Structure, Dynamics, and Electron Transfer of Azurin Bound to a Gold

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Structure, Dynamics and Electron Transfer of Azurin bound to gold electrode Anna Rita Bizzarri, Chiara Baldacchini, and Salvatore Cannistraro Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01102 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Structure, Dynamics and Electron Transfer of Azurin bound to gold electrode Anna Rita Bizzarri1, Chiara Baldacchini1,2, Salvatore Cannistraro1 1 2

Biophysics & Nanoscience Centre, DEB, Università della Tuscia, Viterbo, Italy IBAF-CNR,Porano, Italy

ABSTRACT

Blue copper redox protein Azurin (AZ) constitutes an ideal active element for building bio-nanooptoelectronic devices based on the intriguing interplay among its electron transfer (ET), vibrational and optical properties. A full comprehension of its dynamical and functional behaviour is required for efficient applications. Here, AZ bound to gold electrode via its disulphide bridge was investigated by a Molecular Dynamics simulation approach taking into account for gold electron polarization which provides a more realistic description of the protein-gold interaction. Upon binding to gold, AZ undergoes slightly changes in its secondary structure with a preservation of the copper-containing active site structure. Binding of AZ to gold promotes new collective motions, with respect to free AZ, as evidenced by essential dynamics. Analysis of the ET from the AZ copper ion to the gold substrate, performed by the

Pathways model, put into evidence the main

residues and structural motifs of AZ involved in the ET paths. During the dynamical evolution of the bio-nano system, transient contacts between some lateral protein atoms and the gold substrate occurred; concomitantly the opening of additional ET channels with much higher rates was registered. These results provide new and detailed insights on the dynamics and ET properties of the AZ-gold system, by also helping to rationalize some imaging and conductive experimental evidences and also to design new bio-nano-devices with tailored features.

Keywords Azurin; Electron Transfer Redox proteins; Molecular Dynamics Simulation; bionanodevices

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Introduction The blue copper protein Azurin (AZ) has gained a large interest thanks to its peculiar redox, optical and vibrational properties,1–4 coupled to biorecognition capabilities towards its physiological partner Cytochrome c551, or other biomolecules including the oncosuppressor p53.

5–8

More

specifically, AZ is characterized by a very efficient and fast electron transfer (ET) , through a pathway encompassing the polypeptide chains and involving the lone tryptophan residue at position 48 (Trp48).1,9 The active site of AZ contains a copper ion (Cu2+) which is coordinated to five aminoacid residues according to a characteristic distorted trigonal bipyramidal geometry;10,11 with such an ion being able to switch between two stable oxidation states (Cu1+/Cu2+) during the ET process. This peculiar symmetry gives rise to the ligand field energy levels responsible for both the intense Ligand-to-Metal Charge Transfer (LMCT) absorption band and to the possibility of a fine vibrational tuning of the protein redox potential.2 Notably, an intriguing interplay among the ET process, the optical absorption transitions, and the active site vibronic modes characterizes the AZ functionality

4,12–15

. Furthermore, AZ is endowed with both a remarkable robustness and a native,

exposed disulphide bridge which allows the protein to be quite strongly bound to gold surfaces; while its active site is facing upwards, being thus available to possible interactions with other molecules or physiological partners. 16 All these features make AZ an ideal active element for building bio-nano-devices, suitable for a variety of applications in biosensing, memory storage, and bio-opto-eletronics.14,17–21 Accordingly, large efforts have

been devoted to investigate the structural, ET, conductive, and optical

properties of single AZ molecules bound to bare or functionalized gold, or even to other conductive surfaces, such as Indium Tin Oxide or Highly Oriented Pyrolytic Graphite, by Scanning Tunnelling Microscopy (STM) and Atomic Force Microscopy (AFM), also under electrochemical control (ECSTM, EC-AFM), and Conductive-AFM.22–33 However, the relationship

among

the

protein

structure and dynamics, the topographical arrangement on the substrate and the corresponding ET and conductive properties have not been fully elucidated, even though it represents a long standing issue for a reliable implementation of AZ in nano-devices. In this respect, atomistic Molecular Dynamics (MD) simulations, allowing to follow the dynamical evolution, at atomic resolution, of a biomolecule bound to gold, could constitute an extremely useful tool, even to investigate some physical properties that may not be experimentally accessible. 34 Although MD simulation approach has been widely applied to investigate AZ in water (see e.g. refs.35–37), a few studies have been performed on the AZ-gold system because of the difficulty to treat the protein-gold interaction which, may play a crucial role in the regulation of both the structural and conductive properties of the immobilized biomolecules.38,39 Rigorous protein-gold dynamical simulations would require


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quantum-mechanical ab-initio approaches, whose implementation is however

practically

prohibitive as due to the complexity of the involved systems and consequent huge computing time. A reasonable compromise could be the introduction of the electron polarization of the gold atoms in the framework of the classical MD simulations.40,41 Implementation of such an approach in the GROMACS package has allowed us extract

a collection of physical parameters

from five

independent 100 ns long trajectories of hydrated AZ bound to an Au(111) surface, through its unique disulphide bridge. Analysis of the data has provided a significantly novel set of structural, dynamical and ET properties of AZ bound to gold. The overall results are discussed also in connection with the corresponding experimental data available in the literature; with some of them being still debated. Moreover, the results could be rewarding for a deeper understanding of the AZ behaviour when it is anchored to gold, also in the perspective of an efficient embedding of the biomolecule into bio-nano-devices with tailored features.

Materials and Methods Modelling of AZ bonded to the gold substrate Initial atomic coordinates of AZ were taken from the X-ray structure at 1.93 Å resolution (chain B of PDB entry 4AZU) (Fig.1A).11 The Cu2+ ion is coordinated by three strong equatorial ligands (N of His46 and of His117 and S of Cys112) and two weaker axial ligands (S of Met121 and the backbone oxygen of Gly45) in a peculiar distorted trigonal bi-pyramidal geometry.10,11 According to previous works, the active site of AZ was modelled by introducing covalent bonds between the Cu2+ ion and S of Cys112, N of His46 and of His112; the elastic constant having been fixed to 110 kJ/(mol*nm2) for all the three bonds. The partial charges of the Cu2+ ion and of its ligands were set as in refs. 42,43 The Au(111) flat substrate was modelled by hexagonally arrangement the gold atoms in a cluster of three layers, each one of 22 x 25 atoms; with the nearest neighbour distance being fixed at 2.88 Å.39,44 AZ was anchored to the gold substrate through a covalent bond between the sulphur (S ) atom of Cys26 belonging to the AZ disulphide bridge (Cys3-Cys26) and an Au atom located at the centre of the first layer (Fig.1B). The choice of binding the S of Cys26 to gold, was dictated by the higher accessibility of this atom to the external solvent with respect to the S of Cys3, as found by MD relaxation. The initial distance of 6.1Å between the S and the Au atoms was progressively reduced by a MDbased docking procedure to reach the required final distance value of 2.5 Å. To reliably treat the interaction between the protein and the gold substrate, polarization of gold was introduced by implementing the Rod Model40 in the GROMACS Force Field.45 Briefly, a virtual site, free to rotate, was added to each Au atom at a fixed distance l of 0.05 nm. Opposite electric charges q and -q (with q = 0.1 e) were assigned to the gold and to the virtual site atoms, respectively. Accordingly, ACS Paragon Plus Environment

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a dipole moment with fixed modulus given by q*l =8∙10-31 Cm and free to re-orient in space upon its interaction with the surrounding atoms was generated.

MD simulations of free and bound AZ MD simulations of hydrated AZ (free AZ) and hydrated AZ bound to a polarisable gold substrate (bound AZ) were carried out by the GROMACS 4.6.5 package,46,47 including GROMOS96 43a1 Force Field for the protein (van Gunsteren, et al., 1996), modified as above mentioned, and the SPC/E for water 48. MD runs of free AZ and bound AZ were performed by centering the protein in a rectangular box with dimension of 7.98 x 7.48 x 7.48 nm3, and of 7.10 x 5.50 x 7.80 nm3, respectively, filled with water molecules. Water molecules, whose distance from any atom of the protein molecules was less than 0.23 nm, were deleted. To ensure the neutrality of the system, three water molecules were replaced by three Na+ ions. The final free AZ system contains 1234 protein atoms, a Cu2+ ion, three Na+ ions and 14191 water molecules.

The final bound AZ-gold system

contains 1234 protein atoms, a Cu2+ ion, three Na+ ions, 1650 gold atoms, 1650 virtual sites, and 8375 water molecules. In all the simulations, the cut-off radius of both electrostatic and van der Waals interactions were set at 0.9 nm and the neighbour list relative was updated every 10 steps. The Particle Mesh Ewald (PME) method49,50 was used to calculate the electrostatic interactions with

a lattice constant of

0.12 nm and using fourth order cubic spline interpolation. H bonds were constrained with the LINCS algorithm.51 Periodic Boundary Conditions in the NPT ensemble with T=300 K and P = 1 bar, with a time step of 1 fs were used. The Nosé-Hoover thermostat method was used to control the system temperature, with a coupling time constant T =0.1 ps,52 while constant pressure was imposed using the Parrinello-Rahman extended-ensemble,53 with a time constant P = 1.0 ps. After energy minimization, the systems were heated from 50 K to 300 K by using two intermediate steps at 150 K and 250 K. Five 100 ns-long simulations were carried out by using different initial conditions for the velocity distribution. The analysis of the secondary structure of the protein was carried out by DSSP.54 The corresponding figures were created with Pymol and VMD.55,56

Essential dynamics analysis The essential dynamics (ED) approach, derived from on the Principal Component Analysis (PCA), allows to extract from MD simulations the dimensional subspace describing the most relevant motions. 57,58 Briefly, the ED method is based on the diagonalization of the covariance matrix built from atomic fluctuations in a trajectory from which the overall translation and rotations were removed:


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𝐶𝑖𝑗 = 〈(𝑋𝑖 − 𝑋𝑖,0 )(𝑋𝑗 − 𝑋𝑗,0 )〉

(1)

where X are the separate x, y, and z coordinates of the atoms fluctuating around their average positions X0; the < > brackets representing the average time over the entire trajectory.

The

covariance matrix was calculated by using the protein C atom trajectory, which contains all the information for a reasonable description of the protein large concerted motions.

57

From the

diagonalization of the covariance matrix, a set of eigenvalues and eigenvectors was obtained. The motions along a single eigenvector correspond to concerted fluctuations of atoms. Additionally, the eigenvalues represent the total mean square fluctuation of the system along the corresponding eigenvectors. ED was carried out by using the utilities in the GROMACS package 4.6.5. 46

Analysis of the ET properties The ET properties of AZ were analyzed in the framework of the Pathways

model.59–61 Starting

from the classical Marcus theory, the ET rate between a Donor (D) and an Acceptor (A), in the weak coupling limit, can be expressed by: 62 k ET

2 2    VR  h  k BT

1/ 2

  



e

 G   2 4  k BT

(2)

where VR is the electronic tunnelling matrix element between D and A,  is the reorganization energy G° is the driving force of the reaction, T is the absolute temperature, h the Planck constant and kB the Boltzmann constant. The dependence of kET on the medium between D and A is mainly reflected by VR, which can be cast in the form: VR2  VRo 2 f M2 , where VRo represents the electronic coupling between D and A in van der Waals contact, and fM is a dimensionless attenuation factor directly taking into account for the dependence of the ET process on the medium. Under the assumption of an activation-less process (G°=-), an estimation of the maximum ET rate can be easily obtained from Eq.2, which assumes the form k ET ~ A f M2 , where the pre-factor A includes the Franck-Condon term and ranges in the (1014 -1016) s-1 interval.63 In the framework of the Pathways model, the ET path from D to A in a given molecule, is assumed to be a combination of three types of steps a covalent bond (C) between atoms, a hydrogen bond (H), and a jump through free space (S). 59 Accordingly, fM can be expressed as: NC

NH

NS

i

j

k

f M    iC   Hj   kS

(3)

where NC, NH, NS are the number of C, H and S paths, respectively; i being the corresponding attenuation factors. Semi-empirical expressions for the -factors are:60

 C  0.6;

 H  0.6e1.7( R  0.28) ;

 S  0.6e1.7( R  0.14) 


(4)

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where R is the distance between D and A; generally, a path through a covalent bond providing a higher attenuator factor, and then a higher ET rates. Accordingly, the best D to A pathway for a given snapshot of the analysed molecule can be extracted by evaluating fM for all the possible paths through C-H-S steps, and finding out the path that maximizes the fM term and, then, the electronic coupling. The pre-factor was fixed to be 1014 s-1.

64,65

Calculations of the ET properties

were

carried out by using the HARLEM program.66 Results and discussion Structure and topological arrangement of AZ on the gold substrate Fig.2 (B-F) shows AZ bound to gold

at the end of the five independent 100 ns-long runs, together

with a representative structure of free AZ after a 100 ns long trajectory (Fig.2A). The temporal evolution of the protein during the dynamics has been monitored by following the Root Mean Square Displacement (RMSD) from the initial structure, the gyration ratio, the Surface Accessible Surface (SAS), and the number of protein-protein H-bonds. We found that that after the initial relaxation from the crystallographic structure, all the analysed quantities assume almost constant values indicating that the simulations are quite stable (see Figs.S1-S4). The -helix and the -sheet percentage contents, evaluated from the structure obtained by averaging the conformations over the last 100 ps, are also shown. We note in free AZ a slight reduction of the -helix content with respect to the X-ray structure (see Fig.1A), and a substantial preservation of the -sheet content. These small changes in the secondary structure of free AZ find a correspondence with what is commonly found in the MD simulations of proteins and can be ascribed to the relaxation of the crystal constraints.35,67 Both the -helix and the -sheet contents of bound AZ are found smaller than both those of the X-ray structure and free AZ. Additionally, a significant variability from run to run is observed. In particular in Runs 1, 4 and 5, a reduction of the -helix content and, to a lesser extent, of the -sheet one occurs. In Run 2, the -sheet content is reduced and the -helix is preserved. Finally, in Run 3, both the -helix and the -sheet contents are almost the same as those of free AZ. The

trajectory variability of bound AZ

can be traced

back to the interaction of the protein atoms with the gold dipoles whose orientation can rapidly change according to the protein dynamics, affecting in turn the protein structure. Fig.2 also shows that AZ can assume different vertical positions with respect to the gold plane. Since the orientation of the protein on gold could have a direct impact on its ET response, we have monitored the angle formed by a virtual axis ideally joining the Cu2+ ion and the almost opposite S atom of Cys26 bound to the gold plane (see Fig.2B). Indeed, such an angle may provide a schematic representation of the overall orientation of the protein with respect to gold: to a lower


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value of this angle corresponds a higher tilting of the biomolecule towards the surface, and consequently a lower height of the protein on gold. The temporal evolution of the angle for the five runs is shown in Fig.3. In Run 2,  remains constant around the initial value of about 50°, while in the other runs, it undergoes an initial decrease followed by a trend which differs from run to run. Interestingly, in Run 3, and less evidently in Runs 1 and 4, quick tilting of the axis is observed during the simulation run, while concomitant contacts among protein atoms with gold atoms from the substrate occur. Surprisingly, the temporal evolution of the angle well correlates with that of the electrostatic energy of the protein-gold interaction. A bistable trend between a lower energy value (about -360 kJ/mol) for the lying-down configuration and a higher value (about -330 kJ/mol) for the standup configuration has been observed (see Fig.S5). Even if an energy difference of 30 kJ/mol, is much lower than the total energy, which is of the order of -4.5·105 kJ/mol for Run 3 (and almost the same for the other runs (see Table S1)), it is, however, one order of magnitude higher than the hydrogen bond energy, which is about 3 kJ/mol.68 Accordingly, it is reasonable to assume that such a difference could play a role in driving the preferential orientation of the protein with respect to the gold substrate. The orientation variability of AZ with respect to the gold plane can give rise to different overall heights of the AZ molecules on the gold substrate, whose values can be directly compared with experimental data from AFM imaging. Accordingly, we have determined the height of AZ by evaluating the distance (DThr61-Au) between the Thr61 residue located at the top of the protein (see green spheres in Fig.2B) and the gold plane. Such an approach is different from that used in previous works where the molecule was represented as an ellipsoid;

39,44

however it appears more

suitable for comparison with AFM measurements, in which an interaction force between the tip and the highest portion of the molecule is considered. The DThr61-Au distance, as sampled from snapshots collected every 0.5 ps for all five runs, ranges from 3.2 nm, corresponding to an almost stand-up configuration, to 2.0 nm which complies

with a partially laying down configuration. The

corresponding histogram of the simulated DThr61-Au distances (Fig.3, bottom panel), is characterized by a single mode distribution, centred at about (2.6 ± 0.5) nm; with the slight skewing towards low values pointing out a higher probability for a partially laying down configuration. It is worthy to mention that an AFM imaging work32 on AZ bound to bare gold in fluid has put into evidence the presence of two populations of molecules, one

centred at 2.7 nm, consistent with the protein

molecules in an almost stand-up configuration, and another one at 1.1 nm corresponding to molecules almost lying-down on gold, very likely attributable to a denatured form of the protein. On the other hand, an AFM experiment has revealed the presence of a single mode height


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distribution centered below 1.7 nm with a standard deviation of 0.5 nm which was traced back analogously to the presence of partially denatured biomolecules on gold.25,69 Therefore, our data are essentially consistent with the experimental AFM data referring to bound AZ molecules which substantially preserve their native structure, but assuming a variety of different orientations with respect to the substrate. The occasional occurrence of a direct contacts between AZ and gold atoms may have some effect on the conductance of the AZ-gold system, as it will be analysed in the following. We have previously monitored the distance between the gold substrate and the atoms located in the bottom region of AZ which are more likely candidates to come into a direct contact with the gold. Indeed, we found that the C atoms of both Gln8 and Glu91 (pink and blue spheres, respectively, in Figs.1 and 2)

come into a close contact with the gold substrate during the dynamical run. Fig.4 shows

the temporal trend of these two distances in the five runs. After an initial reassessment of the protein on gold, in Runs 1, 2 and 4, the DGlu91-Au distance (blue line) is found to be always higher than 0.5 nm. Instead, at about 15 ns in Run 5, the DGlu91-Au distance reaches a

0.25 nm value,

which maintained in time together with reduced fluctuation amplitudes. Such a value, which roughly corresponds to the sum of the van der Waals radii for the two atoms, indicates that C(Glu91) is practically in contact with gold. On the other hand, in Run 3, DGlu91-Au exhibits rapid jumps between about 0.25 nm and 0.55 nm, reflecting a switching of the C (Glu91) atom from a contact to a non-contact position, respectively. Globally, a ratio of 2.2 between the total time that the protein spends in the lying-down configuration and that in the stand-up one has been calculated. We found moreover that the residence times of the protein are about LD=4.9 ps and SU=2.9 ps, for the lying-down and the stand-up configurations, respectively. By using these times in framework of the Arrhenius equation (LD-1=A e-ELD/kT and SU-1=A eESU/kT, where ELD and ESU are the activation energies for the lying-down to the stand-up transition and viceversa, respectively), we found a slightly higher activation energy for

the transition from lying-down to stand-up

transition with respect to the opposite one. This result would likely imply a more favourable interaction of the protein with gold with respect to that of the protein with the solvent. 68 Concerning the DGln8-Au distance (pink line), we observe a temporal trend rather similar to that of DGlu91-Au, however, with slightly higher values and amplitudes, indicating that an effective contact of this atom with the gold surface is not reached.

Local and global dynamics


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Anchoring of AZ to gold could affect both the local and global dynamics of the molecule. To investigate the dynamics, at atomic level, we have analysed the RMSF of the AZ C  atoms, by considering the last 50 ns of Run 1, during which AZ is in an almost stand-up configuration, and the last 50 ns of Run 5, where the molecule is almost lying-down on gold; for comparison the RMSF of free AZ

has been also followed. Fig. 5 shows that the three RMSF curves possess

rather similar profiles, although with different peak intensities. Higher RMSF values are observed at turns and bends, consistently with the higher degrees of freedom of these regions with respect to those characterized by secondary structure motifs. The C atoms close to the anchoring region (see the arrows in Fig.5) are characterized by low RMSF values, as

due to the presence of the

disulphide bridge which stabilizes the protein structure.70 The most marked differences in the RMSF of free and bound AZ are found in the proximity of the -helix; with this suggesting a stronger dynamical response of this region upon binding the protein to gold. Information on the collective motions of the biomolecule has been from the ED analysis of the eigenvalues and the eigenvectors of the covariance matrix (Eq.1) of the C atom fluctuations, by taking into account the same trajectories used for the previous RMSF analysis. 58,71 Fig.6 shows the RMSF of the protein (free and bound) C atom, projected on the eigenvectors corresponding to the first five eigenvalues and ranked in a decreasing order; the percentages of the eigenvalues to the total being also reported. Globally, the first five eigenvalues indicate that the amount of collective motions correspond to 52% and 54% of the total for Run1 and Run 5 of bound AZ, respectively, and to 49% for free AZ. This means that a slight effect on the collective dynamics is registered upon anchoring AZ to the gold surface. Progressively less marked differences among the RMSF curves for eigenvalues from 1 to 5 are observed. For Run 1, , the first eigenvalue corresponds to collective motions involving residues located far from the anchoring region, as sketched in Fig.7A (blue line); with these motions being enhanced with respect to free AZ. For Run 5, the first eigenvector corresponds to collective motions involving a large portion of AZ located at the opposite region with respect to the anchoring one and including the region close to the -helix portions, as sketched in Fig.7B (blue lines). In summary, when the AZ molecule is in a lying-down configuration, the collective motions of residues close to gold appear to be restricted with a concomitant enhancement the motions of residues located far from gold, suggesting that anchoring to a substrate can slightly affect the intrinsic dynamics of the biomolecule.

Vibrational properties of the AZ active site


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To check the preservation of the AZ active site, we have focussed our attention on the vibrational features of the copper-ligand bonds, which directly reflect the peculiar geometry of the active site and regulate both the LMCT absorption band and the fine vibrational tuning of the characteristic redox potential.2 We have followed the approach previously developed for another blue copper protein, Plastocyanin, and based on the extraction of these vibrational features from the power spectrum of the temporal fluctuations of the Cu-S(Cys) bond length.72,73 It was shown that the corresponding spectral content encodes the vibrational properties of the active site, and is strongly reminiscent of the experimental resonance Raman spectrum obtained by exciting the strong LMCT band,

73,74

Accordingly, we have calculated the power spectrum of the Cu-S(Cys112) bond length

fluctuations for both the free and bound AZ, through the Fourier Transform (FT) of the correlation function, according to: 𝑇

𝑆(𝑓) = ∫0 〈𝐷(0)𝐷(𝑡)〉 𝑒 2𝜋𝑖𝑓𝑡 𝑑𝑡

(6)

where the brackets < > indicate the correlation function, f is the frequency, T represents the integration time interval and D(t) is the distance between the Cu and S(Cys112) atoms as a function of time t. The power spectrum has been calculated by the Maximum Entropy Method (MEM).75,76 Fig.8 shows the result obtained by averaging a collection of 100 power spectra from the five runs of free AZ (red dashed line), and bound AZ (black continuous line), respectively. Remarkably, the general features of two averaged simulated spectra appear quite similar. The slight difference in both the intensity and peak positions can be attributed to the different sampling of the vibrational modes in the two systems, likely due to the different coupling with the surrounding matrix. However, the

good qualitative agreement between the these two spectra indicates that the main

features of the AZ active site geometry

are largely preserved upon anchoring AZ to gold; with

such a result deserving significant relevance for applicative aims based on the involvement the AZ active site. Additionally, the two simulated power spectra are closely reminiscent of the experimental resonance Raman spectrum of free AZ in solution, shown in the inset of Fig.5. In particular, we note a close correspondence of the main peaks of the simulated spectra with the intense Raman bands in the 350-450 cm-1 region, arising from the Cu-S(Cys112) stretching, and also with the weaker band at 220-270 cm-1 assigned to Cu-N stretchings.4,74We should note that a straightforward comparison between the experimental and simulated spectra cannot be done for several reasons: i) the Cu-S bond vibrations are, in the real AZ active site, somewhat coupled with the surrounding matrix; ii) the average over the ensemble is far from being completely reached in the simulated spectra, as investigated in refs.44,72,73 Nonetheless, the rather good qualitative similarity between the

simulated and experimental spectra indicates that the simulation ACS Paragon Plus Environment

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generation of the active site vibrational modes is able to capture the essential vibrational features; i.e. the main peaks in the 250-450 cm-1 region that characterize the experimental Raman spectrum (see inset in Fig.9). ET properties of AZ bound to the gold substrate To pave the way towards an efficient implementation of AZ in bio-nanodevices, the ET properties of the biomolecule bound to a conductive electrode should be better investigated.31,77 Generally, these properties are strictly related to the protein structure, dynamics, conformational fluctuations and interaction with the substrate. Therefore, appropriate MD simulations may greatly help to disclose these properties which, in turn, can be compared with those inferred from experimental data at level of single molecule. We have investigated the intramolecular ET

between the Cu2+ ion, which is assumed to be the

electron acceptor (A), towards that atom of AZ being in contact with the gold substrate, which acts as the electron donor (D). More specifically, the Cu2+ ion constitutes an intermediate step of the ET process, with the electron being transferred to the biomolecular partner, or to a metallic tip when a physiological ET process is mimicked and investigated experimentally by STM (with a negative bias of the gold electrode with respect to the tip).14,16,19,20,32,78 Accordingly, the electron will flow through the protein matrix towards the gold substrate, by eventually following different pathways 1,79,80

depending on both the instantaneous AZ structure and its topological arrangement on gold. As

possible donors we have taken into consideration two different atoms: i) the S atom of Cys26 belonging to the disulphide bridge (Fig.1B,orange sphere), which is covalently bound to a gold atom of the electrode; with the latter constituting

the expected source for the electron from the

metal electrode; ii) the C atom of Glu91 (Fig.1B, blue sphere) which is located at the bottom part of the protein; with this atom having been observed to frequently come into contact with the gold substrate during the protein dynamics and thus representing a possible alternative source for the transferred electron (see the above Section). Analysis of the ET process has been carried out by the Pathways model describing the electron flow through the protein matrix in terms of a combination of covalent and hydrogen bonds and jumps through free space (see Materials and Methods). For the Cu-S(Cys26) couple, we have analysed the ET process for all the runs, while for CS(Cys91) couple we have taken into consideration only snapshots in which the donor is in contact or near-contact with gold. Analogously to what has been done for the ED analysis, we compare the results from Run 1 (the last 50 ns), in which the protein is in an almost stand-up configuration, and from Run 5 (again the last 50 ns), in which the protein is lying-down on gold. Results of the Cu-S(Cys26) couple for the other runs, reported in Table S2, are in average almost the same as that of Run 1. ACS Paragon Plus Environment

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For the two analyzed D-A couples, Table 1 reports: i) the average and the standard deviations of the D-A distances (column 2); ii) the corresponding number of steps from the collected snapshots (column 3); iii) the minimum and maximum value of the ET rates (column 4). It can be noted that the ET rates related to the paths from Cu2+ to C(Glu91) are much higher than those corresponding to the Cu2+-S(Cys26) couple; a much smaller variability of the ET rate

throughout the

simulation being also registered for the former couple. Both effects can be traced back to the much shorter distance from the Cu2+ to C(Glu91) with respect to that connecting Cu2+ to S(Cys26) (see column 2). Indeed, for a shorter D-A distance, a lower number of steps together with a reduction of the number of accessible paths are expected, according to data reported in column 3. Notably, the difference in the ET rates for the two analysed D-A couples, finds a correspondence with the MD simulation results from ref.38 in which AZ, forced to lay flat on a gold surface, exhibits an enhanced ET rate with respect to AZ in an almost stand-up configuration. It should be remarked that the highest ET rate found for the Cu-S(Cys26) couple (kET =12 s-1) is of the same order of both the value determined by electrochemical impedance spectroscopy (kET =30 s-1),16 and that found by pulse radiolysis in bulk AZ solutions (kET = 44 s-1). 1 The large variability observed in the ET rate when the protein is in a stand-up arrangement (Run 1), could be due to a high sensitivity of the ET process on the protein structural details, as largely observed in redox molecular systems.81

64,82

More generally, such a feature might be related to

an “encounter complex” description according to which, redox proteins undergo a reassessment in their relative conformations before an optimized ET process with the partner occurs.83 Analysis of the ET paths from the Cu2+ to the S(Cys26) has revealed that Tpr48 is involved in about 40% of the total possible paths. Such a rather high recurrence is in agreement with experimental data indicating the important role played by Trp48 in the intramolecular ET pathway of AZ, 1,9 and also with fluorescence experiments showing quenching of Trp48 upon ET between AZ and its physiological partner cytochrome c551.8 Indeed, Trp48, which is located at about 10 Å away from the Cu2+ ion,

84

has been suggested to undergo a quenching of its fluorescence through either

energy transfer or ET from Trp48 to the metal ion. 85–88 Furthermore, the extensive involvement of Trp48 in the ET process is also consistent with recent STM measurements in which optical excitation of AZ anchored to a conductive substrate, in resonance with its LMCT absorption band, resulted in a light-induced current tunnelling within the protein milieu.14 We found that the AZ -sheet strands are largely involved in the ET paths, while the -helix region is almost absent. In particular, the Ser4, Val5, Asp6, Ile7 aminoacid residues, belonging to the S1 strand, 11 have been found to participate in more than 40% of the total number of paths. At least a free-space jump is present in almost all the ET paths between the Cu2+ and S(Cys26),


while

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paths through the H-bonds are always absent (see an example in Fig.9A); on the other hand, two or three free-space jumps are observed in about 30% of the total paths. This means that the structure and arrangement of the protein on gold do not allow a direct transfer of the electron to gold only through the chemical bonds within the protein matrix; explaining thus the rather lower values of the ET rates registered for this DA couple. When the dynamical behaviour of bound AZ leads to the establishment of a tight contact of the C(Glu91) atom

with the gold substrate, a

corresponding rate values paths

being registered

huge increase of the ET rate is observed; with

about 100 times higher than those observed for the Cu2+-S(Cys26) (Column 4). Remarkably, on the basis of these results, it could be

hypothesized that during cyclicvoltammetry measurements, the protein could more likely assume a lying-down configuration , probably at variance with what observed with impedance spectroscopy and pulse radiolysis.89 In other words, these results indicate that the dynamical evolution of AZ on gold may lead to the establishment of new ET paths characterized by very fast ET process, and then to a higher overall conductivity. The ET path corresponding to the D-A Cu2+-C(Glu91) couple, involves almost always Ile87, Gly88, Ser89, and Gly90; an example of ET path being shown in Fig.9B. In this case, paths through H-bonds are also absent, while free-space jumps are only rarely detected; with this probably contributing to the observed higher ET rate. Our results clearly point out that the ET process of AZ toward the electrode, may be strongly affected by the protein dynamical configuration sampling, according to the evidence that the frequent intermittent contacts to gold of protein atoms located closer to the copper ion can give rise to ET paths characterized by much higher rate, with a resulting increased conductivity. Opening of new ET channels characterized by high ET rate, might be at the basis of the intriguing current blinking behaviour measured by STM, on the top of single AZ molecules anchored to gold. While such a blinking was attributed to the formation of a direct contact between the tip and the molecule,

78,90

it could instead reflect the occurrence of jumps of the AZ molecule between

almost lying-down and stand-up configurations on the gold electrod with a concomitant switching between high and a low ET rates. Such a picture may be also consistent with the recent experimental evidence that the height of AZ on gold is modulated by the applied potential, which likely determines conformational and orientational changes of the protein. 28

Conclusions An atomistic MD simulation approach, encompassing an updated gold electron polarization model to take into account the protein-gold interaction, has been applied to investigate the structural, dynamical, spectroscopic and ET properties of the redox blue copper protein AZ bound to a gold


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substrate through its native disulphide bridge almost opposite to the active site. Five 100 ns long runs have allowed us to assess the stability of the simulation and to explore the

various

conformations assumed by the AZ protein with respect to the gold substrate. Although the AZ secondary structure motifs can be slightly modified upon binding to the gold electrode, we found that the active site vibrational modes are largely preserved suggesting that the protein could retain its biological functionality. The sampling of different topological arrangements of the AZ molecule with respect to gold, from an almost stand-up to a lying-down configuration, likely driven by electrostatic interaction between the protein and gold, gives rise to a distribution of the molecular height, in a good agreement with the experimental AFM imaging data. The ED analysis has put into evidence that anchoring to gold yields a reassessment of collective motions of the protein, with more marked effects when the molecule assumes an almost lying-down configuration on the gold substrate. The investigation of the ET process, performed in the framework of the Pathways model, has both provided a detailed mapping of the ET pathways between the Cu2+ ion and the gold electrode, and highlighted the aminoacid residues which are mostly involved in the ET paths. The occasional tilting of the protein onto the gold substrate as observed during the dynamical evolution of the system leads to the establishment of tight contacts between some protein lateral atoms with the gold electrode. This suggests the opening of new ET channels characterized by much higher ET rates with respect to those detected in AZ in stand-up configuration where an ET between the copper ion and the sulphur atom anchored to gold is found to be favourite. Such a finding offers a new ground for the explanation of conductive experimental data of the AZ-gold systems which are still debated and suggests ad hoc immobilization strategies for the design and development of new bio-nano-devices with tailored ET features. Finally, the heterogeneous structural and dynamical behavior of AZ anchored to gold, also opens the possibility to selectively exploit these features, in a controlled way, to enlarge the applicative fields of the AZ-gold system.

Acknowledgments This work was partly supported by a grant from the Italian Association for Cancer Research (AIRC No IG 15866) and by a PRIN-MIUR 2012 Project (No. 2012NRRP5J).

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Legends to Figures Figure 1. A) Graphical representation of the X-ray structure of the AZ; the percentages of the helix and -sheet secondary structure motifs, evaluated by DSSP program, being reported. B) Initial configuration of AZ anchored to Au(111) substrate through the sulphur sphere) from the disulphide group.

of Cys26 (orange

His46, Cys112 and Hys117, ligands of the Cu2+ ion in the

active site, are marked as red sticks. Water molecules are not shown.

Figure 2. A) Graphical view of free AZ at the end of a 100 ns run. B-F) Graphical view of AZ bound to gold

at the end of

the five

S(Cys(26) atom, anchored to gold,

100 ns long runs. The virtual axis connecting the

and the Cu2+ ion, is marked by a red arrow; with the

corresponding angle  formed by the virtual axis with the gold plane being also indicated as a dashed curve in panel B. The percentages of the -helix and -sheet motifs are reported. Water molecules are not shown. Figure 3. A-E) Temporal evolution of the  angle formed by the virtual axis S(Cys(26) atom, anchored to gold, and the Cu2+ ion

connecting the

(see Fig.2B) during the five runs.

F)

Histogram of the distance, DThr61-Au, between the Thr61 residue (green spheres in Figs.1 and 2) and the gold substrate, collected from the snapshots sampled every 0.5 ps during all the runs; the average value and the corresponding standard deviation being reported.

Figure 4. Temporal evolution of the distance, DGlu91-Au between the gold substrate and the C of Glu91 (blue line) and of the distance, DGln8-Au, between

the gold substrate and the Cof Gln8

(pink line) (see Fig.2) for the five runs.

Figure 5. RMSF of free AZ (black dashed line), AZ- gold Run 1 (olive line) and AZ- gold Run 5 (orange line) plotted as a function of C atoms; analysis having been carried out using a 50 ns long trajectory. Secondary structure elements are also shown. Figure 6. RMSF, projected in the subspace of the first five eigenvectors, of free AZ (black dashed line), AZ- gold Run 1 (olive line) and AZ- gold Run 5 (orange line) plotted as a function of C


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atoms; for both cases, analysis having been carried out using the last 50 ns of the trajectory. Secondary structure elements are also shown. Figure 7. Snapshots of backbone structure of AZ anchored to gold at the end of 100 ns trajectories, with the regions involving the collective motions of the first eigenvector marked in blue, for: A) AZ-gold, Run 1 and B) AZ-gold Run 5. The name of some residues along the blue marked regions are labelled.

Figure 8. Power spectra of the Cu-S(Cys 112) bond length of for AZ bound to gold (black line) and of free AZ (dashed red line), obtained by averaging 100 spectra from 5 ps time intervals with a time step of 2 ps. Inset experimental Resonant Raman spectrum of AZ in solution, for details see ref. 4. Figure 9. A) ET path (pink spheres) as evaluated by the Pathways model for: A) the Cu2+ ion and the S(Cys26)

(orange sphere) D-A couple; B) the Cu2+ ion and the C(Glu91) (blue

sphere) D-A couple; the Trp48 residue involved in the ET path being shown as a black sphere. The corresponding maximum ET rates are shown.


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FIGURES

FIGURE 1


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FIGURE 2


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FIGURE 3


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FIGURE 4


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FIGURE 5


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FIGURE 7


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FIGURE 8


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FIGURE 9


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TOC


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Figure 1. A) Graphical representation of the X-ray structure of the AZ; the percentages of the α-helix and βsheet secondary structure motifs, evaluated by DSSP program, being reported. B) Initial configuration of AZ anchored to Au(111) substrate through the sulphur of Cys26 (orange sphere) from the disulphide group. His46, Cys112 and Hys117, ligands of the Cu2+ ion in the active site, are marked as red sticks. Water molecules are not shown 81x60mm (300 x 300 DPI)


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Figure 2. A) Graphical view of free AZ at the end of a 100 ns run. B-F) Graphical view of AZ bound to gold at the end of the five 100 ns long runs. The virtual axis connecting the S(Cys(26) atom, anchored to gold, and the Cu 2+ ion, is marked by a red arrow; with the corresponding angle θ formed by the virtual axis with the gold plane being also indicated as a dashed curve in panel B. The percentages of the α-helix and β-sheet motifs are reported. Water molecules are not shown. 99x45mm (300 x 300 DPI)


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Figure 3. A-E) Temporal evolution of the θ angle formed by the virtual axis connecting the S(Cys(26) atom, anchored to gold, and the Cu2+ ion (see Fig.2B) during the five runs. F) Histogram of the distance, DThr61-Au, between the Thr61 residue (green spheres in Figs.1 and 2) and the gold substrate, collected from the snapshots sampled every 0.5 ps during all the runs; the average value and the corresponding standard deviation being reported. 68x50mm (300 x 300 DPI)


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Figure 4. Temporal evolution of the distance, DGlu91-Au between the gold substrate and the Cα of Glu91 (blue line) and of the distance, DGln8-Au, between the gold substrate and the Cα of Gln8 (pink line) (see Fig.2) for the five runs. 50x35mm (300 x 300 DPI)


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Figure 5. RMSF of free AZ (black dashed line), AZ- gold Run 1 (olive line) and AZ-gold Run 5 (orange line) plotted as a function of Cα atoms; analysis having been carried out using a 50 ns long trajectory. Secondary structure elements are also shown. 288x201mm (300 x 300 DPI)


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Figure 6. RMSF, projected in the subspace of the first five eigenvectors, of free AZ (black dashed line), AZgold Run 1 (olive line) and AZ- gold Run 5 (orange line) plotted as a function of Cα atoms; for both cases, analysis having been carried out using the last 50 ns of the trajectory. Secondary structure elements are also shown 108x60mm (300 x 300 DPI)


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Figure 7. Snapshots of backbone structure of AZ anchored to gold at the end of 100 ns trajectories, with the regions involving the collective motions of the first eigenvector marked in blue, for: A) AZ-gold, Run 1 and B) AZ-gold Run 5. The name of some residues along the blue marked regions are labelled. 108x60mm (300 x 300 DPI)


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Figure 8. Power spectra of the Cu-S(Cys 112) bond length of for AZ bound to gold (black line) and of free AZ (dashed red line), obtained by averaging 100 spectra from 5 ps time intervals with a time step of 2 ps. Inset: experimental Resonant Raman spectrum of AZ in solution, for details see ref. 4. 288x200mm (300 x 300 DPI)


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Cu2+

Figure 9. A) ET path (pink spheres) as evaluated by the Pathways model for: A) the Cu2+ ion and the S(Cys26) (orange sphere) D-A couple; B) the ion and the Cα(Glu91) (blue sphere) D-A couple; the Trp48 residue involved in the ET path being shown as a black sphere. The corresponding maximum ET rates are shown. 81x60mm (300 x 300 DPI)


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Structure, Dynamics, and Electron Transfer of Azurin Bound to a Gold

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