Abstract
Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β–β8 and β7–β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined β4–β7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.
Similar content being viewed by others
Introduction
In general, protein folding and unfolding are complex processes governed by the underlying energy landscape and may exhibit parallel pathways, multiple intermediates and transition states (TSs)1,2,3. Further, the diversity of cooperative effects which create delocalized or collective TSs present formidable challenges for experimental characterization of protein energy landscape. The advent of single-molecule techniques has made it possible to probe molecules one at a time and visualize their energy landscapes4,5,6,7. Molecular dynamics simulations have proven to be an extremely useful complement, providing high resolution atomistic level structural insights of processes probed by the single-molecule techniques8,9,10,11.
Metalloproteins or proteins with metal as a cofactor make up one-third of all known proteins12. Despite representing a significant fraction of the proteome, studies of metalloprotein dynamics and folding/unfolding transitions remain scarce13,14 and assessing the effect of metal cofactors on protein stability remains a fundamental challenge in the field. Single-molecule force spectroscopy (SMFS) and steered molecules dynamics (SMD) have previously been used to study metalloproteins and probe the effect of metal on the mechanical stability of proteins. Metalloproteins which have been studied include sodium/proton antiporter15, GB1 with engineered metal-binding sites16, calmodulin3, cadherin17, rubredoxin18, betaine symporter19, crystallin20, and cupredoxins21. The previous studies found diverse effects of the bound metal on the protein energy landscape. For instance, while metal-binding increased the mechanical stability of sodium/proton antiporter15, rubredoxin18, and crystallin20, the bound metal added new pathways for cadherin17 and rigidified betaine symporter19. Studies on GB1 with engineered metal-binding sites on the protein surface demonstrated how to map the mechanical unfolding transition state. The metal-binding sites that are positioned at the transition state enhance the mechanical stability of the protein whereas the sites away from it do not affect the mechanical stability16. In the case of azurin and plastocyanin, Beedle et al.21 reported complex unfolding features comprising of 2-state and 3-state pathways. The 3-state pathways were stochastic wherein the unravelling of the protein occurred from either termini. The rupture of intermediates in the 3-state pathways has been attributed to the breaking of Cu-Scys and Cu-NHis bonds. However, since the authors investigated a mixture of apo- and copper-bound (holo) azurin proteins, the effects induced by copper-binding on the protein unfolding energy landscape could not be directly ascertained. The authors interpreted their results using a model wherein the 2-state pathway was assigned to the apo-protein and the 3-state and 4-state pathways were attributed to the holo-azurin21.
Azurin, found in the periplasmic space of gram negative bacteria Pseudomonas aeruginosa, is a small 128-residue protein from the cupredoxin family22. It has a β-barrel structure, with 8 β-strands arranged in a double-wound Greek-key topology and one α-helix22. The structure contains a native disulfide bond between Cys3 and Cys26. Azurin is a metalloprotein with a type-I copper centre exhibiting a trigonal bipyramidal geometry with a central copper bound to two histidine imidazoles (His46 and His117), one cysteine thiolate (Cys112), two weaker axial ligands, sulphur of methionine (Met121) and carbonyl of glycine (Gly45)23. Proteins containing type-I copper centre exhibit bright colors in their absorption spectra. Azurin, in particular, shows a strong absorption band with maximum around 625 nm due to S(cys)→Cu2+ ligand-to-metal charge transfer transition24. The exact function of azurin and the role of copper are not known, although in vitro studies suggest that azurin may function as a redox shuttle, facilitating electron transfer in denitrification and/or respiration chains25,26. Azurin is an interesting metalloprotein system, where copper-binding does not change the protein structure22,23. It appears that the polypeptide fold of apo-azurin has a preformed copper binding site and the binding of copper induces very little change in both, the positions of residues at the metal binding site, and the overall structure of protein (see Fig. S1)26. Despite the negligible perturbation in protein structure, we demonstrate using SMFS and SMD that the copper ion (Cu2+) alters the unfolding energy landscape of azurin.
To better understand the full complexity of mechanical unfolding pathways of azurin, and the effect of the copper on metalloprotein stability, it is necessary to directly compare protein unfolding in the presence and absence of bound copper. Here, we prepare individual samples of apo- and holo-azurin polyproteins as verified using absorption, steady-state fluorescence and time-resolved fluorescence spectroscopy. Through a study of the mechanical unfolding of apo- and holo-proteins separately using SMFS measurements and SMD simulations, we directly probe the copper-dependent changes in the mechanical unfolding pathways of azurin. We show that apo-azurin unfolds through both 2-state and 3-state pathways, wherein the intermediate in the latter case ruptures through a structurally collective TS. In contrast, holo-azurin unfolds through 2-state, 3-state, and 4-state pathways. While copper-binding does not change the first TS, it makes the protein unravel through different intermediates. The first intermediate unfolds via a structurally more localized TS relative to that for the apo-azurin intermediate. In the 4-state unfolding pathway, a second intermediate with copper-sequestered β-strands is formed, which then unravels to produce the third TS leading to complete unfolding of the protein.
Results
Apo-azurin unfolds via 2-state and 3-state pathways
To study the mechanical properties of azurin using SMFS, we followed the chimeric polyprotein approach. Polyproteins provide unambiguous mechanical fingerprints in SMFS experiments5,6,27,28,29,30. We synthesized a heptamer azurin chain, wherein seven azurins are covalently attached to each other, through N-C termini, using protein engineering tools (see Materials and Methods). Steady-state fluorescence and time-resolved fluorescence spectroscopy data confirmed that the biophysical properties of azurin were unchanged in the heptamer form (Figs S2–S3 and Table S1). SMFS experimental data on the heptamers of apo-azurin is shown in Fig. 1 (see also Fig. S8). A representative force-versus-extension (FX) trace of apo-azurin showing a saw-tooth pattern of unfolding force peaks is given in Fig. 1B. Azurin molecules in the heptamer unfolded with a total contour length change of ΔLc = 37.2 ± 0.9 nm as determined by worm-like-chain (WLC) model fits to the FX data (Fig. 1B,C). This is consistent with the expected ΔLc ~ 36.4 nm [(128–23) aa*0.36 nm/aa-1.4 nm] of the protein with a folded protein end-to-end length of 1.4 nm (calculated from the 4AZU PDB structure) and with a disulfide bond (Cys3-Cys26) sequestering 23 residues. In the trace shown in Fig. 1B, three azurins (1, 3, and 4 from left in the saw-tooth pattern) have two force peaks indicating that these azurin molecules unfolded through the intermediate Iapo. The ΔLc from the native state (Napo) to the intermediate (Iapo) was determined to be 7.5 ± 1.5 nm (Fig. 1B,C). The mechanical rupture forces of Napo and Iapo were 56 ± 10 pN and 46 ± 10 pN, respectively (Fig. 1D). A correlation analysis showed that the rupture force of Iapo was consistently lower than the rupture force of the corresponding Napo (Fig. 1E). Analysis of the apo-azurin FX traces revealed that Napo unfolded via a parallel 3-state unfolding pathway in 56 ± 3% of the events. The errors were estimated using bootstrap procedure as described in Methods. To see if the 3-state unfolding flux is dependent on the pulling speed, we varied the pulling speed by two orders of magnitude (40–2000 nm/s) and observed that the 3-state flux with an intermediate varied from 32–85% (see Table S7). It is possible that the metastable intermediate is resolved in the unfolding traces more efficiently at higher pulling speeds and its detection at low pulling speeds is limited by the resolution of the instrumentation.
SMD simulations reveal both structurally localized and collective TSs for apo-azurin unfolding
We carried out multiple in silico constant velocity pulling simulations on fully solvated and atomistic equilibrated models of apo-azurin structures (see Materials and Methods). In these simulations the Cα atom of one terminal residue was held fixed while pulling the Cα atom of the other terminal residue to unfold the protein. We carried out both C-terminal as well as N-terminal pulling simulations (Figs 2 and S9). From the simulations we extracted FX traces which could be compared with those from the experiments. The FX traces obtained from simulations clearly show two force peaks, confirming the existence of the Iapo seen in experiments (Fig. 2). The close overlap of the FX traces from multiple simulations indicated reproducible results which capture the unfolding behaviour of the protein (Fig. 2B,C). We carried out a detailed analysis of structures underlying each FX trace from SMD simulations to extract unfolding pathways of apo-azurin and associated TS structures. We analyzed the sequence of unfolding events in terms of changes in the distance between geometric centres (GCs) of adjacent β-strands in the azurin topology (Figs 2A,C, S9 and Table S5). We find that β2B–β8 and β7–β8 contacts are ruptured at the first TS, followed by the unravelling of β8. Thus, the structural changes leading to the first TS are localized to β8 and its flanking strands. In contrast, the unfolding at the second TS is due to a collective rupture of interactions between all remaining β-strand pairs (β1–β3, β3–β6, β4–β7 and β5–β6). At the second TS, β4–β7 pair ruptured (~1.5 nm GC-GC distance change for a 2 nm change in molecular extension) prior to the other β-strand pairs (<1.0 nm GC-GC distance change for the same 2 nm change in molecular extension) in 8 out of 10 instances. In the remaining cases, β1−β3 pair ruptured (~0.5 nm GC-GC distance change) prior to the response of β4−β7, which followed the response of other pairs. These simulation results suggest a structurally collective TS at the second unfolding force peak, wherein the β-strand interactions ruptured at the TS are spread over a large part of the protein.
Copper-binding shifts the position of the second TS and number of intermediates during azurin unfolding
To obtain direct evidence of the effect of copper on the mechanical properties of azurin, we carried out SMFS experiments on Cu2+-bound azurin heptamer (holo-azurin). We prepared holo-azurin in buffered solution containing 1 mM CuSO4 to ensure that azurin was present in the copper-bound form and confirmed it independently by well-established spectroscopic methods (see Materials and Methods, Figs 3, S2 and Table S1). The absorption spectrum of the holo-azurin heptamer has a charge transfer band centered at 628 nm, which confirms the copper binding (Fig. S2). This band is absent in the apo-azurin heptamer indicating the absence of copper. We have also carried out steady-state fluorescence spectroscopy which shows that the spectral shape of fluorescence emission of Trp48 in azurin is unchanged between monomer and heptamer forms. This result indicates that the apo-azurin structure is preserved in the heptamer form, as the binding of copper is expected to quench the fluorescence emission of Trp4831. We further analysed the decrease in Trp48 fluorescence by more sensitive time-resolved fluorescence spectroscopy measurements which showed a decrease in the mean fluorescence lifetime (τm) from 3.45 ns to 0.30 ns due to copper binding (Fig. 3). Moreover, these measured lifetimes of heptamers are comparable with that of the azurin (monomer) (see Table S1). The properties of heptamer are in concurrence with that of the monomer and that of the literature confirming that the apo-protein is free of copper and holo-form contains bound copper. Once, the holo-form heptamer was confirmed with copper binding, we proceeded to SMFS experiments.
The SMFS pulling experiments on holo-azurin showed that the native state (Nholo) unfolded with a total ΔLc = 37.0 ± 0.7 nm analogous to Napo (Figs 4 and S10). We quantified the unfolding pathways captured by the experimental traces by estimating the percentage flux and associated errors. Errors were estimated using a bootstrap procedure described in Methods. While 77 ± 2% of molecules of holo-azurin exhibited an intermediate (Iholo), a small fraction (16 ± 2%) of these molecules further unfolded through a second intermediate (I’holo), in a 4-state manner with three rupture force peaks (Fig. 4A,B). Such 4-state unfolding was rare in the case of apo-azurin (3% flux). The 2-state unfolding pathway flux for holo-azurin was lower (23 ± 2%) as compared to that for apo-azurin (44 ± 3%). As in the case of apo-azurin, to see if the 3-state unfolding flux was dependent on the pulling speed, we varied the pulling speed (40–2000 nm/s) and observed that the 3-state flux varied between 39–86% (see Table S8). Notably, both apo- and holo-azurins show parallel pathways at all pulling speeds. The rupture forces of Nholo, Iholo, and I’holo were 63 ± 9 pN, 54 ± 9 pN and 51 ± 9 pN, respectively (Fig. 4D). Correlation analysis of the rupture forces showed that Iholo is mechanically weaker compared to Nholo, as also seen in the case of apo-azurin (Fig. 4E). Interestingly, the ΔLc from Nholo to Iholo (6.7 ± 1.2 nm) is about ~1 nm shorter than that for apo-azurin. The statistical p-value for the difference in ΔLc of the apo- and holo-azurin intermediates is <0.00001, implying that the observed ΔLc difference is significant. The ΔLc of the second intermediate from Iholo to I’holo is 5.3 ± 2.0 nm. The large spread in the contour lengths suggests heterogeneity in the unfolding pathways which can arise, for instance, due to the rupture of different interactions in the β1-β3-β6 motif. The structural changes behind the contrasting features in the mechanical properties of apo- and holo-azurins are further revealed in SMD simulations.
Presence of copper-coordination sphere sequesters β4–β7 pair from rupture and changes the unfolding pathway of azurin
For SMD simulations on holo-azurin, we considered a model for the copper coordination sphere which included non-bonding interactions (electrostatic and van der Waals potential terms) and a description of copper-ligand coordination bonds as harmonic springs with equal spring constants (see Materials and Methods). The representation of a metal-coordination sphere by a classical potential is a gross oversimplification and may introduce modelling artifacts in the SMD generated FX traces. Therefore, in order to obtain unbiased insights, we carried out systematic tests by varying the coordination bond spring constant over two orders of magnitude (1–100 kcal/mol/Å2), spanning conventional bond strength ranges exhibited by hydrogen bonds and covalent bonds. Multiple SMD simulations were performed at each spring constant value (Fig. S11). The results showed that for spring constant values <25 kcal/mol/Å2, FX traces resembled those of apo-azurin. Figure 5 shows results for C-terminal SMD pulling simulations with spring constant 25 kcal/mol/Å2 (similar results were obtained for N-terminal pulling simulations and higher spring constant values; Figs S11 and S12). These FX traces of holo-azurin produced two force peaks within a 10 nm extension (Fig. 5C) confirming the presence of Iholo observed in experiments. While the first TS peak position is unaltered with respect to that of the apo-azurin simulation trajectory; the peak position of the second TS is shifted to lower molecular extensions (Fig. 5C). Notably, the average peak separation for the holo-azurin traces (4.5 nm) is ~1.7 nm shorter than that of apo-azurin simulation, in accord with experimentally observed shortening of ΔLc between apo- and holo-forms (Fig. 4C). Formulating the sequence of unfolding events in terms of distance changes between GC of adjacent β-strands reveals that the different peak separations for apo- and holo-azurins reflect different unfolding pathways for the two proteins (Fig. 5C). At the second TS, copper sequesters β4–β7 pair from rupture because of the constraints imposed by the copper-ligand bonds. The unfolding of Iholo is due to a collective rupture of interactions between the β-strands pairs: β1–β3, β3–β6, β4–β5 and β5–β6. In almost all traces, the β1–β3 and β5–β6 pairs ruptured (GC-GC distance changed by ~1 nm for a 4 nm change in molecular extension) prior to the other β-strand pairs (<0.5 nm GC-GC distance change for the same 4 nm change in molecular extension). In one instance, β3–β6 pair ruptured prior to the response of β1–β3 (Fig. 5C). While the second TS of holo-azurin is still collective, involving many interactions spread over the protein, it is structurally more localized than that for apo-azurin as it excludes the β4–β7 pair.
To highlight the imprint of the copper coordination sphere in the FX trace of holo-azurin, we further carried out SMD simulations constraining only the equatorial copper-ligand bonds (i.e., Cu–Cys112:S, Cu–His117:N and Cu–His46:N) and no harmonic constraints on the axial bonds (i.e., Cu–Gly45:O, Cu–Met121:S). In these simulations, the second TS occurred at extensions intermediate to that seen for apo-azurin, and holo-azurin with all copper-ligand bonds constrained. From our analysis (Figs 5C,D, and S13), the shift in the second TS of holo-azurin relative to that for apo-azurin can be understood as follows. In the case of apo-azurin, with no metal coordination sphere, the protein is free to unravel after the first TS till the end of β7 (i.e., up to Cys112). In contrast, in holo-azurin with ‘no axial’ copper coordination, the protein extension is curtailed to His117. For holo-azurin with full copper coordination, the extension is further curtailed to Met121. Figure 5D compares the sequence of residue separations (Cα–Cα distance change) in the immediate vicinity of the copper binding site and β4−β7 along the unfolding pathways of different forms of azurin (see Fig. S13). From Fig. 5D (thin-lines), it is clear that the change in separation of the Gly45-Met121 pair at the second TS between apo- and holo-azurins accounts for the ~ 1 nm shortening of ΔLc between apo- to holo-forms observed in experiments (Fig. 4C).
Controlled rupture of copper-coordination bonds in holo-azurin and the manifestation of third TS
Unlike the case of apo-azurin, the FX traces from our classical holo-azurin SMD simulations do not capture the complete unravelling of azurin (Figs 2B and 5C) due to the imposition of harmonic constraints on the copper coordination sphere. We therefore carried out new simulations wherein the harmonic constraints on the copper-ligand bonds were removed at different extensions along the holo-azurin unfolding pathway (Fig. 6). From the FX traces in Fig. 6 it is clear that a rupture of the copper coordination sphere at 4 nm (i.e., before the second TS) produces a FX trace identical to that seen for apo-azurin simulations (Fig. 2C). This result highlights the selective imprint of the copper on the second TS of holo-azurin. When the rupture of the copper coordination sphere is delayed past the second TS a broad second force peak is produced at an extension ~1.7 nm shorter than that for apo-azurin (Fig. 5C). If the copper coordination sphere rupture occurs at or beyond the second TS, our simulations predict (bottom three panels in Fig. 6) a third force peak corresponding to the separation of the β4−β7 pair whose position depends on the extension at which the copper coordination sphere breaks. Rupture at 6, 7 and 8 nm extensions, where β4–β5 and β5–β6 elongated to different extents, produces a third force peak ~2.7, 4.7, and 5.8 nm away from the second force peak respectively (Table S6 in the Supplementary Material). In Fig. 6, the GC-GC distance change plots for the simulated FX traces attribute the third force peak to the β4–β7 separation (Fig. 6). We note that in the simulations presented in this section, all copper-ligand bonds rupture simultaneously, producing a sharp, almost vertical drop in force with extension in all FX traces. Since such features are absent in the experimental FX traces, we reason that the copper-ligand bonds must rupture sequentially. Our simulations do not resolve contributions from copper-ligand bonds to the third TS. The results presented here show that the copper-ligand bonds have to rupture at or after the second TS in the holo-azurin unfolding pathway. Rupture of the copper-ligand sphere along with the sequestered β4–β7 pair can contribute to the formation of a third TS. Interestingly, a small population of Iholo (~16%) in experiments showed a third TS leading to I’holo.
Discussion
Our view of the unfolding pathways of apo- and holo-azurins derived from single-molecule mechanochemistry experiments and non-equilibrium MD simulations is depicted as a schematic in Fig. 7. In 56 ± 2% of unfolding events, apo-azurin (Napo) unravels via a structurally localized TS with the rupture of interactions between β2B–β8 and β7–β8, to a well-defined intermediate (Iapo). In contrast, Iapo unfolds through a structurally collective TS due to the rupture of interactions spread over the remaining protein structure. Ensemble unfolding experiments have classified TSs into polarized (or local) and diffuse TSs mainly based on ϕ-value analysis32,33,34,35,36,37,38. A local TS is defined as one where the high ϕ-value residues are clustered over a small region of a protein39,40. In contrast, for diffuse TS, most of the residues of the protein have similar ϕ-values41,42. The structurally localized and collective TSs captured in azurin unfolding pathways are analogous to the picture of local and diffuse TSs based on ϕ-value analysis. To the best of our knowledge, our study is the first to provide fingerprints of structurally collective TSs along the mechanical unfolding pathways of proteins. Upon copper-binding, the first TS of holo-azurin (Nholo) remains unchanged from that of apo-azurin, and leads to Iholo with a copper-coordination sphere. Further unravelling of the protein differs from that in Napo, with elongation curtailed by ~1 nm relative to that for apo-azurin due to constraints imposed by the copper coordination sphere. As the copper coordination sequesters β4−β7, it has to rupture first for β4−β7 elongation. If the copper coordination of Iholo ruptures early at the second TS, then the β4−β7 rupture and subsequent extension is only separated by ~2–3 nm from the second TS position and cannot be resolved as a separate force peak in SMFS experiments. Thus, early copper rupture at the second TS results in 3-state pathways as observed for 84% of Iholo population. However, if the rupture of the copper-coordination sphere is delayed then Iholo unfolds to I’holo through a structurally more localized second TS (relative to that of apo-azurin) with copper sequestering the β4−β7 strand pair. Subsequent rupture of the copper coordination sphere along with separation of β4–β7 pair through a structurally localized third TS results in a 4-state pathway as observed for 16% of Iholo population in SMFS experiments. Diffuse TSs in protein folding have earlier been reported in ensemble experiments38. However, in SMFS experiments, the canonical view is that rupture of interactions at the TSs is structurally local in nature. For example, mechanical unfolding of titin I27 or ubiquitin has been attributed to the rupture of H-bonds between terminal β-strands in43,44.
Earlier studies by Beedle et al.21 on the mechanical unfolding of a mixture of apo- and holo-azurin polyproteins using constant-velocity pulling experiments and constant-force SMD simulations showed complex unfolding pathways with multiple intermediates. The authors observed 20% molecules unfolding through a 2-state pathway, which they attributed to be coming from apo-azurin molecules. The remaining 80% population showed 3-state and 4-state pathways. The 3-state pathways were stochastic and the unfolding originated either from the N-terminus or from the C-terminus, which is based on their observation of two different unfolding contour length values for the intermediates. In 4-state pathways, both of these intermediates were observed one after the other before complete unfolding. Interestingly, their constant-force SMD simulations suggested that the majority of the population (90%) unfolded from the C-terminus and the remaining population unfolded either from the N-terminus or from both the termini21. Beedle et al.21 also attributed the rupture of intermediates to the breaking of Cu-Scys112 and Cu-NHis117 bonds. Our study differs from that by Beedle et al.21 in two crucial aspects. First, we carry out independent SMFS measurements on apo- and holo-azurin polyprotein samples. Secondly, we carry out constant-velocity SMD simulations which directly mimic the experiments. Though some of the aspects of complex unfolding pathways of holo-azurin from Beedle et al.21 agree with our results, there are also notable differences. Our results show that 2-state pathways are possible for both apo- and holo-azurins. Further, the unfolding of native states always occurred from the C-terminus in our constant velocity SMD simulations. This result is in agreement with our experiments where we observed only one unfolding contour length for the intermediate and the native state along each unfolding pathway. Constant-force simulations by Beedle et al.21 also show that azurin predominantly unfolds from C-terminus. Most importantly, the copper-binding effects manifest in terms of a subtle 1 nm change in the unfolding contour length of the native state to the intermediate. We stress that the copper induced subtle 1 nm shift of the intermediate could not have been observed without independent measurements on apo- and holo-azurins. Our findings suggest that apo-azurin follows 2-state and 3-state unfolding pathways. Copper-binding, shortens the contour length leading up to the second TS and may produce a third TS (4-state unfolding). This picture differs from the proposal by Beedle et al.21 that apo-azurin unfolding occurs exclusively via a 2-state pathway, whereas holo-azurin unfolds via 3-state and 4-state unfolding pathways. Furthermore, Beedle et al.21 propose that the intermediate force peak observed during azurin unfolding is entirely due to copper-ligand bond rupture. In contrast, we show clearly that apo-azurin also unfolds via 3-state pathway, suggesting that the rupture at the second TS observed in holo-azurin by Beedle et al.21 may not solely be due to the rupture of copper-ligand bonds.
Our systematic SMFS constant-velocity experiments together with constant-velocity SMD simulations of apo- and holo-azurins unambiguously provide the evidence for copper-binding induced changes in the mechanical unfolding pathways of azurin. Copper-binding modulates the unfolding energy landscape specifically at the second TS leading to create parallel unfolding pathways in holo-azurin which differ in the sequence of β-strand separation events and number of intermediates from that in apo-azurin. Such a kinetic partitioning of parallel pathways observed here for single domain apo- and holo-azurins has been reported previously only for multi-domain proteins5,7,45,46,47,48,49.
Conclusions
Our results significantly further our understanding of the effect of metal ions on the unfolding energy landscape and pathways of metalloproteins. Earlier studies on the unfolding of proteins with ligands have highlighted direct changes in the mechanical stability of proteins17,18,28. Recently, it was shown for ferridoxin that metal coordination leads to an intermediate50. In the present study, however, the mechanical stability of azurin in terms of rupture force remains largely unaltered upon copper binding. Rather, new unfolding pathways are introduced to make the energy landscape of protein more rugged. While previous studies on metalloproteins have revealed the role of metal containing active site and the individual metal-ligand bonds on the mechanical properties of proteins, the proteins were themselves mechanically compliant with the entire mechanical resistance due to the coordination bonds18,51. Further studies on other metalloproteins are necessary to establish the generality of the metal-induced sequestering mechanism during the mechanical unfolding of proteins. However, the effect of metal in azurin is complex, creating no major change in the rupture force, yet altering unfolding pathways dramatically in terms of sequence of rupture events as well as intermediates. Our studies add to recent reports of copper-induced changes in mechanochemical reactions within single-molecules52. The analytical framework using combined force spectroscopy experiments and SMD simulations presented here should be useful to study metal induced changes to protein energy landscapes of other metalloproteins and enhance our understanding of the roles of metal on protein mechanical stability and conformational flexibility.
Furthermore, azurin is a periplasmic protein. It is synthesized in cytosol and then undergoes translocation into the periplasm. Forced unfolding of proteins prior to their insertion into the narrow pores of protein channels in the membrane is a key step in the translocation process53,54. Hence, the mechanical unfolding pathways of azurin and the intermediate states captured here might be important in understanding its translocation process in vivo.
Materials and Methods
Polyprotein engineering
The azurin gene cloned in pGK22 vector was taken for polyprotein construction55. The heptameric construct of azurin was cloned in pQE80L expression vector (Qiagen Valencia, CA) between the restriction endonuclease sites BamHI and HindIII using the iterative cloning technique described previously27.
Azurin-heptamer containing pQE80L plasmid was over-expressed in E. coli BL21 (DE3) cells using 1 mM IPTG induction at an optical density (OD600) of 0.6 of the cell culture and then incubating at 37 °C for 6 hrs. The cell pellet expressing the apo-protein was resuspended in Tris-HCl (20 mM, pH 7.4) in the presence of 1 X protease inhibitor cocktail (Sigma Aldrich) and lysed by sonication at 4 °C. Lysate obtained after centrifugation (17,000 rpm for 45 min at 4 °C) was incubated with Ni-NTA resin (Roche) for 16 hrs at 4 °C on rotaspin. The resin was washed with Tris-HCl (20 mM, pH 7.4) and Tris-HCl (20 mM, pH 7.4) containing 20 mM imidazole after which azurin heptamer with His6–tag at the N-terminus was eluted using Tris-HCl (20 mM, pH 7.4) containing 250 mM imidazole. Apo-protein was further purified using superdex200 columns (GE Healthcare) on Bio-Rad Biologic Duo-Flow FPLC system. Purity of the protein was checked using SDS-PAGE (see Fig. S2).
Holo-azurin heptamer was prepared in 1 mM CuSO4 containing Tris-HCl (20 mM, pH 7.4) buffered solution, and copper-binding was confirmed by absorbance at 628 nm (A628), steady-state fluorescence, and time-resolved fluorescence spectroscopy (Figs 3, S2 and Table S1)56. Purification procedure of azurin monomer is also given in Supplementary Material.
Spectroscopic characterization of apo- and holo-azurin heptamers
Purity of protein and copper-binding was further confirmed by absorption and fluorescence spectroscopy (see Figs 3 and S2). Fluorescence measurements were carried out at a protein concentration of 10 μM. All the steady-state measurements were carried out on a SPEX fluorolog (T-format) fluorimeter by exciting the protein samples at 295 nm wavelength. The fluorescence emission spectral properties of apo-azurin remained unaltered in the heptamer (Fig. S3).
Time-resolved fluorescence intensity measurements were carried out using a time correlated single photon counting (TCSPC) instrument coupled to a microchannel plate photomultiplier (Model 2809 u; Hamamatsu Corp), as described previously56. Time-resolved fluorescence intensity was measured by employing CW-passively mode-locked frequency-doubled Nd:YAG laser (Vanguard, Spectra Physics, USA) driven Rhodamine 6 G dye laser. Dye laser pulses at 590 nm frequency-doubled to 295 nm were used for excitation. The repetition rate of the laser pulses was 4 MHz. Time per channel of the collected time-resolved data was 48.9 ps. The half-width of the instrument response function (IRF) at 295 nm was ~80 ps. The fluorescence emission was collected through a 305 nm cutoff filter followed by a monochromator set at 310 nm. The cutoff filter was used to prevent scattering of the excitation beam from the samples. Fluorescence was collected with the emission polarizer oriented at the magic angle (54.7°) with respect to the excitation polarizer. For all the time-resolved fluorescence measurements data were collected such that the peak count is >5000.
Experimentally measured time-resolved fluorescence intensity data, F(t), is a convolution of the instrument response function, R(t), and the intensity decay function of the sample, I(t):
where, \(\delta \) is the shift parameter, which is a fraction of the time per channel. R(t) is experimentally determined. I(t) is a function assumed to describe the fluorescence dynamics of the sample. Decay data analysis involves the determination of the best values for the unknown parameters in I(t). We fitted the fluorescence decay data from the peak position to the time it decayed to 0.1% of the peak count. Time-resolved fluorescence decay data were fitted to a function that is a sum of discrete exponentials,
where, τ i and α i are the lifetime and the corresponding amplitude, respectively. Also Σiαi = 1.0. There parameters were optimized by the iterative reconvolution method57. Correction factors for the parameters (αi and τi.) in successive iterations were determined by the application of Marquardt’s method in nonlinear least-squares analysis58. Numerical calculation of the convolution integrals for intensity and partial derivatives were done using the Grinvald-Steinberg recursion equations57. The mean lifetime \({\tau }_{m}=\,{\sum }^{}{\alpha }_{i}{\tau }_{i}\), which is the area under I(t) vs t curve, gives us information on the fluorescence quantum yield of the system. Time-resolved measurements of holo- forms of heptamer and monomer azurin were carried out in the presence of 1 mM CuSO4 in the buffered solution. Reduction by more than ten times in the τm of holo-azurin heptamer compared to apo-azurin heptamer, in a manner similar to that of the azurin monomer, confirmed copper-binding (Figs 3, S4 and Table S1).
Single-molecule force spectroscopy (SMFS)
Single-molecule pulling experiments were carried out using a custom-built atomic force microscope, whose details have been described elsewhere5. In SMFS pulling experiments, a 50 μl of ~10 μM protein in Tris-HCl (20 mM, pH 7.4) was added on to a glass coverslip. We used gold-coated reflective cantilevers with silicon nitride tip from Bruker, CA, USA. Before acquiring force-versus-extension (FX) traces, spring constant of the cantilever was measured using equipartition theorem as previously suggested by Florin et al.59. The spring constant of the cantilever that we used was ~40 pN/nm. All the experiments were performed at room temperature and with 5 kHz data sampling rate. All the pulling experiments were performed at a pulling speed of 400 nm/s except for the pulling speed-dependent measurements where it was varied in the range 40–2000 nm/s.
Data analysis
FX traces were fitted to worm-like chain (WLC) model of polymer elasticity using the Equation 3 given below60:
Where, L c and p are contour length and persistence length, respectively. T is absolute temperature and kB is Boltzmann constant. In the analysis, we considered FX traces that contain complete unravelling of at least four azurin molecules from the heptamer chain.
Bootstrap method
To estimate the standard error in our experimentally obtained percentage of flux through the intermediate, we carried out the nonparametric bootstrap method that was earlier implemented in analyzing the SMFS data5,61. For a given set of experimental FX traces containing a mixture of unfolding pathways (e.g. 2-state and 3-state unfolding for apo-azurin), a sample data set of equal size comprising of randomly chosen FX traces from the experimental set was created. We measured the percentages of traces exhibiting an intermediate for 500 such sample data sets constructed from the original data set. This procedure resulted in a distribution that provided the standard error of the mean for the percentage of traces exhibiting an intermediate and is reported in Fig. 7.
Azurin models and equilibrium molecular dynamics (MD) simulations
Crystal structures of apo-azurin (PDB code: 1E65) and holo-azurin (PDB code: 4AZU) were processed in Visual Molecular Dynamics (VMD)62 to add hydrogen atoms and solvated in a water box of size 153 × 152 × 154 Å3. We neutralized the apo-azurin and holo-azurin systems by adding 3 Na+ and 2 Na+ ions respectively. Subsequently, classical atomistic MD simulations of apo- and holo-azurin protein systems were carried out using the NAMD program (version 2.9)63. The simulations employed the TIP3P water model and the CHARMM27 force field to describe atomic interactions64. In general, classical force field parameters are not available for metal-ligand coordination spheres and a customized procedure (details in the next subsection) was employed to create multiple models of the copper coordination sphere for holo-azurin. We first equilibrated our protein systems using the following protocol. First, the solvated protein structure was optimized in 20000 steps keeping all the protein heavy atoms (including the copper coordination sphere for holo-azurin) fixed. Then the system was heated from 0 K to 300 K over 50 ps followed by equilibration at 300 K for further 50 ps. The optimization and heating steps were repeated with harmonic constraints on the protein heavy atoms. Subsequently three 150 ps constant pressure and temperature (NPT) equilibration simulations were performed with progressively weaker harmonic constraints. Finally, a single 1 ns run was performed with no constraints. The same equilibration protocol was applied for apo-azurin and the 5 different holo-azurin models described in the next subsection. We then obtained production runs for apo-azurin (100 ns) and for the different holo-azurin systems (10 ns) under constant temperature and volume (NVT) conditions.
Modelling copper coordination sphere in holo-azurin
At present, there is no force field to mimic the copper coordination sphere in holo-azurin. The trigonal bipyrimidal coordination of copper in holo-azurin with 5 ligands was modelled by a potential comprising of harmonic bonded interactions and non-bonded electrostatic and van der Waals (CHARMM27 parameters) interactions. Atomic partial charges for electrostatic interactions were adapted from van den Bosch et al.65 (see Table S2). The copper-ligand coordination bonds were modelled as harmonic springs with equilibrium positions set to the crystal structure values (Table S3) and equal spring constants. We varied the spring constant values of the copper-ligand coordination bonds between 1–100 kcal/mol/Å2 (1, 5, 25, 100 kcal/mol/Å2) to create 4 separate models of holo-azurin. We also created an additional model (no axial) with no constraints on the axial bonds. For the apo-azurin and these 5 holo-azurin systems, we carried out equilibration and production runs as described in the previous subsection.
Steered molecular dynamics (SMD) simulations
Structures sampled from the equilibrium production runs for apo- and holo- azurin proteins (Fig. S5) were used to seed 10 different SMD simulations in order to build statistics of the unfolding events. In SMD a dummy atom attached by a spring to the terminal protein atom was pulled with a constant velocity of 0.0001 Å/fs. The dummy spring had a spring constant 7 kcal/mol/Å2 and extension of this spring was used to monitor the force on the protein. During the pulling, the other terminal protein atom was held fixed so that the pulling direction was along the two terminal atoms of the protein. We carried out both N- and C-terminal pulling simulations wherein azurin was extended. Based on the experimental FX traces and preliminary simulations extending azurin to the full unfolding contour length of ~36 nm (shown in Fig. 2B), we first determined that all experimentally resolved transition states and intermediates would be captured within a molecular extension of 14 nm in the SMD simulations. For a molecular extension of up to 14 nm, we verified that the simulation data was free from artefacts arising from periodic boundary conditions by analysing distances between protein atoms in the simulation cell and the nearest protein atoms of neighbouring protein images. Our analysis shows (Supporting Information Fig. S6) that the distances between protein atoms and their nearest periodic images are always greater than the cut-off distance for non-bonded interactions (1.4 nm) for molecular extensions up to 14 nm.
These N- and C-terminal pulling simulations reveal the structural details of proteins at the atomistic level as they undergo mechanical unfolding. SMD simulations were carried out under NPT conditions using the Langevin dynamics and a Nose-Hoover barostat. The Langevin damping constant was set to 0.01 ps−1 after trial SMD runs testing FX traces and temperature controls (Fig. S7). For holo-azurin protein simulations with controlled rupture of copper-ligand bonds, the copper-ligand harmonic bond constraints were removed at different pulling extensions and simulations continued with no constraints to mimic the copper rupture event. Over 100 simulations were carried out on the apo- and holo-azurin proteins (See Table S4 for a listing of systems and simulation timescales).
References
Dill, K. A. & Chan, H. S. From Levinthal to pathways to funnels. Nat Struct Biol 4, 10–9 (1997).
Bryngelson, J. D., Onuchic, J. N., Socci, N. D. & Wolynes, P. G. Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins 21, 167–95 (1995).
Stigler, J., Ziegler, F., Gieseke, A., Gebhardt, J. C. & Rief, M. The complex folding network of single calmodulin molecules. Science 334, 512–6 (2011).
Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–12 (1997).
Aggarwal, V. et al. Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins. J Biol Chem 286, 28056–65 (2011).
Hughes, M. L. & Dougan, L. The physics of pulling polyproteins: a review of single molecule force spectroscopy using the AFM to study protein unfolding. Rep Prog Phys 79, 076601 (2016).
Schonfelder, J., Perez-Jimenez, R. & Munoz, V. A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution. Nat Commun 7, 11777 (2016).
Gao, M., Wilmanns, M. & Schulten, K. Steered Molecular Dynamics Studies of Titin I1 Domain Unfolding. Biophysical Journal 83, 3435–3445 (2002).
Izrailev, S. et al. Steered Molecular Dynamics. In Computational Molecular Dynamics: Challenges, Methods, Ideas: Proceedings of the 2nd International Symposium on Algorithms for Macromolecular Modelling, Berlin, May 21–24, 1997 (eds Deuflhard, P. et al.) 39–65 (Springer Berlin Heidelberg, Berlin, Heidelberg, 1999).
Zhmurov, A. et al. Mechanical Transition from Alpha-Helical Coiled Coils to Beta-Sheets in Fibrin(ogen). Journal of the American Chemical Society 134, 20396–20402 (2012).
Sotomayor, M. & Schulten, K. Single-Molecule Experiments in Vitro and in Silico. Science 316, 1144–1148 (2007).
Shi, W. & Chance, M. R. Metalloproteomics: forward and reverse approaches in metalloprotein structural and functional characterization. Curr Opin Chem Biol 15, 144–8 (2011).
Wilson, C. J., Apiyo, D. & Wittung-Stafshede, P. Role of cofactors in metalloprotein folding. Quarterly Reviews of Biophysics 37, 285–314 (2004).
Arnesano, F., Banci, L. & Piccioli, M. NMR structures of paramagnetic metalloproteins. Quarterly Reviews of Biophysics 38, 167–219 (2006).
Kedrov, A., Krieg, M., Ziegler, C., Kuhlbrandt, W. & Muller, D. J. Locating ligand binding and activation of a single antiporter. EMBO Reports 6, 668–674 (2005).
Shen, T., Cao, Y., Zhuang, S. & Li, H. Engineered Bi-Histidine Metal Chelation Sites Map the Structure of Mechanical Unfolding Transition State of an Elastomeric Protein Domain GB1. Biophysical Journal 103, 807–816 (2012).
Oroz, J. et al. Nanomechanics of the cadherin ectodomain: “canalization” by Ca2+ binding results in a new mechanical element. J Biol Chem 286, 9405–18 (2011).
Zheng, P. & Li, H. Highly covalent ferric-thiolate bonds exhibit surprisingly low mechanical stability. J Am Chem Soc 133, 6791–8 (2011).
Ge, L., Perez, C., Waclawska, I., Ziegler, C. & Muller, D. J. Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+-coupled betaine symporter BetP. Proceedings of the National Academy of Sciences 108, E890–E898 (2011).
Ramanujam, V., Kotamarthi, H. C. & Ainavarapu, S. R. K. Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin. PLOS ONE 9, e94513 (2014).
Beedle, A. E., Lezamiz, A., Stirnemann, G. & Garcia-Manyes, S. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins. Nat Commun 6, 7894 (2015).
Nar, H., Messerschmidt, A., Huber, R., van de Kamp, M. & Canters, G. W. Crystal structure of Pseudomonas aeruginosa apo-azurin at 1.85 A resolution. FEBS Lett 306, 119–24 (1992).
Nar, H., Messerschmidt, A., Huber, R., van de Kamp, M. & Canters, G. W. Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip. J Mol Biol 221, 765–72 (1991).
Solomon, E. I. et al. Spectroscopic studies of stellacyanin, plastocyanin, and azurin. Electronic structure of the blue copper sites. Journal of the American Chemical Society 102, 168–178 (1980).
van de Kamp, M. et al. Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome c551 and nitrite reductase. European Journal of Biochemistry 194, 109–118 (1990).
Wittung-Stafshede, P. A Copper Story: From Protein Folding and Metal Transport to Cancer. Israel Journal of Chemistry 56, 671–681 (2016).
Carrion-Vazquez, M. et al. Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci USA 96, 3694–9 (1999).
Ainavarapu, S. R., Li, L., Badilla, C. L. & Fernandez, J. M. Ligand binding modulates the mechanical stability of dihydrofolate reductase. Biophys J 89, 3337–44 (2005).
Kotamarthi, H. C., Sharma, R. & Koti Ainavarapu, S. R. Single-molecule studies on PolySUMO proteins reveal their mechanical flexibility. Biophys J 104, 2273–81 (2013).
Javadi, Y., Fernandez, J. M. & Perez-Jimenez, R. Protein folding under mechanical forces: a physiological view. Physiology (Bethesda) 28, 9–17 (2013).
Fuentes, L., Oyola, J., Fernandez, M. & Quinones, E. Conformational Changes in Azurin from Pseudomona aeruginosa Induced through Chemical and Physical Protocols. Biophysical Journal 87, 1873–1880 (2004).
Itzhaki, L. S., Otzen, D. E. & Fersht, A. R. The Structure of the Transition State for Folding of Chymotrypsin Inhibitor 2 Analysed by Protein Engineering Methods: Evidence for a Nucleation-condensation Mechanism for Protein Folding. Journal of Molecular Biology 254, 260–288 (1995).
Burton, R. E., Huang, G. S., Daugherty, M. A., Calderone, T. L. & Oas, T. G. The energy landscape of a fast-folding protein mapped by Ala-> Gly Substitutions. Nat Struct Mol Biol 4, 305–310 (1997).
Grantcharova, V. P., Riddle, D. S., Santiago, J. V. & Baker, D. Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain. Nat Struct Mol Biol 5, 714–720 (1998).
Martinez, J. C., Pisabarro, M. T. & Serrano, L. Obligatory steps in protein folding and the conformational diversity of the transition state. Nat Struct Mol Biol 5, 721–729 (1998).
Gruebele, M. & Wolynes, P. G. Satisfying turns in folding transitions. Nat Struct Mol Biol 5, 662–665 (1998).
Sanchez, I. E. & Kiefhaber, T. Origin of Unusual Phi-values in Protein Folding: Evidence Against Specific Nucleation Sites. Journal of Molecular Biology 334, 1077–1085 (2003).
Fersht, A. R. & Sato, S. Phi-value analysis and the nature of protein-folding transition states. Proc Natl Acad Sci USA 101, 7976–81 (2004).
Went, H. M. & Jackson, S. E. Ubiquitin folds through a highly polarized transition state. Protein Engineering Design and Selection 18, 229–237 (2005).
Dao, T. P., Majumdar, A. & Barrick, D. Highly polarized C-terminal transition state of the leucine-rich repeat domain of PP32 is governed by local stability. Proceedings of the National Academy of Sciences 112, E2298–E2306 (2015).
Kelly, S. E. et al. Diffuse transition state structure for the unfolding of a leucine-rich repeat protein. Physical chemistry chemical physics: PCCP 16, 6448–6459 (2014).
Bueno, M., Ayuso-Tejedor, S. & Sancho, J. Do Proteins with Similar Folds Have Similar Transition State Structures? A Diffuse Transition State of the 169 Residue Apoflavodoxin. Journal of Molecular Biology 359, 813–824 (2006).
Marszalek, P. E. et al. Mechanical unfolding intermediates in titin modules. Nature 402, 100–3 (1999).
Carrion-Vazquez, M. et al. The mechanical stability of ubiquitin is linkage dependent. Nat Struct Biol 10, 738–43 (2003).
Peng, Q. & Li, H. Atomic force microscopy reveals parallel mechanical unfolding pathways of T4 lysozyme: evidence for a kinetic partitioning mechanism. Proc Natl Acad Sci USA 105, 1885–90 (2008).
Gao, X. et al. Single-molecule experiments reveal the flexibility of a Per-ARNT-Sim domain and the kinetic partitioning in the unfolding pathway under force. Biophys J 102, 2149–57 (2012).
Kotamarthi, H. C., Sharma, R., Narayan, S., Ray, S. & Ainavarapu, S. R. Multiple unfolding pathways of leucine binding protein (LBP) probed by single-molecule force spectroscopy (SMFS). J Am Chem Soc 135, 14768–74 (2013).
Kotamarthi, H. C., Narayan, S. & Ainavarapu, S. R. Mechanical unfolding of ribose binding protein and its comparison with other periplasmic binding proteins. J Phys Chem B 118, 11449–54 (2014).
Perez-Jimenez, R., Garcia-Manyes, S., Ainavarapu, S. R. & Fernandez, J. M. Mechanical unfolding pathways of the enhanced yellow fluorescent protein revealed by single molecule force spectroscopy. J Biol Chem 281, 40010–4 (2006).
Lei, H. et al. Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy. Journal of the American Chemical Society 139, 1538–1544 (2017).
Wei, W. et al. Structural Insights and the Surprisingly Low Mechanical Stability of the Au-S Bond in the Gold-Specific Protein GolB. J Am Chem Soc 137, 15358–61 (2015).
Ladenthin, J. N. et al. Force-induced tautomerization in a single molecule. Nat Chem 8, 935–40 (2016).
Matouschek, A. Protein unfolding an important process in vivo? Current Opinion in Structural Biology 13, 98–109 (2003).
Prakash, S. & Matouschek, A. Protein unfolding in the cell. Trends in Biochemical Sciences 29, 593–600 (2004).
van de Kamp, M., Hali, F. C., Rosato, N., Agro, A. F. & Canters, G. W. Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli. Biochim Biophys Acta 1019, 283–92 (1990).
Ramreddy, T., Kombrabail, M., Krishnamoorthy, G. & Rao, B. J. Site-Specific Dynamics in TAT Triplex DNA As Revealed by Time-Domain Fluorescence of 2-Aminopurine. The Journal of Physical Chemistry B 113, 6840–6846 (2009).
Grinvald, A. & Steinberg, I. Z. On the analysis of fluorescence decay kinetics by the method of least-squares. Analytical Biochemistry 59, 583–598 (1974).
Bevington, P. R. Data Reduction and Error Analysis for the Physical Sciences, (McGraw-Hill Inc., New York, 1969).
Florin, E. L. et al. Sensing Specific Molecular-Interactions with the Atomic-Force Microscope. Biosensors & Bioelectronics 10, 895–901 (1995).
Marko, J. F. & Siggia, E. D. Stretching DNA. Macromolecules 28, 8759–8770 (1995).
Efron, B. The Jackknife, the Bootstrap, and Other Resampling Plans, (SIAM, Philadelphia, 1982).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J Mol Graph 14(33–8), 27–8 (1996).
Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J Comput Chem 26, 1781–802 (2005).
MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J Phys Chem B 102, 3586–616 (1998).
van den Bosch, M. et al. Calculation of the redox potential of the protein azurin and some mutants. Chembiochem 6, 738–46 (2005).
Acknowledgements
The azurin gene is a kind gift from Prof. G.W. Canters, Leiden, Netherlands. The authors thank Department of Atomic Energy (DAE) and TIFR for financial support and their lab members for critical reading of the manuscript.
Author information
Authors and Affiliations
Contributions
S.R.K.A. and R.V. conceived and coordinated the study. A.Y. performed the experiments. A.Y. and S.P. conducted computer simulations. All authors analyzed the data, reviewed the results and approved the final version of the manuscript.
Corresponding authors
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Yadav, A., Paul, S., Venkatramani, R. et al. Differences in the mechanical unfolding pathways of apo- and copper-bound azurins. Sci Rep 8, 1989 (2018). https://doi.org/10.1038/s41598-018-19755-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-018-19755-7
This article is cited by
-
Steered molecular dynamic simulations of conformational lock of Cu, Zn-superoxide dismutase
Scientific Reports (2019)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.