous reports. 14,52 Computational Methods</p><p>The [Re(CO) 3 (dppz)(Py)] + structure was obtained from the Cambridge Crystallographic Data Center (CCDC) 53 (CSD: NAXQOS) 33 in CIF file format, which was converted to PDB format with Mercury-3.5.1 software. 54 Molecular-docking procedures were performed on Ab 1-40 fibril structures generously provided by Robert Tycko 55 with Autodock Vina 1.5.6 software. 56 Parameters for rhenium were obtained from a previous work by Fayer and coworkers. 57 YASARA 13.2.2 software 58 was used to make single bonds between the metal and ligand so that the Autodock Vina program would treat the complex as a whole. The grid size was chosen to contain the entire ligand-peptide complex, and the spacing was kept to a standard value (1.00 A ˚) in Autodock Vina. An exhaustiveness value of 20 was used, which produced 20 docked poses with the highest relative binding affinities. In the rigid docking, the flexibility of the receptor (the Ab fibril) was minimal. Ab fibril flexibility of the docked structures was compared by two methods: (1) flexible docking and (2) rigid docking on the different structures of the Ab fibrils derived from short-term (5 ns) MD simulations in an aqueous solution.</p><p>The docking procedures produced over 140 poses and were analyzed utilizing PyMol software. All three docking procedures produced similar results. Therefore, rigid docking to different conformers of the Ab fibrils was used and 20 top-ranking poses were chosen for MD runs. The MD simulations of [Re(CO) 3 (dppz)(Py)] + bound to the Ab peptide were performed with AMBER 03 force field 59 as implemented in the GROMACS program 39 in explicit aqueous solution. The box was filled with TIP3P water molecules, and the sodium and chloride ions were added to simulate a physiological concentration of 0.154 mM. The docked poses obtained in the earlier step were used as starting structures and placed in a cubic box with dimensions of 70 3 70 3 70 A ˚. A matrix of distance restraints (semi-rigid model) with a high energetic penalty (R1,000 kJ/mol) was added to the [Re(CO) 3 (dppz)(Py)] + complex in order to accurately maintain the geometry as well as the coordination to the metal center. The dppz ligand was kept at 180 because it was required to remain planar throughout the simulations. The pyridine ring was also locked at 180 with respect to the opposite CO ligand, but was allowed to rotate during the simulations. During the MD simulations, C a atoms of the Ab 1-40 fibrils were frozen in place to maintain the stability of the fibril structure. These atomic constraints helped to maintain the secondary structure of these fibrils without affecting the flexibility of their side chains within the binding sites. In addition, MD simulations of molecular oxygen bound to the Ab peptide were performed according to the aforementioned protocol in explicit aqueous solution. These simulations provided potential binding or entrapment modes of oxygen molecules within the fibril's hydrophobic core. MD parameters for molecular oxygen were</p>