New Insights into Metal Interactions with the Prion Protein: EXAFS

Article pubs.acs.org/JPCB

New Insights into Metal Interactions with the Prion Protein: EXAFS Analysis and Structure Calculations of Copper Binding to a Single Octarepeat from the Prion Protein Alex McDonald,‡ M. Jake Pushie,*,† Glenn L. Millhauser,‡ and Graham N. George† †

Department of Geological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E2, Canada Department of Chemistry, University of California, 1156 High Street, Santa Cruz, California 95064, United States

‡

S Supporting Information *

ABSTRACT: Copper coordination to the prion protein (PrP) has garnered considerable interest for almost 20 years, due in part to the possibility that this interaction may be part of the normal function of PrP. The most characterized form of copper binding to PrP has been Cu2+ interaction with the conserved tandem repeats in the N-terminal domain of PrP, termed the octarepeats, with many studies focusing on single and multiple repeats of PHGGGWGQ. Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used in several previous instances to characterize the solution structure of Cu2+ binding into the peptide backbone in the HGGG portion of the octarepeats. All previous EXAFS studies, however, have benefitted from crystallographic structure information for [CuII (Ac-HGGGW-NH2)−2H] but have not conclusively demonstrated that the complex EXAFS spectrum represents the same coordination environment for Cu2+ bound to the peptide backbone. Density functional structure calculations as well as full multiple scattering EXAFS curve fitting analysis are brought to bear on the predominant coordination mode for Cu2+ with the Ac-PHGGGWGQ-NH2 peptide at physiological pH, under high Cu2+ occupancy conditions. In addition to the structure calculations, which provide a thermodynamic link to structural information, methods are also presented for extensive deconvolution of the EXAFS spectrum. We demonstrate how the EXAFS data can be analyzed to extract the maximum structural information and arrive at a structural model that is significantly improved over previous EXAFS characterizations. The EXAFS spectrum for the chemically reduced form of copper binding to the Ac-PHGGGWGQ-NH2 peptide is presented, which is best modeled as a linear two-coordinate species with a single His imidazole ligand and a water molecule. The extent of in situ photoreduction of the copper center during standard data collection is also presented, and EXAFS curve fitting of the photoreduced species reveals an intermediate structure that is similar to the Cu2+ form with reduced coordination number.

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INTRODUCTION The prion protein (PrP) plays a central role in a host of neurodegenerative diseases in humans and animals, most notably Creutzfeldt-Jakob disease (CJD), Grestmann-Straussler-Scheinker syndrome, variant CJD and bovine spongiform encephalopathy (BSE, also known as Mad Cow disease).1,2 These diseases are characterized by the accumulation of a misfolded and infectious form of the native host-encoded PrP, which are capable of transmitting disease to new hosts. The considerable rise in interest and investigations of PrP over the past two decades has led to many discoveries relating to the importance of PrP in physiological function, including the first reports in the 1990′s of its copper binding competence.3−6 PrP appears to play a myriad of subtle roles, ranging from neuronal growth and differentiation,7 to maintaining robust synaptic transmission,8 brain metal homeostasis,9 transport of metals from the gut10 and modulating NMDA receptor susceptibility − a function mediated in a copper-dependent manner.11 PrP is expressed in many non-neuronal tissues,12 possibly hinting further at a significantly more subtle and wide-ranging role in © 2013 American Chemical Society

normal physiological function and development; although its highest levels of expression in mammalian tissue is found at the presynaptic membrane of neurons.12−14 The prion protein is anchored to the exterior cell surface by a modified lipid anchor attached at the C-terminal end of the protein.2 The PrP C-terminus is largely α-helical, with a small proportion of β-sheet content,15 however, the N-terminal region (approximately half of the PrP primary amino acid sequence) is disordered in solution and does not adopt any regular ordered structure. The N-terminus of mammalian PrP contains multiple tandem repeats of the same eight amino acids (human sequence shown in Figure 1A), which is comprised of four repeats with the sequence PHGGGWGQ (bovine and manatee PrP possess five octarepeats), which are highly conserved. Although the number of repeats may differ between species the presence of the His residues, which constitute the Received: August 18, 2013 Revised: October 4, 2013 Published: October 8, 2013 13822

dx.doi.org/10.1021/jp408239h | J. Phys. Chem. B 2013, 117, 13822−13841

The Journal of Physical Chemistry B

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number of amide nitrogens adjacent the coordinating His residues deprotonate and serve as ligands to the Cu2+ center, coordinating the metal through the amide N-atom lone pair, such that the Cu2+-atom lies along the previous N−H bond vector, while maintaining the normal planarity and conformation of the peptide backbone. This intermediate coordination form appears to involve at least two His residues, however, due to the high degree of flexibility within this region of the protein this form is the least-well characterized of all the Cu2+ coordination modes to PrP. It is also likely that this coordination mode (termed Component 2)28 encompasses more than one local Cu2+ coordination environment32,33 and multiple protein conformations may also be accessible, most of which are likely in equilibrium with one another. The final coordination mode for Cu 2+ -binding to PrP, termed Component 1, occurs under high Cu2+-occupancy conditions and at physiological pH. Anchoring by a single His residue is maintained, with the equatorial coordination of the metal center completed by coordination from the protein backbone (Figure 1C). Binding to the octarepeat region of PrP involves coordination by atoms from the HGG region of a single repeat, specifically the Nδ-atom atom from the His imidazole ring, two deprotonated amide nitrogens from the two adjacent Gly residues as well as the carbonyl of the second Gly in the sequence (Figure 1B and C). Component 3 presents the tightest binding for Cu2+, over Components 2 and 3, such that the N-terminal domain presents negative cooperativity for binding Cu2+.22,23 Component 1 type coordination is the most extensively characterized form of Cu2+-binding to PrP, as it is an easily reached end-point in titrations with short peptide fragments of PrP, such as PHGGGWGQ. There is a wealth of experimental data published on this specific Cu2+ coordination mode, most notably, Burns, et al. previously published the only crystal structure of copper interacting with PrP, using the peptide AcHGGGW-NH2.34 Extended X-ray absorption fine structure (EXAFS) spectroscopy is a powerful technique for elucidating chemical and structural information from an element of interest, typically providing highly accurate bond lengths within 5 Å or less of the element of interest for bioinorganic molecules in solution. X-ray diffraction-based methods are the most widely utilized and informative of techniques for elucidating structural information and can provide information at, or approaching, atomic resolution. Compared with diffraction-based methods, EXAFS generally cannot provide three-dimensional information, and has a comparatively limited radial field of view about the central absorbing atom, but can provide improved precision in atomic positions over diffraction techniques, and can also provide structural information on solutions.35 Several groups have performed EXAFS characterizations of Cu2+ binding to PrP, or related peptide fragments, however, despite the wealth of published data in this area to-date much of the previous EXAFS work in this area has not been rigorously analyzed or reported transparently.36−39 As component 1 binding is the predominant coordination form for Cu2+ at physiological pH under saturating Cu2+ conditions, this abrogates the possibility of additional major copper coordination species from contributing significantly to the total EXAFS spectrum. Recent potentiometric titration work by Di Natale, et al. indicates that at physiological pH under high Cu2+ occupancy conditions the protein loses two protons for each copper atom bound (Cu4L−8H),32 which is

Figure 1. (A) The octarepeat sequence from human PrP with the metal-coordinating His residues emphasized, (B) schematic view of the metal-free PHGG region of a single octarepeat, and (C) the consensus component 1 coordination mode for Cu2+.

primary metal-anchoring sites, is highly conserved across essentially all cordate species characterized to date.16,17 Most PrP proteins also contain a number of nonoctarepeat His residues further along the sequence (not shown in Figure 1A), sometimes referred to as the fifth (or fifth and sixth) copperbinding sites. Mammalian PrP (mouse and human in particular) have been the most extensively studied and the high sequence similarity among mammalian PrPs means that biochemical and structural characterizations are widely applicable. The resting copper concentration at the synapse appears to be ∼1 μM, while the maximum concentration may range from 3 μM to 250 μM.18,19 Copper is exocytosed via synaptic vesicles during depolarization and it has been proposed that copper release may be an integral component of neurotransmitter release.20 The affinity for PrP to bind other cations notwithstanding, the protein displays a range of binding affinities for Cu2+, each corresponding to a distinct coordination mode. Dissociation constants derived for Cu2+-PrP complexes, which take into account these distinct coordination modes, range from the low nM to ∼12 μM.21−23 Copper binding to PrP has been reported to induce endocytosis of the CuPrP complex via clathrin-coated pits.24 PrP can bind from one to perhaps as many as six equivalents of Cu2+, and it has been postulated that the endocytotic mechanism may serve to buffer synaptic content following depolarization.20 Copper is a well-known pro-oxidant. Despite the hypothesis offered by Brown, et al. that PrP may function as a superoxide dismutase (SOD)-mimetic,25 follow-on studies have been unable to verify this claim.26,27 As there are no known SOD molecules which function within the synaptic space the clearance of copper from within the synaptic junction through PrP endocytosis may indirectly provide some measure of protection against oxidative damage. The N-terminal domain of PrP is selective for binding Cu2+ (and Zn2+) over other divalent metal cations, however, PrP displays considerably more complex behavior in copper binding than any other metals, and copper has been the focus of most metal-PrP studies. At low copper concentrations the N-terminal domain of PrP can bind a single equivalent of Cu2+ through multiple His residues (Figure 1B), and has been termed component 3 in detailed electron paramagnetic resonance (EPR) spectroscopic characterizations.28 At physiological pH component 3-type coordination is best represented as a CuII(His)4 complex, with each His imidazole coordinating via the Nε-atom.29−31 At intermediate copper concentrations a 13823



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copper sample was prepared from a stock solution of Cu(II)OR with an additional 20 mM sodium ascorbate in degassed buffer as a mild reducing agent. The chemically reduced solution was left to equilibrate for 30 min and was then loaded into a cuvette and frozen as above. X-ray Absorption Spectroscopy. The XAS measurements were conducted at the Stanford Synchrotron Radiation Lightsource (SSRL) with the SPEAR storage ring containing 500 mA at 3.0 GeV. Copper K-edge data were collected on the structural biology XAS beamline 7−3 operating with a wiggler field of 2 T and employing a Si(220) double-crystal monochromator. Beamline 7−3 is equipped with a rhodiumcoated vertically collimating mirror upstream of the monochromator. To minimize radiation damage and minimize thermal fluctuations contributing to the Debye−Waller factor for EXAFS analysis, samples were maintained at a temperature of approximately 10 K in a liquid helium flow cryostat (Oxford Instruments, Abingdon, UK). X-ray absorption spectra were measured as the Cu Kα fluorescence excitation spectra using a 30-element germanium array detector41 with analog electronics (Canberra Corporation, Meriden CT, USA) employing an amplifier shaping time of 0.8 μs. To avoid problems with nonlinearity of the detector due to high count-rates, 3absorption unit Ni X-ray filters were used to preferentially absorb scattered radiation and silver Soller-slits (EXAFS Co., Pioche NV, USA) were optimally positioned between the sample and the detector to reduce filter fluorescence registered by the detector. Incident and transmitted X-ray intensities were measured using nitrogen-filled ionization chambers. Spectra were energy-calibrated with reference to the K-edge spectrum of a copper foil, measured simultaneously with each spectrum. The lowest energy inflection of the copper K-edge was assumed to be 8980.3 eV. Successive spectra were compared using both the Cu K near edge and EXAFS data, which revealed increasing contribution from photoreduced copper species the longer the samples were exposed to the incident beam. To minimize photoreduction of the Cu(II) species a sample volume of 1.5 mL was prepared without glycerol, flash frozen, and loaded into a specially designed sample holder with a height of 15 mm, allowing multiple acquisitions from frozen sample on areas which had not previously been exposed to the incident beam. Rapid EXAFS data acquisition scans of c.a. twenty-five minutes per scan were also used for a k-range of 16.2 Å−1, as opposed to ∼45 min per scan we have employed previously. The shorter acquisition time traded improved statistics per scan with minimizing the contribution from photoreduced copper species. A total of 12 scans of Cu(II)OR were collected, representing the minimally photoreduced copper species. Two scans of the chemically reduced Cu(I)OR sample were collected (k-range of 14 Å−1). For the photoreduced EXAFS data a total of 6 scans were collected on a glycerol-containing sample, without repositioning the sample between scans. The final five scans were averaged as they appeared to overlay well and were assumed to be representative of the photoreduced species (k-range of 12 Å−1). EXAFS Data Analysis. The EXAFS oscillations χ(k) were quantitatively analyzed by curve-fitting using the EXAFSPAK suite of computer programs42 as described by George et al.43 Fourier transforms were phase-corrected for Cu−N backscattering. The threshold energy E0 was assumed to be 9000 eV, and ab initio theoretical phase and amplitude functions were calculated using the program FEFF version 8.25.44 FEFF

consistent with component 1-type coordination. Based on this data, at pH 7.5 component 1 accounts for ∼90% of the species present in solution, while other species present, such as Cu3L−6H, and Cu4L−6H, are likely to present very similar copper coordination environments as the Cu4L−8H species.32 The potentiometric titration experiments were carried out on a four octarepeat peptide fragment of PrP. Smaller fragments, such as a single octarepeat, will present a less complex picture (i.e., fewer copper coordination modes), which will further simplify the system for spectroscopic characterization. Component 1-type binding presents a relatively planar coordination environment for the Cu2+ center, and this planar geometry extends away from the central copper atom for several Ångstroms to include the adjacent peptide backbone. Our experience with EXAFS spectroscopy leads us to the conclusion that component 1-type binding should yield exceptionally good EXAFS multiple scattering interactions, however, beyond references to observing His imidazole multiple scattering in the EXAFS Fourier transform, and minimal references to peptide backbone contributions, which were implemented with questionable fitting parameters (or too few multiple scattering paths), such results have not been explicitly detailed in the literature.36−39 Our approach serves to i) significantly improve the solution characterization of component 1 binding of Cu2+ to a single octarepeat, ii) apply results from density functional theory (DFT) structure calculations to add a thermodynamic component to the structures being assessed, and iii) raise the profile of EXAFS data fitting and analysis through detailed and rigorous structural models that include full multiple scattering interactions for the EXAFS curve fitting procedure, and transparently present the rich structural information contained in the EXAFS spectrum. The chosen methods provide an efficient framework for detailed EXAFS curve fitting and affords the means to characterize the complex coordination structure of the AcPHGGGWGQ-NH2 peptide (abbreviated OR) binding environment for copper in solution. We also present a detailed analysis of the reduced form of copper with a single octarepeat as well as the photoreduced form, which, without prior precautions, can be generated in situ by the incident X-ray beam during data collection.29,40 Analysis reveals that the coordinating peptide backbone provides significant enhancement to the multiple scattering interactions in the EXAFS data which have not been fully exploited in previous work.

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EXPERIMENTAL AND THEORETICAL METHODS Sample Preparation. All reagents were purchased from Sigma-Aldrich, and were of the best quality available. The N-terminal acetylated and C-terminally amidated peptide Ac-PHGGGWGQ-NH2 was prepared by solid-phase synthesis using standard fluorenylmethoxycarbonyl (Fmoc) methods, purified using reverse phase HPLC and characterized by electrospray ionization mass spectrometry (ESI-MS). Samples for X-ray absorption spectroscopy (XAS) were prepared in aqueous solution with 5 mM peptide in degassed buffer containing 20 mM MOPS buffer and 30% glycerol (v/v) as a glassing agent. The copper stock solution was prepared from the sulfate salt, titrated to a final concentration of 4.9 mM Cu2+ (∼0.98 Cu: 1 peptide). The solution was adjusted to pH 7.5 using concentrated hydrochloric acid (HCl) and potassium hydroxide (KOH) solutions. Solutions were loaded into 2 × 3 × 25 mm acrylic cuvettes and frozen in liquid nitrogen immediately prior to data collection. The chemically reduced 13824



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energy. The gas-phase entropy at 1 atm standard state is also provided in the harmonic frequency output and is corrected for the volume change upon going from 1 atm standard state in the gas-phase to 1 M standard state in solution (−26.6 J K−1 mol−1). The free energy for each species is corrected for the stabilizing effect of solvation through the continuum solvation procedure, which describes the stabilizing electrostatic component as well as an entropic term, describing the work required to generate the molecular cavity and exclude solvent from that volume. For calculations with gain or loss of explicit solvent molecules, the free energy of solvation for a water molecule in bulk water is taken as −16.5 kJ mol−1, which is derived from the experimental free energy of formation in the gas-phase (corrected to 1M) and in solution (corrected to 55 M). In some instances the entropic contribution contains an additional term arising from the contribution of multiple potential conformations, such as in the case of an extended flexible region of the peptide, calculated as Rln(n), where n is the number of conformers (assumed to be energetically equivalent), and R is the gas constant 8.314 J K−1 mol−1. For peptide structures (or portions of structures) which were constrained by H-bonds these regions were assumed to be rigid (n = 1). In all other instances the backbone His and Trp ϕ and ψ angles, were assumed to be 2-fold rotamers, while the Gly ϕ and ψ and His and Trp χ1 and χ2 angles were assumed to be 3fold rotamers. Assuming unhindered rotation about Gly ϕ and ψ this would give an rough estimate of n = 9 conformations, and the Rln(n) term would contribute 18.3 J K−1 mol−1 to the total entropy of the molecule. Collectively, the above terms are sufficient to calculate an aqueous free energy difference for the structures modeled herein.

multiple scattering calculations from the heavy atom framework, as defined by optimized geometries from DFT calculations (see below), used a k-range of 15.8 Å−1, although data was fit to 15.75 Å−1, because increasingly poor S/N persisted above 15.8 Å−1. Calculated scattering paths included a maximum Nleg path of 4 and an Rmax of 5.5 Å. A minimum cutoff of 7% of the mean amplitude of the largest amplitude path was used for the curved wave calculation as well as the plane-wave approximation multiple scattering paths, while scattering paths with amplitudes below this cutoff were discarded. The correlated Debye model in FEFF 8.25 was used to calculate the EXAFS Debye−Waller parameters (σ2). The σ2 value for the first shell was allowed to float during the fit procedure, with all other Debye−Waller parameters scaled to this value. In some cases the Debye−Waller factor for longer range scattering interactions (i.e., scattering paths attributable to solvent bound in an apical position) were scaled up by ∼150% − the rationale behind this procedure and the increased σ2 value is discussed in detail in the Cu2+ EXAFS fitting section. Structure Calculations. Density functional calculations were carried out with the Gaussian09, revision C.01, suite of software.45 Spin unrestricted calculations of Cu2+ complexes, and spin-restricted Cu+ calculations, were optimized without constraints using the B3LYP hybrid functional method. Simplified models of [CuII(Ac-HGGG-NH2)−2H] were used to assess the accessible stable backbone conformations of the peptide backbone, while the larger [CuII(Ac-HGGGWNH2)−2H]·n(H2O) system, optimized in the presence of a polarizable continuum, was used to assess possible configurations of water molecules and the Trp side chain. Geometry optimizations on the simplified structural models used the 631G(d) or LANL2DZ basis sets for geometry optimizations. Harmonic frequency calculations were used to identify incomplete optimizations, and were calculated at the same level of theory as the geometry optimization. Geometry optimizations and harmonic frequency calculations for the more elaborate models, which included the Trp residue and explicit water molecules, were carried out at the B3LYP/631G(d)-level with the CPCM solvation method,46 to mimic the effects of bulk solvation during geometry optimization. Due to instabilities in the geometry optimization procedure that we have encountered in G09 when using other reaction fields, the CPCM method was chosen for geometry optimizations. All structures were considered optimized when the change in energy between subsequent optimization steps fell below a negligible predefined cutoff (0.05 J mol−1). The stabilizing effect of bulk solvation on all optimized structures was modeled using the integral equation formalism variant of the polarizable continuum model, IEFPCM,47 with united atom radii defining the molecular cavity and a dielectric representing water (ε = 78.39), calculated at the B3LYP/6-31G(d) level. Single point energies for all structures were calculated with the 6311+G(2df,2p) basis set to obtain more accurate relative energies between structures. Molecular structures were rendered with Chemcraft 1.6.48 Free Energy Derivation. Estimates of the free energy changes in aqueous solution are constructed from various calculated parameters for each of the DFT-optimized structures, as described previously.49,51 Briefly, the gas-phase enthalpy at zero Kelvin is calculated at the large basis set level, with additional terms from the harmonic frequency output to correct for the small temperature dependence in the absolute enthalpy upon going from 0 to 298 K, as well as the zero-point

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RESULTS AND DISCUSSION Copper Coordination to the Octarepeat Region of PrP. Herein we examine the high-occupancy coordination mode, (Component 1 form) using EXAFS spectroscopy. The most effective EXAFS fitting procedures utilize an iterative fitting procedure, where the EXAFS Fourier transform is used to guide initial construction of a structural model. The model is then used to calculate multiple scattering paths, which can be used in further rounds of EXAFS curve fitting. The heavy atom framework from optimized DFT structures is used for the EXAFS fitting. EXAFS generally provides exceptionally good bond length precision for the primary coordinating atoms (also referred to as primary backscattering atoms in EXAFS), however, resolving longer-range structural information can be problematic. First, the EXAFS oscillations are dampened by a 1/R2 term in the EXAFS equation,50 with the result that atoms further from the absorbing atom contribute less to the total EXAFS amplitude. Second, the Debye−Waller factor (represented by the parameter σ2) used in the EXAFS fitting procedure contains a description of the static and vibrational disorder of backscattering atoms. The combination of larger σ2 and the 1/R2 terms means that the contribution to the total EXAFS amplitude for long-range backscattering interactions is often dampened beyond detection (or beyond the ability to fit to the experimental data). Multiple scattering interactions, where the outgoing photoelectron wave scatters off adjacent atoms, occurs with the highest probability when atoms lie within relatively rigid arrangements where the scattering path is close to linear. As we will demonstrate, component 1-type coordination of copper gives rise to multiple scattering contributions from the 13825



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Figure 2. continued

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Figure 2. DFT optimized structures for [CuII(Ac-HGGG-NH2)−2H] (structures G1−G6), [CuII(Ac-HGGGW-NH2)−2H]·n(H2O), where n = 1 or 3 (structures G8−G10), and the reduced copper models [CuI(MeIm)ε(OH2)]+ (G11) and [CuI(Ac-HGGG-NH2)−2H]−·2(H2O) (G12).

Density Functional Structure Calculations of Cu2+ Binding to a Single Octarepeat. To supplement the

His imidazole as well as the much of the local peptide backbone. 13829



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Table 1. Parameters from Density Functional Structure Calculations for Various Octarepeat Fragments with Cu2+ a Simplified Model (Ac-HGGG-NH2) name

# H2O

G1 G2 G3 G4 G5 G6

0 0 0 0 0 0

G1 G2 G3 G4 G5 G6

0 0 0 0 0 0

name G7 G8 G9 G10 OH2

# H2O 1 1 3 3 n/a

SCRF

Eb

H° − H0°

ZPE

S

B3LYP/6-311+G(2df,2p)//B3LYP/6-31G(d) −2945.401452 0.356588 0.384741 188.623 −2945.401494 0.356446 0.384717 190.834 −2945.383156 0.355957 0.384538 191.284 −2945.385719 0.356416 0.384717 188.822 −2945.393679 0.356714 0.384717 187.705 −2945.384459 0.356317 0.384717 187.946 B3LYP/6-311+G(2df,2p)//B3LYP/LANL2DZ −2945.375106 0.356756 0.384597 187.543 −2945.390550 0.357224 0.384849 185.661 −2945.370690 0.356630 0.384534 186.947 −2945.379229 0.356858 0.384793 188.240 −2945.379273 0.356785 0.384769 189.175 −2945.372891 0.356754 0.384617 188.067 Expanded Model (Ac-HGGGW-NH2) SCRF yes yes yes yes yes

Eb

H° − H0°

ZPE

S

B3LYP/6-311+G(2df,2p)//B3LYP/6-31G(d) −3632.017759 0.575873 0.618569 254.558 −3632.017982 0.574820 0.618953 273.139 −3784.975771 0.627999 0.675631 278.675 −3784.979407 0.628557 0.676346 281.965 −76.4624202 0.021177 0.024956 45.135

# confc

ΔG(solv)

ΔΔG(aq)

18 9 9 18 9 18

−361.87 −344.39 −369.74 −365.85 −342.75 −365.51

0.0 +16.3 +38.1 +37.0 +42.3 +41.8

18 9 9 18 9 18

−385.35 −362.38 −389.49 −377.40 −377.27 −384.93

0.0 +0.7 +23.3 +12.0 +10.8 +20.9

# confc

ΔG(solv)

ΔΔG(aq)

2 648 2 2 1

−489.40 −491.08 −504.30 −483.25 −16.4d

+38.7 0.0 +54.5 +63.8 n/a

Energies, unscaled zero-point energies (ZPEs), and H° − H0° are in hartrees; entropy (S) is listed in J K−1 mol−1; ΔG(solv) and ΔΔG(aq) are in kJ mol−1. bLarge basis set energies are calculated at the B3LYP/6-311+G(2df,2p) level. cA full description of the selection criteria for the number of conformers is given in the Supporting Information. dDerived from the experimental data; see the Experimental and Theoretical Methods section.

a

spectroscopic data, as well as the EXAFS curve fitting, numerous Cu2+ complexes of a truncated portion of the OR peptide, Ac-HGGG-NH2, and Ac-HGGGW-NH2 were constructed and optimized. The shorter peptide models were optimized in the gas-phase, and using various basis sets, to assess the various deformations of the local coordination environment about the metal center, in particular the 7membered chelate ring. The larger peptide model was optimized in the presence a dielectric continuum, mimicking the stabilizing effect of bulk solvation, and also included a number of explicit solvent molecules. The simplified models were also used to survey basis set effects on the optimized geometries. [CuII(Ac-HGGG-NH2)−2H] Structure Calculations. Initial DFT calculations were carried out on a simplified model of the PrP octarepeat region with the sequence HGGG (Figure 1A). The peptide termini were capped with an N-terminal acetyl and C-terminal amide to preserve the charge-neutral amide linkages. The peptide sequence was deprotonated at the backbone amide N-atoms from the first and second Gly residues and the structure was coordinated to Cu2+ in agreement with the consensus copper coordination mode for this region of PrP under high occupancy conditions.28,34,51,52 A series of alternate [CuII(Ac-HGGG-NH2)−2H] geometries were generated and optimized to assess the preferred peptide conformation for Cu2+ binding (Figure 2, G1 through G6), with calculated parameters summarized in Table 1. The first two structures, G1 and G2, are structurally analogous to the crystal structure reported by Burns et al.,34 and differ in the orientation of the first peptide moiety (rotation about the His ϕ angle), which also has an effect on the orientation of the His imidazole ring at the B3LYP/6-31G(d) level. The most stable geometry G1 has the His imidazole ring

very nearly coplanar with the copper coordination plane, whereas G2 is +16.3 kJ/mol higher in energy than G1. Distorting the peptide backbone such that the N-terminal end is oriented below the copper coordination plane (G3 and G4) imposes a considerable energetic penalty of nearly 40 kJ/mol relative to G1, despite both imidazole rings being nearly coplanar with the copper site. Coordinating the first amide carbonyl O-atom to the axial position below the coordination plane (G3) does not result in any net stabilization of the complex. In the G1 and G2 geometries the His Cβ moiety is directed below the coordination plane, whereas in G3 and G4 this group lies roughly in the copper coordination plane. In G5 and G6 the Cβ group is directed above the coordination plane, which significantly alters the orientation of the His imidazole ring, bringing it nearly perpendicular with the copper coordination plane, with both structures lying slightly more than 40 kJ/mol higher in energy than the G1 geometry. Overall, G1 appears to be the preferred conformation of the peptide backbone and the Cu2+ coordination site are in agreement with the crystal structure for this portion of the peptide.34 Basis Set Effects on Cu2+ Coordination Geometry. The basis set chosen for geometry optimizations does display some deviation from the expected geometries for Cu2+, most notably a D2d-type distortion in some 4-coordinate square planar coordination complexes.29,51 Based on recent successes with the LANL2DZ basis set for reproducing the expected coordination geometries for Cu2+ complexes53 we have also assessed each of the G1 through G6 geometries by performing geometry optimizations at the B3LYP/LANL2DZ level. Based on our methods for calculating relative free energies, these structures can be related to the B3LYP/6-31G(d)-optimized structures as well, however, comparisons are most meaningful if 13830



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Aside from the placement of the apical solvent and orientation of the Trp residue there are no other significant differences between the G7 and G8 structures. The calculated large basis set single point energy (SPE) for G8 is only −0.59 kJ/mol below the large basis set SPE of G7. The increased stability by 38 kJ/mol of the extended G8 conformation arises from a combination of vibrational entropy (contributing −21.58 kJ/mol to the total free energy, as -TΔS), and the larger number of anticipated conformations (R ln(n)), which contributes −14.33 kJ/mol to the calculated free energy difference. Comparing these two complexes further, the free energy of solvation (ΔΔG(solv)), as well as the translational and electronic components of the total entropy are equivalent, whereas the rotational entropy of the elongated conformation contributes a further −1.60 kJ/mol in favor of G8. Examining an extended network of waters, either above or below the coordination plane, both involving Trp indole Hbonding, reveals that the above-plane water network is more favorable by 9.3 kJ/mol. This structure may be preferred due to the more favorable local H-bonding environment above the coordination plane. Below the coordination plane the most readily available H-bond partners from the peptide are the deprotonated amide N-atoms and the carbonyl O-atoms from the first and second Gly residues, and these interactions destabilize the apical interaction of water with the Cu2+ site, resulting in their dissociation (3.98 Å for the closest solvent in G10 versus 2.29 Å in G9). Including the Trp residue in the DFT calculations, effectively calculating the same structure used in the original X-ray crystallography experiments,34 provides a somewhat more favorable binding environment for a water molecule in the apical position. Inclusion of continuum solvation was required in order to obtain the desired structure as the solvent readily dissociates in the gas-phase and H-bonds elsewhere. Many of the optimized geometries which included additional explicit water molecules gave rise to significant distortions in the Cu(II) coordination environment (not shown) as a result of direct Hbonding interactions with the deprotonated amide nitrogens. Furthermore, initial geometry optimization of a deprotonated water (hydroxyl) to the Cu(II) coordination sphere (not shown) results in a tetrahedral Cu(II) coordination environment, which was sufficiently different from the expected square planar coordination geometry that further such models were not pursued. Aside from structures that include explicit water molecules directly coordinated to the Cu(II) center we did not pursue more elaborate models involving further explicitly defined waters. Cu+ Structure Calculations. The reduced form of copper binding to the single OR peptide fragment was modeled using a combination of 4-methylimiadzole (MeIm) donors and explicit solvent. The in silico coordination chemistry derived in related work29 was used to inform the choice of models herein. The previous work was supplemented with additional models, including models of coordination via the His Nε-atom of the imidazole ring. As will be presented, the best candidate structure matching the EXAFS data corresponds to a 2coordinate complex, with a single peptide coordinating the Cu(I) center through the His Nε-atom, with the metal coordination completed by a water molecule: [CuI(MeIm)ε(OH2)]+ (G11, Figure 2). Without additional stabilizing ligand interactions for the metal center, coordination via the Nε-atom is preferred over Nδ coordination. Comparing the free energies for

made relative to structures generated with the same level of theory. In some cases the B3LYP/LANL2DZ-optimizations converged to structures slightly different from those shown in Figure 2, but for the most part the major conformational changes are maintained. At the B3LYP/LANL2DZ level almost all of the structures had smaller distortions in the Cu2+ coordination environment (smaller D2d-type distortions) and the His imidazole moiety was very nearly coplanar in almost all cases, even for structures optimized at the B3LYP/6-31G(d) level that showed notable out-of-plane orientations. Both optimization methods find that the G1 geometry is the most stable, whereas the B3LYP/ LANL2DZ optimized structures tended to have a smaller spread in relative free energies. At this level the coordination of the first amide carbonyl was still unfavorable by +23.3 kJ/mol, while reorientation of the His Cβ moiety in G5 and G6 required 10.8 and 20.9 kJ/mol, respectively, relative to G1 at the same level of theory. As was found for the B3LYP/6-31G(d)-optimized structures, the B3LYP/LANL2DZ-optimized G1 structure remained the most favorable, whereas comparing the G1 structures generated from each of the optimization methods finds that the B3LYP/631G(d) is the best overall structure. This is likely due to the 631G(d) basis set performing better overall for optimizing the peptide geometry, than the LANL2DZ basis set, as judged by the large basis set single point energies calculated for each structure (Table 1). [CuII(Ac-HGGGW-NH2)−2H]·n(H2O) Structure Calculations. With the core structure of the Cu2+ coordination environment established (G1, Figure 2) the involvement of additional apical solvent and the role of the adjacent Trp indole were modeled using the G1 structure as a starting point. The [CuII(Ac-HGGGW-NH2)−2H]·n(H2O) structures included 0, 1, 2, or 3 explicit water molecules, various potential conformations and H-bonding interactions for the Trp side chain, and each was optimized in the presence of a reaction field (e.g., mimicking the long-range stabilizing effect of solvation), with representative structures G7 through G10 shown in Figure 2. Attempts to optimize these structures with 1 or 2 explicit waters occupying the axial positions was problematic, due primarily to the fact that they are relatively weakly bound. Dissociation of the waters from the apical coordination sites and formation of H-bonding interactions elsewhere within the models is more favorable and occurs readily during geometry optimizations, however, within bulk solvent these other Hbonding sites would likely be occupied and the remaining sites would most likely be the less favorable and weakly coordinating apical positions. Nevertheless, it was possible to obtain stable optimized geometries with solvent occupying the apical coordination sites, however, only one such water could be maintained during geometry optimizations and attempts to stabilize additional water to the second axial site always resulted in one of the water molecules fully dissociating. The 2 water structures, therefore, were not optimized to completion and are not shown. Optimization of [CuII(Ac-HGGGW-NH2)−2H]·(H2O) in a coordination geometry closely resembling the crystal structure, including the apically bound solvent and H-bonded Trp indole (G7) generated a structure that is 38.7 kJ/mol higher in energy than G8, which has the water molecule occupying the same position, albeit at a slightly shorter Cu−O distance, and with the peptide backbone and Trp side chain fully extended away from the Cu2+ coordination site (i.e., fully solvent exposed). 13831



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[CuI(MeIm)δ(OH2)]+ versus [CuI(MeIm)ε(OH2)]+ the latter is preferred by 6.3 kJ/mol. As we have presented elsewhere,29 exchange of the coordinating solvent for an additional MeIm ligand via its Nε-atom affords a net stabilization of 46.3 kJ/mol, however, the OR peptide employed herein has only one His residue available to coordinate copper, and as equivalent concentrations of copper and peptide were used there are no further stabilizing ligands available to coordinate to the reduced metal center. Upon photoreduction of the Cu(II) center to Cu(I) it is expected that the central copper atom will tend to favor a reduced coordination number, thereby affecting significant reorganization of the coordination environment. The EXAFS experiments are carried out on solution samples at 10 K, and therefore only minimal reorganization of the system is possible from the initial Cu(II) coordination geometry. Partial spinrestricted geometry optimization of the Cu(II) model G1 was performed with Cu(I) as the central atom and two apical water molecules included in the starting structure. The geometry optimization was not completed, as the water molecules tend to dissociate during the optimization, leading to SCF convergence errors before reaching completion. The partial optimization effectively represents the structural preference of the system in the gas phase at zero K, and is expected to be indicative of how the surrounding coordination environment would relax in the presence of Cu(I) (G12, Figure 2). From the partially relaxed Cu(I) structure the most significant changes from the Cu(II) structure occur within the primary coordination sphere. The His Nδ- and the deprotonated Gly N−-atom trans to the imidazole lie at similar distances of 1.87 Å and 1.88 Å, respectively, while the intervening Gly N−-atom and the second Gly carbonyl Oatom are 1.90 Å and 3.06 Å from the metal center, respectively. The Cu(I) complex G12 appears to tend toward a 3-coordinate T-shaped complex, which may be an intermediate geometry on the way to a diagonal 2-coordinate complex. In the T-shaped geometry the intervening Gly N−-atom is maintained due to structural constraints imposed by coordination at His Nδ and the second amide N−-atom. The position of the other imidazole heavy atoms are 0.11 Å (∼3%) closer than they are in the corresponding Cu(II) complex G1, while the adjacent atoms in the peptide backbone for the second and third Gly are 0.2 Å − 0.9 Å further away. The partial optimization also reveals that any solvent which may lie at the apical positions of the central copper are readily lost and would therefore be unlikely to lie in an orientation consistent with metal-coordination and would more likely reorient themselves to maximize stabilizing Hbonding interactions in their immediate environment. X-ray Absorption Near Edge Spectroscopy. Initial monitoring of Cu(II) XAS data collected at SSRL beamline 7−3 revealed that photoreduction of Cu(II) was occurring in situ during data acquisition. The effect is readily apparent in the Cu K-edge near edge spectrum when comparing the first scan of Cu(II)OR with scans after exposure for ∼4 h (Figure 3A). The initial spectrum shows no apparent signs of photoreduction and the distinctive 1s→3d pre-edge peak at 8979.4 eV is indicative of a 3d9 Cu(II) species. Subsequent scans of the same region of the sample reveals increasing contribution of a dipole-allowed 1s→4p transition, with concomitant loss of the well-defined 1s→3d peak (Figure 3A). The photoreduced spectrum is similar to the near edge spectrum reported by Pushie et al.,29 resembling a 3-coordinate Cu(I) species.54 To determine whether photoreduction contributed to significant

Figure 3. Comparison of the copper K-edge near edge spectrum (A), EXAFS spectrum (B), and EXAFS Fourier transform (C) for the Cu(II)OR and photoreduced CuOR species.

changes in the EXAFS spectrum the minimally photoreduced EXAFS spectrum was compared with the photoreduced spectrum. The EXAFS spectrum (Figure 3B) reveals subtle changes in the intensity and shape the EXAFS oscillations, which manifests as a marked decrease in the magnitude of the primary backscattering peak in the EXAFS Fourier transform (Figure 3C), as well as more subtle shifts in the position of the multiple scattering peaks between 2.5 Å and 4.7 Å. These results indicate that structural changes within the sample occurred, concomitant with photoreduction, signaling a need 13832



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Figure 4. Deconvolution and curve fitting of Cu(II)OR EXAFS data, showing only the primary backscattering EXAFS (A) and EXAFS Fourier transform (B), as well as the EXAFS and EXAFS Fourier transforms for the longer range scattering contributions without the primary backscattering contributions (C and D, respectively).

data by r = π/2Δk, where Δk is the k-range of the data used in curve fitting analysis. By way of example, for a k-range of 15.75 Å−1, where the EXAFS curve fitting uses the k-range from 1.0 − 15.75 Å−1, Δk is 14.75 Å−1 and r is nearly 0.11 Å. This value of r means that similar backscattering distances would have to be greater than 0.11 Å to potentially resolve them during data analysis, while backscattering distances smaller than this should be represented using a single backscattering shell with a higher coordination number (N) instead. Adding a scattering path for a longer range light atom at ∼2.3 Å (e.g., representing an apical water molecule) slightly improves the fit to the data, however, the σ2 parameter for both shells remains relatively large (∼0.0065 Å2). The σ2 parameter represents the combined static (σ2stat) and vibrational (σ2vib) disorder associated with the scattering path. Apical solvent is at a longer distance and is also more weakly bound than the equatorial ligands, which would justify larger σ2stat and σ2vib parameters. Conversely, the spread in bond lengths for the more tightly bound equatorial donor atoms would also justify a smaller σ2vib and a relatively larger apparent σ2stat. By way of example, calculating the vibrational contribution to σ2, described in reference 50, for the Cu−Nε, based on a force constant of 85,000 kJ/mol/nm2 gives σ2vib ≈ 0.0020 Å2, which is a reasonable value for a primary backscattering shell. Examining the individual EXAFS contributions, however, it is apparent that some of the improvement in the fit with the

for improved sample preparation and data acquisition to minimize these contributions. More detailed analysis of the photoreduced species is described following the Cu(I) EXAFS results. Beating the Theoretical Resolution Limit of the EXAFS Data. Abrogation of significant photoreduction allowed the acquisition of EXAFS spectrum that best represents the Cu2+ form of component 1. In order to simplify the initial fitting of the EXAFS data the primary backscattering contributions were separated from the longer range scattering interactions by windowing on the EXAFS Fourier transform above 2.3 Å, backtransforming the data, and subtracting the contributions from the parent EXAFS data. The transforms isolated the EXAFS contribution arising from the primary backscattering interactions alone (Figure 4 A and B). Initial EXAFS curve fitting of the primary backscattering contributions yielded 4 light atom backscatterers at a distance of 1.96 Å from the metal center, with a relatively large Debye− Waller parameter (σ2) of 0.0063 Å2 (Table 2). The fitted distance is a reasonable approximation for the average N/O coordination distance for the coordinating atoms that lie in the equatorial positions,29,34,51 and is in close agreement with data published by Morante, et al.36 The theoretical resolution (r) of EXAFS data describes the ability to differentiate backscattering distances for similar backscattering atoms (C, N and O, or P, S and Cl, for example) and is related to the k-range of the EXAFS 13833



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Table 2. EXAFS Curve-Fitting Parameters for CuII(AcPHGGGWGQ-NH2)−2Ha pathb

N

R

σ2

ΔE0

backscatterers be damped, whereupon we chose to explicitly model the individual backscattering paths based on distances from the DFT structures in Figure 2. By curve fitting the primary backscattering EXAFS contributions with individual paths corresponding to the anticipated bond lengths for component 1 (Figure 4 A and B) it is apparent that the longer equatorial Cu−O bond from the backbone carbonyl also destructively interferes with other backscattering paths, affording significantly improved σ2 parameters for these shells. Further addition of a fifth backscattering atom at a distance consistent with representing an apical water molecule continued to improve the overall fit, albeit with a relatively large σ2 as might be expected for a weakly coordinated group in an apical position. Due to the cancellation between the long Cu−O interaction and the closer backscattering ligands the contribution of the apical water is not apparent in the EXAFS Fourier transform and the primary backscattering peak also has diminished amplitude, and is slightly less than would otherwise be expected for a 4-coordinate complex (hence the large σ2 fit parameters). Based on the informed choice of backscattering distances from the DFT calculations, which are also consistent with the published crystal structure (Table 3), it was possible to significantly improve the fit to the EXAFS data, as well as obtain more reasonable fit parameters (Table 2), using individual backscattering paths which were below the theoretical EXAFS resolution limit of 0.11 Å. Deconvoluting EXAFS Multiple Scattering Contributions. Multiple scattering interactions are most probable when adjacent atoms are colinear or very nearly colinear, with angles in the 160−180° range. The Cu−N−−C−O and Cu−O−C−N dihedral angles for the three interacting amide groups in the OR peptide were found to be 174.4 ± 2.4°, 166.7 ± 6.4° and 177.6 ± 2.1° for the first, second and third amide C-terminal to the anchoring His residue (Figure 1C), respectively.55 The coordinating amide moieties are within the range where multiple scattering interactions would be expected, and a significant portion of the surrounding peptide environment can be reasonably fitted to the EXAFS data. The longer range backscattering interactions were separated from the parent EXAFS data by windowing on the EXAFS Fourier transform from 0 to 2.3 Å, backtransforming the data, and subtracting the contributions. The EXAFS contributions arising from the longer range single and multiple scattering interactions, without the primary backscatterer single scattering path contributions, are shown in Figure 4 C and D. In order to assess whether there was an appreciable contribution to the EXAFS multiple scattering interactions from the peptide backbone coordinated to the Cu2+ center a multiple scattering calculation was performed on the immediate coordination environment of the G8 DFT structure. From the list of scattering paths that were generated those corresponding to single or multiple scattering paths involving the primary coordination sphere were removed, as were the scattering paths which included the imidazole ring. The remaining peptide backbone scattering paths were fit to the backtransformed data in Figure 4 C, with the EXAFS Fourier transform (Figure 4 D) clearly demonstrating that portions of the multiple scattering interactions can be attributed to the peptide backbone, particularly the backscattering peak at ∼4.6 Å in the EXAFS Fourier transform, which cannot be modeled without backbone coordination.56

F

Cu(II)OR, Simplified Model Cu−N/O 4 1.958(3) 0.0063(2) −2.6(5) 0.4215 Cu(II)OR, Model 1, with Apical Coordination Cu−N/O 4 1.958(3) 0.0065(3) −2.8(6) 0.4034 Cu−O 1 2.34(1) 0.0065 Cu(II)OR, Model 2, with Apical Coordination Cu−N/O 3 1.949(3) 0.0049(5) −1.8(6) 0.3984 Cu−O 1 2.03(2) 0.0067 Cu−O 1 2.34(3) 0.014 Cu(II)OR, Model 3, with Apical Coordination Cu−N/O 4 1.957(3) 0.0055 −10.5(7) 0.3747 Cu−O 1 2.305(6) 0.0032 Cu···C 3 3.33(4) 0.018 Cu·N·C·N 2 3.12(3) 0.003 Cu·N·C·C 2 4.03 0.003 Cu(II)OR, Imidazole and Primary Coordination (Figure 5A,B) Cu−N 1 1.922(3) 0.0035(2) −7.0(6) 0.3092 Cu−N 1 1.948 0.0037 Cu−N 1 1.954 0.0037 Cu−O 1 2.015 0.0033 Cu−O 1 2.303(7) 0.0052 +31 Additional unlisted single and multiple scattering shells Cu(II)OR, Full Multiple Scattering Model (Best Fit, Figure 5C,D) Cu−N 1 1.938(2) 0.0027(2) +1.7(4) 0.2667 Cu−N 1 1.974 0.0028 Cu−N 1 1.929(7) 0.0028 Cu−O 1 2.028 0.0025 Cu−O 1 2.326(9) 0.0083 +63 Additional unlisted single and multiple scattering shells Cu(I)OR, Imidazole and Primary Coordination (Best Fit, Figure 6B,C) Cu−N 1 1.85(3) 0.003(2) −8.1(7) 0.4126 Cu−O 1 1.89(3) 0.004 +18 Additional unlisted single and multiple scattering shells Photoreduced CuOR, Full Multiple Scattering Model (best fit, Figure 7) Cu−N 1 1.884(3) 0.0021(4) +1.2(5) 0.2683 Cu−N 1 1.942 0.0021 Cu−N 1 1.989 0.0021 Cu−O 1 2.046 0.0021 Cu−O 1 2.301 0.0092 +51 Additional unlisted single and multiple scattering shells a Coordination numbers N, interatomic distances R (Å), Debye− Waller factors (the mean-square deviations in interatomic distance) σ2 (Å2), and threshold energy shifts ΔE0 (eV). The values in parentheses are the estimated standard deviations in the last digit obtained from the diagonal elements of the covariance matrix. The fit-error function F is defined by F = (∑k6(χ(k)calcd − χ(k)expt)2/∑χ(k)expt2)1/2, where χ(k) are the EXAFS oscillations and k is the photoelectron wavenumber given by k = ((2me/ℏ2)(E − E0))1/2. bAll paths terminate at the originating Cu atom (not indicated).

additional 2.3 Å scattering interaction is due to cancellation of a portion of the EXAFS oscillations arising from the closer backscattering shell. Regardless of the fit parameters it was not possible to obtain an improved fit for the primary backscattering interactions and σ2 remained relatively large throughout the preliminary EXAFS curve fitting procedure. The combination of large σ2 and improved fitting with the destructively interfering longer range backscattering path indicated that the best fit to the experimental data required that the EXAFS oscillations arising from the N = 4 13834



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Table 3. Summary of Key Copper−Peptide Bond Lengths from Experiment and Calculationa bond

crystal structureb

EXAFSc

EXAFS,d this work

G6, no apical ligand

G9,e apical H2O

G2, n − 1 C(O)-bound

G12, copper(I)

Cu−NεHis Cu−NGly1 Cu−NGly2 Cu−OGly2 Cu−Oapical

1.991 1.996 1.921 2.066 2.380

1.957 1.957 1.957 1.957 2.305

1.974 1.938 1.929 2.028 2.326

1.973 1.928 1.878 2.102

2.023 1.960 1.924 2.128 2.292

2.024 1.957 1.892 2.145 2.283

1.870 1.967 1.896 3.057

a

Bond lengths reported in Å. bBurns et al.34 cMorante et al.36 dFrom this work, with the caveat that the initial scattering paths were generated from a DFT-optimized structure with inequivalent primary coordination sphere bond lengths. eDFT geometry optimization in the presence of a reaction field (ε = 78.39) and three water molecules.

Figure 5. Results of the EXAFS curve fitting for Cu(II)OR, using the inset structures in full multiple scattering calculations for the primary backscattering and imidazole atoms (A and B), where arrows indicate under fitted regions of the EXAFS spectrum, and the more complete structural model which gave the best overall fit to the data (C and D).

EXAFS Curve Fitting of Cu(II)OR. The EXAFS spectrum of Cu(II) bound to the single octarepeat peptide fragment (Figure 5 A) is dominated by a single backscattering peak at ∼1.96 Å in the EXAFS Fourier transform (Figure 5 B), as well as numerous multiple scattering interactions from ∼2.5 Å to ∼4.9 Å. Scattering contributions not only from the backbone are likely to contribute to the multiple scattering, as shown in Figure 4 C and D, but the coordinated His imidazole is also expected to contribute many single and multiple scattering paths. Full multiple scattering calculations were carried out using the heavy atom core of structure G8, which included the primary coordinating atoms and the intact methyl imidazole

ring, which coordinates via the Nδ-atom. Fitting the truncated coordination model to the EXAFS data (Figure 5 A and B), which includes individual scattering paths for each of the primary backscattering paths and reasonable parameters (Table 2) gives a weighted F-factor of 0.3092. This fit is already significantly better than the other initial fits to the data shown in Table 2; however, some of the scattering contributions to the EXAFS are not well fit, as indicated by arrows and the residual EXAFS oscillations in Figure 5A. It is also apparent from the EXAFS Fourier transform that the backscattering peaks at ∼2.8 Å and ∼4.5 Å are represented with the chosen structural model. Using the heavy atom framework from G8 and performing a full multiple scattering calculation from the backbone His 13835



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Figure 6. Comparison of the Cu(I) K-edge near edge spectra for OR and OR4 (A) as well as the best fits for the EXAFS (B) and EXAFS Fourier transforms (C), generated from the inset structure.

reasonable values for primary coordinating atoms. As described above, for Figure 4A, the primary backscattering atoms are not all in phase with one another and this leads to some slight cancellation in amplitude, which would otherwise force a large σ2 value if these bond lengths were grouped into a single shell. The fifth backscattering path, attributed to a bound water at ∼2.3 Å, was also best modeled with a relatively large Debye− Waller parameter of 0.0083 Å2, with the justification that this ligand is relatively weakly bound and therefore its position is not well-defined. The inclusion of the fifth backscattering path, however, always improved the EXAFS fit even though the presence of this atom is not apparent in the EXAFS Fourier transform (Figure 5B and D) due to significant cancellation with the EXAFS oscillations from the other primary backscattering atoms. The combination of more realistic Debye− Waller parameters for the primary coordinating atoms, as well as an informed selection of bond lengths from DFT calculations, suggests that the improved structural description is valid. This improved fit to the EXAFS data also yielded the smallest residual from any of the curve fitting attempts (Figure 5 C). To rule-out the possibility that the EXAFS curve fitting procedure was overfitting the data, by virtue of the additional scattering paths presented by the larger peptide model, all single and multiple scattering interactions involving the peptide backbone can be selectively removed. This simplified model is

amide N-atom to the Cα-atom of the third Gly residue, as well as an apical solvent molecule generates 68 scattering paths within the imposed criteria (see Methods). Before curve fitting with such a large number of scattering paths careful consideration must be given to the number of degrees of freedom afforded with the collected EXAFS data. The maximum number of independent relevant points (NI) can be calculated by NI = [(2 Δk ΔR)/π] +2, where Δk and ΔR are the k-range and R-space over which the data is modeled, respectively. In our EXAFS curve fitting analysis it is the EXAFS photoelectron wave vector that is fit, while the data in R-space is inspected visually for goodness of fit; ΔR, on the other hand, represents the spatial extent of the fitted EXAFS model. The k-range of the data is 1.02−15.75 Å−1, while a reasonable range of R-space is 1.5−5 Å, giving an estimate of 34 for NI. In the full multiple scattering fit of the EXAFS data only select groups of distances (R) were allowed to vary, along with σ2 for the first backscattering shell (all subsequent Debye− Waller parameters scaled uniformly as a function of σ2 for the first shell), and a single ΔEo for the experiment, which yields a total of 15 variables used in the fit procedure. Using the larger G8-derived structure for curve fitting gave the best fit overall to the EXAFS data (Figure 5 C and D), with a weighted F-factor of 0.2667. The fitted model includes the separate scattering paths for the primary backscattering atoms, with σ2 values in the range of 0.0025−0.0028 Å2, which are 13836



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Figure 7. Results of EXAFS curve fitting, generated using full multiple scattering from the inset structure, showing the EXAFS (A) and EXAFS Fourier transform (B).

identical to the fit shown in Figure 5 A and B. The EXAFS Fourier transform backscattering peak at ∼2.9 Å in Figure 5 D that was most difficult to fit with the full model. This peak arises primarily due to the combined contributions from imidazole multiple scattering and single scattering interactions with the first and second Gly Cα-atoms. These numerous scattering interactions contributing to the peak at 2.9 Å both constructively and destructively interfere, which substantially complicates the curve fitting analysis. Further substantiating the choice of a relatively large structural model is the backscattering peak at ∼4.6 Å in the Fourier transform (Figure 5 B). As described for Figure 4 D, this backscattering contribution can only be modeled with a combination of scattering paths involving atoms in the peptide backbone and is absent in the EXAFS Fourier transform of the model that excludes these portions of the structure (Figure 5 B). The EXAFS data clearly contains contributions from peptide backbone single and multiple scattering interactions, while the parameters obtained from EXAFS curve fitting, shown in Table 2, are all within justifiable ranges, as are the remaining 63 additional scattering paths used in the model (listed in Supporting Information). The relatively large σ2 for the long Cu−O scattering path, attributed to an apical water molecule, is consistent with the relatively weaker binding in this position. The small molecule X-ray crystal structure clearly demonstrates electron density attributable to a solvent molecule in the apical position, whereas solution EPR experiments suggest that any such apical solvent is weakly bound and highly exchangeable.34 X-ray Near Edge and EXAFS Curve Fitting of Chemically Reduced Cu(I)OR. Using sodium ascorbate as a mild reducing agent with Cu(II)OR led to formation of Cu(I)OR, as evidenced by the intense 1s→4p pre-edge peak in the Cu Kedge near edge spectrum (Figure 6A). The 1s→4p peak is a dipole allowed transition which gains intensity in centrosymmetric environments such as 2-coordinate digonal complexes of Cu(I).29 The intensity of the 1s→4p transition for Cu(I)OR in Figure 6A is compared with the intensity arising from Cu(I) bound to the Ac-(PHGGGWGQ)4-NH2 peptide (abbreviated OR4),29 with the latter pre-edge clearly more intense than the former. This implies that the Cu(I)OR coordination environment has lower symmetry than Cu(I) bound to the OR4 peptide.

Elsewhere we report that Cu(I)OR4 is best modeled as a digonal 2-coordinate complex bound by two His imidazole rings via their Nε-atoms.29 The EXAFS Fourier transform in Figure 6C demonstrates somewhat ambiguous multiple scattering peaks from 2.3 to 4.5 Å, however, the first two EXAFS oscillations in Figure 6B clearly demonstrate the diagnostic pattern arising from His imidazole multiple scattering contributions. As the OR peptide only contains a single His imidazole the multiple scattering calculations were performed using a single imidazole moiety and the second coordination site occupied by solvent, with the best fit provided by the [CuI(MeIm)ε(OH2)]+ structure, G11, from DFT calculation. The primary backscattering distances from curve fitting were slightly longer than those from the DFT structure calculations (Table 2), however, this trend has been observed elsewhere for digonal Cu(I) complexes29 and may be due to solvation effects, alternate coordination modes, or facile interactions with nearby solvent that are not represented in the gas-phase DFT optimizations. Attempts to fit the EXAFS data with other structural models, such as [CuI(MeIm)δ(OH2)]+ generated poorer fits to the EXAFS data (weighted F-factor =0.4336 compared with 0.4126 for G11), and was ruled out as not significantly contributing to the EXAFS spectrum. EXAFS Curve Fitting of the Photoreduced CuOR Species. Applying the same methods described above for Cu2+ coordinating to the OR peptide the spectrum of the photoreduced species was analyzed in an analogous manner. A total of six ∼45-min scans of the photoreduced sample were collected (k-range 1−12 Å−1), with the first scan omitted from averaging as the copper near edge demonstrated a moreclearly defined 1s→3d transition, indicative of 3d9 Cu(II) (Figure 3A). The photoreduced EXAFS data (Figure 7 A) shows a very similar environment to that of the minimally photoreduced data (Figure 3B). The primary backscattering peak in the EXAFS Fourier transform shows a marked decrease in magnitude but little change in peak width (Figure 3C).57 This comparison indicates that the Cu−L bond lengths are comparable between the two forms, within the precision of the data (r = 0.14 Å), and that the photoreduced species has a reduced coordination number. Photoreduction is common for Cu(II) species in these types experiments, which is largely the result of radical species 13837



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generated within the ice matrix.40 The exact conditions for species to be more or less susceptible to photoreduction are not fully understood, however, the incident energy, flux density, and duration of exposure to the X-ray beam are prime factors. For EXAFS curve fitting of the photoreduced data, Figure 7A and B, the best fit is obtained with the partially DFT-optimized Cu(I) structure G12, shown in Figure 2, where the apical solvent molecules were removed from the structure for multiple scattering calculations. The multiple scattering interactions were calculated using a maximum Nleg of 4, Rmax of 5 Å, with a plane wave approximation cutoff of 15% and 10% for curved wave, generating a total of 56 single and multiple scattering paths. The EXAFS curve fitting gives very close agreement to the G12 Cu−N bond lengths from DFT calculation, however, the other single scattering distances attributed to Cu−O interactions were at shorter distances than expected from the DFT calculation and more consistent with the Cu(II)OR form. The subtle structural changes that are associated with relaxation of the peptide structure in the presence of Cu(I) (described in the DFT section above) are also well matched to the EXAFS Fourier transform in Figure 7B. Overall the DFT model of the photoreduced CuOR species yields an exceptionally good fit to the EXAFS data (weighted F-factor of 0.2683), with reasonable backscattering distances and σ2 parameters (listed in Table 2 and Table S1 in Supporting Information). Putting the Current EXAFS Results in Context. There have been several previous investigations of similar Cu2+ coordination to PrP fragments using XAS, including characterizations of the component 1 form.36−39 Like the current investigation, all of the previous EXAFS studies of Cu(II) coordination to the PHGGGWGQ region of PrP has benefitted from the X-ray crystal structure as a target in EXAFS data analysis, however, none of the previous work unequivocally extracts the structural information indicative of component 1type coordination. The current EXAFS data does not assume this type of coordination a priori, and the work clearly elucidates the characteristic structure of Cu2+ coordination into the local peptide backbone through rigorous DFT structure calculations and EXAFS curve fitting, as opposed to inferring that component 1-type coordination is represented by the data. Each of the previous studies will be briefly reviewed, chronologically, in order to place the current methods of analysis and the results in context. The first EXAFS characterization of Cu(II) coordination to the OR peptide was described by Morante, et al. nearly a decade ago.36 In their experiments, the copper-to-peptide ratio was 1:1 and conditions were maintained at pH 7.4 (7.5 herein), with a k-range of ∼2.5−14 Å−1 (with a theoretical resolution of 0.14 Å). The authors report that Cu K-edge near edge spectra were compared for consistency, and although the pre-edge regions of their near edge spectra are difficult to discern it appears that there is no appreciable contribution from photoreduced species in their data.36 The EXAFS data was best fit with 4 Cu−N/O backscattering interactions at 1.97 Å, with σ2 of 0.006 Å−1,36 which is very close to our initial model presented in Table 2. Their choice to group these backscattering paths into a single shell is the default standard (and correct) procedure for EXAFS curve fitting within the theoretical resolution limit of the data. Morante et al. attribute the EXAFS Fourier transform peaks beyond the primary backscattering peak as being largely attributable to His scattering interactions.36 There is, however, no detailed

inclusion of scattering contributions from the peptide backbone included in their analysis, aside from 3 nleg = 3 scattering interactions that the authors attribute to paths involving “Cu...C(Gly)” at ∼3.3 Å. From their curve fitting, however, these scattering paths have an exceptionally large σ2 (0.009 − 0.018 Å2), although further justification for this significantly damped contribution are not given. Based on FEFF scattering calculations and EXAFS curve fitting our results reveal 4 scattering paths between ∼3.2 and ∼3.4 Å, although these are not all attributable to interactions with the Gly Cα or carbonyl carbons. Although Morante et al. are correct, that such scattering paths are possible,36 this only touches on the full extent of the scattering contributions of the local peptide backbone to the EXAFS data. Their data is also k2-weighted and will tend to under-emphasize the higher k-range compared to our presented data, which is k3-weighted. The lower kweighting also makes the residual from their curve fitting look slightly better and de-emphasizes the high k-range noise compared to the data presented herein. The series of publications by Parak and colleagues37−39 will be considered collectively here as each paper presents the identical EXAFS spectrum for Cu(II) coordination to AcPHGGGWGQ-NH2. In their initial study, EXAFS data is collected on the Cu(II)OR species at pH 7.0, as opposed to pH 7.5 herein, with a k-range of ∼3−11 Å−1.37 The lower pH likely means there are additional contributions from N2O2 equatorial Cu2+ coordination, in addition to the expected N3O1 primary coordination environment (excluding consideration of apical solvation).32 As the Cu K-edge near edge spectrum is not shown it is not possible to determine whether photoreduction contributes to their Cu(II) EXAFS data. Only minimal EXAFS curve fitting parameters are given, such as bond lengths and the coordination number, but no additional parameters to judge the goodness of fit or the validity of the fit parameters. The author’s fit the EXAFS data to 2 Cu−N backscattering interactions at 1.95 Å, one of which they attribute to belonging to the His imidazole, 1 Cu−N interaction at 1.91 Å, 1 Cu−O interaction at 2.00 Å and one additional Cu−O interaction at 2.35 Å − again, no additional fit parameters are given such as σ2. We have conclusively demonstrated that improved fits are indeed obtained by separating the primary coordination shell into contributions from multiple backscattering paths beyond the theoretical EXAFS resolution limit (Table 2 and Figure 4 A and B), however, the authors in the 2005 paper give no justification for working beyond the theoretical resolution of their data (r = 0.20 Å).37 These previous studies do not identify the specific multiple scattering contributions from the local peptide backbone in the EXAFS data and only make brief mention of the backscattering peaks at ∼3 and ∼4 Å as characteristic of imidazole coordination. The multiple scattering peaks in the EXAFS Fourier transform, as well as the higher frequency oscillations, which add structure to the first two oscillations in the EXAFS spectrum, are indeed indicative of His imidazole coordination, however, we clearly demonstrate that the peptide backbone contributes additional structure to the EXAFS spectrum. As this is the case, the assertion in del Pino, et al.38 and Weiss, et al.39 that the number of additional coordinating His can be fit to their EXAFS data may also need to be revisited. Using EXAFS fit parameters (i.e. R) to deduce angles for a coordinating imidazole is highly likely to give misleading structural information. We have investigated the thermodynamics of various orientations for a coordinated imidazole and found that only very small distortions are 13838



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energetically feasible and that these distortions are unlikely to give rise to EXAFS contributions that can be fit reliably and are much closer to the inherent noise in most EXAFS data.

Structure calculations revealed that there was no preference for the interaction of the Trp side chain with the metal binding site in the DFT calculations, and the extended conformation G8 was preferred over the more compact H-bonded complex represented by G7. The conformation of the Trp residue is likely dynamic and the close association observed in the crystal structure may be attributable to crystal packing forces.34 Although a similar structure has been reported for Trp indole H-bonding to the shell of local water molecules in the vicinity of the Cu2+-binding environment,37 this is also the first work to investigate and directly compare the free energy associated with various Trp orientations. The high occupancy coordination mode for Cu2+ coordination to the PrP octarepeats results in a highly structured, charge neutral, and relatively planar coordination geometry and represents the primary coordination mode in vivo under high copper concentrations for this region of the protein.

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CONCLUSIONS Photoreduction of Cu(II) was identified as a significant problem at the outset of these experiments and contributions to the total EXAFS spectrum from photoreduced copper were minimized through a combination of glycerol-free sample preparation and moving to shorter data acquisition times on multiple sample volumes. Structural characterization of photoreduced species is not routinely done, and we have also provided new structural insight regarding the chemically reduced form of copper bound to the OR region of PrP. We have identified that the in situ photoreduction of Cu(II) during EXAFS data collection results in contributions from an intermediate 3-coordinate Cu(I) center. Photoreduction undoubtedly contributes in other published Cu(II) EXAFS data, which will tend to skew EXAFS curve fitting parameters and give an inaccurate picture of coordination structure. Because in situ photoreduction of aqueous Cu(II) species is dependent on incident photon flux, the high flux densities on modern beamlines suggests photoreduction may contribute in other published Cu(II) EXAFS data and will continue to be an issue on modern high flux EXAFS beamlines. Solvent coordination to component 1 Cu(II) is likely facile, as evidenced from DFT calculations. The best fit to the EXAFS data was obtained by including a single water molecule at 2.30 Å, and this site is best represented as highly disordered (as evidenced by the large σ2 f it parameter). There is no evidence from the EXAFS curve fitting for more than 1 apical water ligand. EXAFS curve fitting cannot differentiate the two apical coordination sites, and an oxygen backscattering atom in either position provides an equally good fit to the experimental data. The DFT structure calculations, however, could only find stable structures for a water molecule above the coordination plane, as represented by G7 and G8. Comparing G8 with the same structure without the explicit solvent molecule (not shown) reveals that in the gas-phase the apical water is bound by 4.3 kJ/ mol, as calculated at the B3LYP/6-311+G(2df,2p) level. With the implemented method for calculating ΔG(aq), however, the formation of G8 from separate species is slightly unfavorable, by 3.4 kJ/mol. The coordination mode of the component 1 form of the PrP octarepeat region is highly amenable to EXAFS analysis, due in large part to the approximately planar coordination environment which facilitates multiple scattering out to ∼5 Å from the copper center. The EXAFS spectrum represents contributions from all copper species present in solution, and fortunately recent potentiometric titration data indicates that component 1-type coordination appears to be the predominant form at pH 7.5.32,33 This work demonstrates the wealth of chemical and structural information underlying the XAS data for Cu(II)OR, which has not been exploited in previous work. The combination of DFT structure calculations, combined with full multiple scattering calculations and EXAFS curve fitting, yields exceptional structural and thermodynamic insight into the Cu2+ coordination to a single PrP octarepeat in solution. To our knowledge this is also the first detailed computational study of His side chain and imidazole orientation to be investigated for component 1-type binding since the early work in this area by the corresponding author.51

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ASSOCIATED CONTENT

* Supporting Information S

Table S1, listing all scattering paths used in EXAFS curve fitting analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +01-306-966-8592. Fax: +01-306-966-8593. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an Operating Grant from the Canadian Institutes of Health Research (CIHR, G.N.G.) as well as the Natural Sciences and Engineering Research Council of Canada (NSERC, G.N.G.) and the Saskatchewan Health Research Foundation (SHRF, G.N.G.). M.J.P. is supported by Fellowships from CIHR and SHRF. M.J.P. is also supported by CIHR-THRUST (CIHR-funded Training in Health Research using Synchrotron techniques) Fellowships. Research at the University of Saskatchewan was supported by a Canada Research Chair award (to G.N.G.), the University of Saskatchewan, the Province of Saskatchewan, and SHRF. Portions of this work were also carried out at the Stanford Synchrotron Radiation Lightsource which is funded by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE, Office of Biological and Environmental Sciences, and by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. Computing resources for DFT calculations were provided by WestGrid and Compute/Calcul Canada.

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ABBREVIATIONS EXAFS = extended X-ray absorption fine structure XAS = X-ray absorption spectroscopy DFT = density functional theory PrP = prion protein OR = a single octarepeat fragment: Ac-PHGGGWGQ-NH2 MeIm = 4-methylimidazole dx.doi.org/10.1021/jp408239h | J. Phys. Chem. B 2013, 117, 13822−13841


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New Insights into Metal Interactions with the Prion Protein: EXAFS

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