proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Rubredoxin refolding on nanostructured hydrophobic surfaces: Evidence for a new type of biomimetic chaperones Matteo Miriani,1 Stefania Iametti,1 Donald M. Kurtz,2 and Francesco Bonomi1* 1 Section of Chemistry and Biomolecular Sciences, DeFENS, University of Milan, Milan, Italy 2 Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas

ABSTRACT Rubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron-uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at “catalytic” NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific “iron-uptake-competent” conformations of apoRd on the NP surface. This bio-mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor-triggered refolding, particularly when overexpressed under limited cofactor accessibility. Proteins 2014; 00:000–000. C 2014 Wiley Periodicals, Inc. V

Key words: metalloproteins; protein folding; rubredoxin; latex nanoparticles; biomimetic chaperones; hydrophobic interfaces; protein-interface interactions.

INTRODUCTION Molecular chaperones either drive the correct folding of nascent polypeptides or restore the native structure of misfolded proteins.1–4 Several molecular chaperones require auxiliary proteins or cochaperones to elicit their functions, and in many cases rely on the activation of conformational changes via ATP hydrolysis.5,6 Given the relevance of protein misfolding also to pathological conditions and to overexpression of proteins in biotechnology, there have been several attempts to use molecular chaperones for therapy of diseases related to protein misfolding7 or for improving the yields in natively folded, active proteins.8 In most cases, chaperones induce unfolding of selected regions of the substrate protein,9–11 which then interact with other regions of the substrate protein, thereby facilitating folding into the native structure. Specialized chaperones have been

C 2014 WILEY PERIODICALS, INC. V

implied in the assembly and folding of proteins containing metals,12,13 metal clusters,6,14 or organic cofactors,15,16 thus allowing cells and organelles to fine tune these assembly processes. Rubredoxins (Rds) are the simplest iron-sulfur proteins, having small size (54 amino acid residues) and low structural complexity.17 In Rds, iron is directly bound to four cysteine residues to form a tetrahedral Fe(SCys)4 site that likely does not require a specialized intracellular machinery for iron incorporation in vivo, unlike the case Additional Supporting Information may be found in the online version of this article. Grant sponsor: University of Milan; Grant number: POR FSE Project 2007-13, ASSE IV. *Correspondence to: Francesco Bonomi, DeFENS, University of Milan, 2 Celoria, 20133 Milan, Italy. E-mail: [email protected] Received 6 June 2014; Revised 4 August 2014; Accepted 11 August 2014 Published online 21 August 2014 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24675

PROTEINS

1

M. Miriani et al.

for sulfide-containing Fe-S clusters.18,19 Rds are, thus, simple prototypes for investigating the relationships between metal binding and native protein structure. Natively folded iron(III)-containing (holo)Rds from both thermophilic and mesophilic bacteria retain their native structural features with no iron loss even in 8M urea. Studies investigating either thermally or denaturantinduced unfolding of holo- and iron-depleted Rds (apo)Rds showed no direct correlation between the stability of iron ligation and that of the native polypeptide structure.20,21 It has also been reported that mesophilic and thermophilic apoRds retain significant secondary and tertiary structure20,22 at room temperature and neutral pH. Iron at the native Fe(SCys)4 site of Rds can be replaced isomorphically by other thiophilic metal ions (Cd21, Zn21) under nondenaturing conditions either by direct displacement of Fe(II)23,24 or on protein over-expression in heterologous systems.25,26 Some of these studies also pointed out a noticeable impairment of the overall structural rigidity in the reduced form of holoRds.23,27 All these studies addressed the issue of iron loss or substitution in this class of proteins, but did not address perhaps more physiologically relevant issues, namely iron uptake by the apoRd polypeptide and formation of the native holo- from apoRd structure. In previous studies, some of us took advantage of the stability of the native holoRd structure at chaotrope concentrations where the apoRd has no detectable secondary structure to assess the role of iron incorporation in the protein folding. We indeed found that refolding of urea- or guanidine hydrochloride-denatured apoRd is iron nucleated, and leads to quantitative formation of the native holoRd structure without dilution of the chaotrope.28 These approaches were also used to chronologically discriminate iron binding and protein folding events, and to elucidate the roles of individual ligand cysteines in iron incorporation and refolding.29 These studies made it evident that structural flexibility of the chaotrope-denatured apoRd facilitated rapid formation of an Fe(SCys)4 site, which in turn nucleated folding into the native holoRd structure. Other studies have demonstrated that several proteins– including proteins with remarkable structural stiffness, such as bovine beta-lactoglobulin–undergo large structural changes when allowed to interact with the hydrophobic surface of polystyrene nanoparticles (NPs) in the absence of chemical or physical denaturation.30 The structural changes ensuing from adsorption resulted in the exposure of buried amino acid side chains.31 Similar unfolding events have been shown to occur with proteins exposed to other types of hydrophobic nanoparticles,32–35 and to be dependent on the geometrical features of the nanosystem.33 On the basis of these precedents, we investigated the possibility that polystyrene nanoparticles (NPs) could

2

PROTEINS

function as an alternative chaotrope that would facilitate iron uptake and folding of the apoRd from Clostridium pasteurianum. Our rationale was that hydrophobic contacts with the NP surface would expose otherwise buried protein regions in the structured but iron-uptakeincompetent apoRd. Solvent exposure of ligand cysteines in the NP-bound apoRd should improve their ability to capture added metals, thereby triggering refolding to the native protein structure and–as a consequence—the subsequent detachment of the folded holoRd (where hydrophobic surfaces are mostly buried the protein interior) from the NP surface. Yields and rates of individual steps of the iron uptake/refolding process induced by denaturing urea were compared with those obtained with various concentrations of NP in the absence of urea. MATERIALS AND METHODS Proteins and chemicals

Clostridium pasteurianum rubredoxin (Rd) was expressed and purified as the iron holoprotein (holoRd) according to published procedures.36 The concentration of holoRd was determined using e492 5 8850 M21 cm21.37 ApoRd was prepared by treating holoRd with 10% (v/v) trichloroacetic acid in the presence of 5 mM 2-mercaptoethanol at 0 C. The apoRd precipitate was washed twice with water, and redissolved in buffer in the absence of 2-mercaptoethanol. ApoRd concentration was calculated using published extinction coefficients.36 Unless otherwise indicated, all the experiments described in the following sections were carried out with protein solutions in 100 mM Tris/HCl, pH 7.4 at 23 C. Chemicals were of reagent grade or better. Ultra-pure urea was from Sigma. NPs (latex NP, nominal diameter, 46 nm and 200 nm) were from Kisker Biotech, and were provided as a suspension in water. Some physical properties of the NP are summarized in the Supporting Information Table S1. In some experiments, 46 nm NP were pre-coated with a sub-saturating amount of bovine betalactoglobulin (0.05 g/L protein–equivalent to 0.003 mM–and 0.625 g/L NP) following procedures reported elsewhere.30 Iron uptake by apoRd and refolding of holoRd

Strict anaerobic conditions were used for preparation of buffers and solutions, and for sample handling. With the exception of ultrafiltration and iron removal, all other manipulations were carried out under an Ar atmosphere in glass test tubes or in quartz cuvettes. A freshly prepared, washed apoRd pellet was dissolved in 0.6–0.8 mL of buffer to give a protein concentration in the 0.05 mM range. When appropriate, the buffer contained either 5M urea or the desired final concentration of NP, as calculated from the nominal concentration

Nanoparticles-Assisted Rubredoxin Refolding

given by the supplier. HoloRd reconstitution was started by adding one equivalent of iron per mol protein as a freshly prepared 50 mM aqueous solution of ferric ammonium citrate. Changes in circular dichroism (CD) at 495 nm and in absorbance (at 492 nm) because of the Fe(III)(SCys)4 site28,29 were monitored either continuously at the given wavelengths in separate mixtures of identical composition, or by obtaining spectra of the same individual mixtures at appropriate time intervals. Yields of holoRd (absorbance and CD) were calculated by comparison with the spectroscopic features of native holoRd. In experiments involving aged reconstitutionimpaired forms of apoRd, a solution of apoRd prepared as mentioned above was kept at 4 C under Ar for at least 2 weeks before being used in reconstitution studies. Metal and protein analyses for all the experiments described above were performed after removal of excess reagents by exhaustive buffer exchange with Centricon devices having a nominal Molecular Weight Cut-Off (MWCO) of 3000 Da under aerobic conditions. Metal content of the native and reconstituted holoRds was assessed by ICP-MS or by ESI-MS as detailed elsewhere.28,29 Spectroscopic studies

UV-Vis absorption spectra and time courses were taken on a Perkin-Elmer Lambda 2 spectrophotometer. CD spectra and kinetics of ellipticity changes were recorded on a J-810 Jasco spectropolarimeter. Both instruments were equipped with computer-controlled, Peltier-driven cell holders for accurate temperature control. Binding to NPs

Binding of apoRd, holoRd, and iron salts to NPs was evaluated by analyzing the permeate from a Centricon device having a MWCO of 10,000 Da after spinning for 30 min at 3000g of mixtures containing NP and protein at specified amounts (see Results). Absorbances at either 280 nm or 492 nm were used to evaluate the apoRd and holoRd content in the permeate, respectively. Iron content of permeates and retentates was assessed spectrophotometrically with bathophenanthroline sulfonate in the presence of 1 mM sodium dithionite, as described elsewhere.38 RESULTS Apo but not holoRd binds to NPs

Ultrafiltration studies on Centricon devices of appropriate MWCO clearly indicated that holoRd was unable to bind to NP in buffer even at the highest NP concentrations used (2–5 g/L). At an apparent mass of 30 MDa even the smallest NP used in our studies (46 nm

diameter) were retained by the 10,000-Da MWCO membrane, whereas holoRd can pass through this membrane quantitatively. The lack of HoloRd binding to NPs is consistent with the absence of exposed hydrophobic patches,17 and with the high negative charge of the protein at neutral pH (pI  3), that would result in electrostatic repulsion with the few residual negative charges that remain on the, as stated by the NP supplier. Titration experiments with apoRd and subsequent separation of the unbound protein through Centricon devices were carried out to assess the amount of protein bound to 46-nm NP. Under the conditions used in this part of the study (i.e.,  50 micromolar apoRd and NP concentrations in the 2–5 g/L range), about 200 apoRd molecules were found to be adsorbed on the surface of each NP (not shown), consistent with that observed with other negatively charged proteins30 that interact with the NP surface through hydrophobic interactions. The number of bound molecules may appear low when considering an estimated “footprint” of 7 nm2 for the native protein, but it seems reasonable to expect this "footprint" to be sensibly larger in the case the protein unfolds on the NP surface, even without considering structural fluctuation and mutual repulsion among the bound proteins. Thus, at the concentrations used in other parts of this study, the amount of apoRd (typically, 0.32 g/L, equivalent to 0.05 mM or 50000 nM) may exceed the binding capacity of NPs even at the highest concentration of NP (2 g/L, corresponding to 60 nM for 46 nm NP, allowing a maximum concentration of NP-bound apoRd in the 12,000 nM range according to the figures given above). Thus, only a fraction of the apoRd in the mixtures used in our reconstitution studies was NP-bound. This, along with the strong background absorbance of the polystyrene NP in the near- and far-UV spectral regions, prevented spectroscopic investigation of possible conformational changes that may occur when the structured apoRd present in solution interacts with the NP surface.

Ferric ammonium citrate does not detectably bind to NPs in the absence of Rd

For iron incorporation, ferric ammonium citrate was used in amounts equimolar to apoRd. A nonlinear dependence on the ferric ammonium citrate concentration was reported for the rate of some of the individual events observed in holoRd reconstitution in the presence of chaotropes,28 although the iron concentration had little effect on the yield of holoRd reconstitution regardless of the presence of chaotropes.29 Ultrafiltration experiments showed that the presence of NPs had little effect on the partition of iron between the permeate and retentate phases (Supporting Information Table 2S). This result indicates that binding of ferric ion or ferric ammonium citrate to the NP surface was PROTEINS

3

M. Miriani et al.

Figure 1 A: CD spectra of 0.05 mM apoRd anaerobically incubated for 24 h at 23 C in the presence of 0.05 mM ferric ammonium citrate in 100 mM TrisHCl, pH 7.6 containing the indicated additional components. B: Spectroscopic yield in holoRd as detected by CD spectroscopy in the presence of the given concentration and type of additional materials.

negligible in the absence of protein, consistent also with the high affinity of citrate for the ferric iron. Thus, in the frame of this particular study, the addition of ferric salts to apoRd under a variety of conditions should be considered as a convenient way of triggering formation of holoRd and its folding. Indeed, iron addition to apoRd starts a sequence of events whose rates and yield can be easily monitored through spectroscopic approaches.28,29

NPs can increase yields of holoRd formation from structured apoRd

Despite most of the apoRd being free in solution even at the highest NP concentrations used in this study, the presence of NP resulted in a significant improvement in the equilibrium yield of folded holoRd, as indicated by the signature visible CD spectra of the Fe(III)(SCys)4 site in holoRd shown in Figure 1(A) with signals at 492 (negative) and 570 nm (positive). As shown in Figure 1(B), the equilibrium yield in holoRd became nearly quantitative at NP concentration around 0.2 g/L, but decreased slightly at lower or higher NP concentrations. Thus, NP appeared to be most effective in promoting holoRd formation at concentrations where only a minor fraction of the apoRd present in the system is bound on their surface, suggesting the occurrence of turnover events. HoloRd prepared in the presence of 0.2 g/L NP had an iron content of 1 6 0.02 mol Fe/mol protein, indicating quantitative reincorporation of iron, as was the case for protein reconstituted in the presence of denaturing concentrations of 5M urea without NPs. Whereas the improvement in holoRd yield observed in the presence as well as the absence of NP may be explained by the hypothesized unfolding of apoRd when binding to the NP surface, the observation of an optimal apoRd:NP ratio for holoRd yield is not so

4

PROTEINS

straightforwardly rationalized. One possible explanation is that steric effects due to molecular crowding of bound apoRd on the NP surface allow binding of apoRd to NP in an orientation and/or structure that favors an ironuptake competent conformation.39 In support of this system geometry effect, the yield of holoRd did not increase more than 1.5–2-fold over that observed in buffer alone when various concentrations of 200-nm NP were used in place of the 46-nm NPs. Binding experiments indicated that these large NP have a binding affinity for apoRd comparable to that of the small ones (on a surface area basis) at similar NP and protein concentrations (data not shown).

NP concentration has a large effect on apoto holoRd conversion rates

On the basis of the time course of holoRd formation, as detected by the appearance of the signature CD signals of the folded and oxidized holoRd (Fig. 2), the fastest rates were observed at 0.2 g/L NP, and increasing or lowering the NP concentration with respect to this optimum value (at 0.05 mM apoRd) was much more detrimental to the rate than yield of holoRd formation [compare Figs. 2(B) and 1(B)]. This contrast supports the turnover and adsorption orientation/structure hypotheses proposed earlier. As reported previously,28 apoRd in solution over several days slowly converts into a conformer with distinctive CD and 1H-NMR features, that has almost lost the ability to take up iron and to refold into holoRd,28 even when 2-mercaptoethanol is added to keep the cysteine residues reduced. However, the “aged” uptakeincompetent apoRd conformer was reported to give fast and quantitative formation of holoRd when unfolded in 5M urea.28 We observed fast and nearly quantitative holoRd formation from “aged,” uptake-incompetent apoRd

Nanoparticles-Assisted Rubredoxin Refolding

Figure 2 A: Semilog plot for the time course of holoRd formation–as monitored by CD–under the various conditions listed in the panels of Figure 1. Circles, buffer only; squares, buffer containing 5M urea. Triangles indicate the addition of NP to the following concentrations (g/L): 0.04 (blue); 0.2 (magenta); 0.4 (green); 2.0 (red). B: Pseudo-first order rates of holoRd formation as a function of the NP concentration.

in the presence of 0.2 g/L NP (Table I). The rates and yields for the “aged” apoRd in the presence of 0.2 g/L NP were nearly indistinguishable from those obtained with the urea-denatured “aged” apoRd. All of these observations are consistent with the hydrophobic and/or cysteine exposure hypothesis for the catalytic role of NP. Precoating NP with beta-lactoglobulin inhibits their ability to accelerate apo to holoRd conversion

The requirement for apoRd contact with the NP surface in the “catalysis” of holoRd formation is underscored by the evidence presented in Figure 3, which compares the time courses of holoRd formation when 46 nm NP were used before and after pre-coating with bovine beta-lactoglobulin, a protein that tightly–but not covalently–adheres to NP through hydrophobic interactions.30 The precoating clearly impairs the NP’s ability to function as a catalyst for holoRd formation. This observation also provides circumstantial evidence of the stability of the interaction between NP and a relatively large (Mr 16000 Da) and hydrophobic protein, such as beta-lactoglobulin, that cannot be displaced from the NP surface by the much smaller and much less hydrophobic

apoRd even when this latter is present in relatively large excess. Increasing ionic strength leads to modest increases in holoRd formation rates

The data in Figure 4 indicate that the presence of NaCl slightly improves the rate of holoRd formation regardless of the presence of other components in the reaction medium. Although this may suggest that ionic strength affects specific steps of the uptake/refolding process, a more thorough study of these effects was not possible, as NP formed insoluble aggregates at NaCl concentrations above 0.25M in the buffer system used in our studies. The increased rates observed in the presence of NaCl in Figure 4 also rules out that charge-dependent iron accumulation in the immediate vicinity of the NP surface may be responsible for the observed accelerating effects of NP stemming from altered local concentration of the interacting species. Increased iron concentrations had been shown to result in increased rate and yield of holoRd formation in refolding studies on chaotropeunfolded apoRds.28 The data in Figure 4 clearly indicate that the presence of competing ions improves rather

Table I Rate and Yield of holoRd Formation from Freshly Prepared and Aged apoRd Refolding rate constant (min21 3 103)

holoRd yield (%) Apo type

Buffer

1 5M urea

1 0.2 g/L NP

Buffer

1 5M urea

1 0.2 g/L NP

Freshly prepared Aged

38 6 7 22 6 6

100 6 6 88 6 7

101 6 8 86 6 9

28 6 4 22 6 7

133 6 16 101 6 12

147 6 11 107 6 18

Rates and yields were calculated from reconstitution experiments using equimolar amounts of apoRd and ferric ammonium citrate, similar to those reported in Figures 1 and 2. The preparation indicated as “aged apoRd” was kept under Ar at 4 C for 4 weeks after iron removal, as a 0.5 mM protein solution in 100 mM Tris-HCl pH 7.4 (Bonomi et al., 2008). Values are given plus or minus standard deviation (n  3).

PROTEINS

5

M. Miriani et al.

Figure 3 Precoating with betalactoglobulin (BLG) impairs the NP-dependent acceleration of holoRd formation. Reconstitution experiments were carried out as in Figures 1 and 2. Where indicated, 46 nm NP were precoated with a subsaturating amount of bovine beta-lactoglobulin (0.05 g/L protein and 0.625 g/L NP), as reported elsewhere.30

than impairing the rate of holoRd formation, thus ruling out the possible role of electrostatic interactions in accumulation of ferric ions on the NP surface. Effect of NP on chronology of iron uptake vs. holoRd formation

As reported in previous studies, appearance of the absorption spectrum typical of a Fe(III)(Cys)4) structure

Figure 4 Effects of ionic strength on the early phases of the time course of holoRd formation. Formation of holoRd was monitored continuously by following ellipticity changes at 495 nm after the addition of 0.05 mM ferric ammonium citrate to 0.05 mM apoRd in buffer (open symbols) or in buffer containing 0.15M NaCl (full symbols). Circles, buffer only; squares, buffer 1 5M urea; triangles, buffer 1 0.2 g/L NP.

6

PROTEINS

Figure 5 A comparison of CD (495 nm) and absorbance (492 nm) time courses. Changes in ellipticity (full symbols) or in absorbance (open symbols) were recorded after the addition of 0.05 mM ferric ammonium citrate to 0.05 mM apoRd in buffer (circles), and in buffer containing 5M urea (squares), 0.2 g/L NP (upward triangles), or 2 g/L NP (downward triangles).

recovered much more rapidly than the signature CD signal of the folded holoRd when reconstitution was carried out in plain buffer (Fig. 5). This observation had been taken as evidence that metal ligation triggers subsequent protein folding, and that refolding of the iron-ligated protein into the native holoRd was the rate-limiting step of the whole process.28,29 As observed in those studies, the time course of metal coordination and of folding events was much closer in 5M urea (although the respective sequence was retained), indicating that the kinetic barriers toward refolding of metal-containing holoRd were lowered by the conformational freedom induced by chaotropes. The data in Figure 5 suggest that a similar situation may occur in the presence of NP, and that–at appropriately high molar ratios protein/NP–the rate of holoRd refolding almost matches that of metal coordination. Figure 5 also provides evidence that the lower rates and yields associated with very high NP concentrations (2 g/ L, see also Figs. 1 and 2) may be due to the fact that the apoRd conformer present on the NP surface at low protein/NP ratios is somehow “iron-capture impaired” with respect to that present at higher protein/NP ratios. The lower ability of this hypothetical conformer at capturing iron translates, out of necessity, in making it also folding impaired. Taken together, the data in Figure 5 indicate that: (1) iron chelation by apoRd in the presence of NP involves the NP-bound species; (2) iron binding to ligand cysteines triggers subsequent folding events as observed for urea-denatured apoRd; (3) the interaction between NP and the iron-ligated NP adhering to its surface are not

Nanoparticles-Assisted Rubredoxin Refolding

Figure 6 A schematic view of the events leading to iron uptake and rubredoxin refolding on the NP surface.

strong enough to impede the structural changes that lead to holoRd refolding when starting from iron-ligated–but yet unfolded–apoRd.

DISCUSSION Taking into account the evidence presented earlier, our current view of the events occurring in the NP “catalysis” of holoRd formation is summarized in Figure 6. This highly schematic presentation tries to account for the

reported turnover of activated apoRd species on the NP surface and for the hypothesized relevance of molecular crowding effects to the rate and yield of metal uptake and protein refolding, that represent sequential but kinetically separated events in holoRd formation. Incorporation of iron and refolding of holoRd in the presence of NP may be a balance between events involving three apoRd species: (a) folded apoRd in solution (that gives very slow rates and low yields also because apoRd over time converts into an uptake-incompetent form (see “aged” in Table I28); (b) a conformer of PROTEINS

7

M. Miriani et al.

unfolded apoRd on the NP surface that still has an impaired ability to convert into holoRd when iron is present; (c) a conformer of unfolded apoRd on the NP surface that can be converted into holoRd as efficiently as that of the urea-denatured apoRd. The results indicate that this latter species is most abundant at relatively high molar ratios of apoRd/NP, where molecular crowding may favour formation of an iron uptake competent conformer rather than an ironuptake incompetent conformer promoted at lower molar ratios apoRd/NP. Alternatively, the same conformer of the bound and unfolded apoRd could be present on the NP surface but with differing orientations dictated by molecular crowding. Finally, the observed decrease in rates and yields at very low NP concentrations [Fig. 2(B)] may be explained as a decrease in the number of iron-uptake competent apoRd species, that is, of the number of protein molecules being bound to NP in the proper orientation or conformation. The data in Figure 5 indicate that the orientation of the apoRd with respect to the NP surface only affects the rate of iron uptake, but has no appreciable effects on the folding events that occur after formation of a tetrahedral Fe(SCys)4 site. This report demonstrates the feasibility of exploiting the structural changes ensuing from protein interactions with the hydrophobic surface of polystyrene NP for “selective” denaturation of protein regions that could be relevant to those protein folding events that are triggered by the binding of nonprotein components to the protein itself. For incorporation of iron into apoRd, our results indicate that the NP may act as genuine “catalyst” in the iron incorporation and/ or refolding process, in a way that seems to be dependent on the geometry of interaction between the protein and the NP surface. In this regard, NPs may be seen as a biomimetic chaperone. Indeed, it has been well established that chaperone/cochaperone systems most enhance folding rates and yields by inducing selective unfolding of specific regions of the target protein.9,10 For instance, chaperone systems relevant to the assembly/transfer of inorganic cofactors have been shown to act through ATP/driven distortion of metal ligation.14 Although most of the “bona fide” chaperone system require ATP hydrolysis for overcoming the thermodynamic barriers associated with either substrate uptake or product release,5,6 there are reports of chaperone-improved refolding of metalloproteins in the absence of cochaperones and of ATP hydrolysis.40 Thus, the concept of turnover folding through protein unfolding on a suitable surface (a protein or–in the present case–a biomimetic nanoparticle) followed by triggering of the refolding through binding of the appropriate cofactor is not completely new. Several metallo-proteins (i.e., proteins containing either single or multiple type of metal cofactor) have been reconstituted in vitro (in variable yield) from solutions of their apoform in chaotropes, most often by adding the required inorganic components to the chaotrope-unfolded protein, followed by dilution of the chaotrope to an extent and at a

8

PROTEINS

rate appropriate with concomitant refolding of the native holoprotein.41–44 This study underscores the possibility that polystyrene NP could serve as the functional equivalent of traditional protein chaotropes. Such a substitution should be used to address practical and mechanistic folding issues related to the use of chaotropes. Practical issues include excessive protein dilution, that may lead to stability or solubility problems, dissociation of multimeric proteins, metal loss during further purification steps when starting from nonpurified apoproteins (such as those often present as inclusion bodies when overexpressing the protein of interest). Structural issues are implicit in the use of high concentrations of chaotropes, that typically unfold vast portions of the (apo)protein tertiary structure in quite nonselective fashion by acting mostly on hydrophobic interaction. Thus, proper reversal of the sequence of events during refolding in the presence of cofactors (as occurs in the case studied here) often had been a matter of chance, or of allowing enough time for equilibration between possible conformers at any given chaotrope/metal/protein concentration. Our observation that polystyrene NPs enhance the formation and/or stability of a specific refolding-prone conformation of the protein could potentially be used to overcome some of the difficulties encountered when reconstituting “random coil” proteins in concentrated chaotrope solutions when the folding process involves incorporation of a native cofactor. In this frame, the sensitivity of rate and yield of the refolding triggered by nonprotein components to the system geometry and to possible protein-specific factors (be that the protein/NP mass ratio, the ionic strength, the NP size and charge, or other physical and chemical factors) may provide opportunities for “engineering” this novel function of nanostructured materials. The possibilities of selective denaturation of specific protein regions on the NP surface, and of using the newly exposed surface for binding of appropriate nonprotein components offer some novel perspectives for understanding the molecular aspects of “assisted folding” events and for engineering novel functions into existing cofactorbearing proteins, possibly including those where folding relates to the uptake/presence of other cofactors.16 ACKNOWLEDGMENTS MM is the grateful recipient of a post-doctoral fellowship from the University of Milan, in the frame of the POR FSE Project 2007-13, ASSE IV, “Gain and loss of function and structure of proteins on the surface of solid nanoparticles and of lipid-based nanostructures". REFERENCES 1. Ellis J. Proteins as molecular chaperones. Nature 1987;328:378–379. 2. Gething MJ, Sambrook J. Protein folding in the cell. Nature 1992; 355:33–45. 3. Todd MJ, Viitanen PV, Lorimer GH. Facilitated protein-folding. Science 1994;265:659–666.

Nanoparticles-Assisted Rubredoxin Refolding

4. Gershenson A, Gierasch LM. Protein folding in the cell: challenges and progress. Curr Op Struct Biol 2011;21:32–41. 5. Jackson GS, Staniforth RA, Halsall DJ, Atkinson T, Holbrook JJ, Clarke AR, Burston SG. Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle–implications for the mechanism of assisted protein folding. Biochemistry 1993;32:2554–2563. 6. Vickery LE, Cupp-Vickery JR. Molecular chaperones HscA/Ssq1 and HscB/Jac1 and their roles in iron-sulfur protein maturation. Crit Rev Biochem Mol Biol 2007;42:95–111. 7. Rochet JC. Novel therapeutic strategies for the treatment of proteinmisfolding diseases. Exp Rev Mol Med 2007;9:1–34. 8. Ventura S, Villaverde A. Protein quality in bacterial inclusion bodies. Trends Biotechnol 2006;24:179–185. 9. Martin J, Langer T, Boteva R, Schramel A, Horwich AL, Hartl FU. Chaperonin-mediated protein folding at the surface of GroEL through a molten globule-like intermediate. Nature 1991;352: 36–42. 10. Brazil BT, Horowitz PM. The hydrophobic properties of GroEL: a review of ligand effects on the modulation of GroEL hydrophobic surfaces. Cell Stress Chaperones 1999;4:177–190. 11. Gomez-Puertas P, Martin-Benito J, Carrascosa JL, Willison KR, Valpuesta JM. The substrate recognition mechanisms in chaperonins. J Mol Recogn 2004;17:85–94. 12. Niemiec MS, Weise CF, Wittung-Stafshede P. In vitro thermodynamic dissection of human copper transfer from chaperone to target protein. PLOS One 2012;7: Article Number: e36102. 13. Kozlowski H, Potocki S, Remelli M, Rowinska-Zyrek M, Valensin D. Specific metal ion binding sites in unstructured regions of proteins. Coord Chem Rev 2013;257:2625–2638. 14. Bonomi F, Iametti S, Morleo A, Ta D, Vickery LE. Facilitated transfer of IscU[2Fe2S] clusters by chaperone-mediated ligand exchange. Biochemistry 2011;50:9641–9650. 15. Mavridou DAI, Ferguson SJ, Stevens JM. Cytochrome c assembly. IUBMB Life 2013;65:209–216. 16. Christiansen A, Wang Q, Cheung MS, Wittung-Stafshede P. Effects of macromolecular crowding agents on protein folding in vitro and in silico. Biophys Rev 2013;5:137–145. 17. Park IY, Eidsness MK, Lin IJ, Gebel EB, Youn B, Harley JL, Machonkin TE, Frederick RO, Markley JL. Smith ET, Ichiye T, Kang CH. Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: Effects of side-chain size on reduction potential. Proteins 2004;57:618–625. 18. Johnson DC, Dean DR, Smith AD, Johnson MK. Structure, function, and formation of biological iron-sulfur clusters. Annu Rev Biochem 2005;74:247–281. 19. Bonomi F, Iametti S, Morleo A, Ta D, Vickery, LE. Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones. Biochemistry 2008;47: 12795–12801. 20. Bonomi F, Fessas D, Iametti S, Kurtz DM, Mazzini S. Thermal stability of Clostridium pasteurianum rubredoxin: deconvoluting the contributions of the metal site and the protein. Prot Sci 2000;9: 2413–2426. 21. Bonomi F, Eidsness MK, Iametti S, Kurtz DM, Mazzini S, Morleo A. Contribution of the [Fe(II)(SCys)4] site to the thermostability of rubredoxins. J Biol Inorg Chem 2004;9:297–306. 22. Zartler ER, Jenney FE, Terrell M, Eidsness MK, Adams MWW, Prestegard JH. Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum. Biochemistry 2001;40:7279–7290. 23. Bonomi F, Burden AE, Eidsness MK, Fessas D, Iametti S, Kurtz DM, Mazzini S, Scott RA, Zeng QD. Thermal stability of the [Fe(SCys)4] site in Clostridium pasteurianum rubredoxin: contributions of the local environment and Cys ligand protonation. J Biol Inorg Chem 2002;7:427–436.

24. Maher M, Cross M, Wilce MCJ, Guss JM, Wedd AG. Metal-substituted derivatives of the rubredoxin from Clostridium pasteurianum. Acta Cryst D 2004;60:298–303. 25. Richie KA, Teng Q, Elkin CJ, Kurtz DM, Jr. 2D 1H and 3D 1H-15N NMR of zinc-rubredoxins: Contributions of the b-sheet to thermostability Prot Sci 1996;5:883–894. 26. Taylor PK, Parks BYA, Kurtz DM, Amster IJ. Analysis of metal incorporation during overexpression of Clostridium pasteurianum rubredoxin by electrospray FTICR mass spectrometry. J Biol Inorg Chem 2001;6:201–206. 27. Bertini I, Kurtz DM, Eidsness MK, Liu GH, Luchinat C, Rosato A, Scott RA. Solution structure of reduced Clostridium pasteurianum rubredoxin. J Biol Inorg Chem 1998;3:401–410. 28. Bonomi F, Iametti S, Ferranti P, Kurtz DM, Jr, Morleo A, Ragg EM. "Iron priming" guides folding of denatured aporubredoxins. J Biol Inorg Chem 2008;13:981–991. 29. Morleo A, Bonomi F, Iametti S, Huang VW, Kurtz DM. Ironnucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events. Biochemistry 2010;49: 6627–6634. 30. Miriani M, Eberini I, Iametti S, Ferranti P, Sensi C, Bonomi F. Unfolding of beta-lactoglobulin on the surface of polystyrene nanoparticles: experimental and computational approaches. Proteins 2014;DOI: 10.1002/prot.24493. 31. Barbiroli A, Bonomi F, Ferranti P, Fessas D, Nasi A, Rasmussen P, Iametti S. Bound fatty acids modulate the sensitivity of bovine betalactoglobulin to chemical and physical denaturation. J Agric Food Chem 2011;59:5729–5737. 32. Deng ZJ, Liang MT, Monteiro M, Toth I, Minchin RF. Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nat Nanotech 2011;6:39–44. 33. Pan H, Qin M, Meng W, Cao Y, Wang W. How do proteins unfold upon adsorption on nanoparticle surfaces? Langmuir 2012;28:12779–12787. 34. Sheng Y, Wang W, Chen P. Adsorption of an ionic complementary peptide on the hydrophobic graphite surface. J Phys Chem C 2010; 114:454–459. 35. Sheng Y, Wang W, Chen P. Interaction of an ionic complementary peptide with a hydrophobic graphite surface. Protein Sci 2010;19: 1639–1648. 36. Eidsness MK, Burden AE, Richie KA, Kurtz DM, Scott RA, Smith ET, Ichiye T, Beard B, Min TP, Kang CH. Modulation of the redox potential of the [Fe(SCys)4] site in rubredoxin by the orientation of a peptide dipole. Biochemistry 1999;38:14803–14809. 37. Lovenberg W, Walker MN. Meth Enzymol 1978;53:340–346. 38. Bonomi F, Iametti S, Kurtz DM, Jr, Ragg EM, Richie KA. Direct metal ion substitution at the [M(SCys)4]2- site of rubredoxin. J Biol Inorg Chem 1998;3:595–605. 39. Mikaelsson T, Aden J, Johansson LBA, Wittung-Stafshede P. Direct observation of protein unfolded state compaction in the presence of macromolecular crowding. Biophys J 2013;104:694–704. 40. Iametti S, Bera AK, Vecchio G, Grinberg A, Bernhardt R, Bonomi F. GroEL-assisted refolding of adrenodoxin during chemical cluster insertion. Eur J Biochem 2001;268:2421–2429. 41. Hong JS, Rabinowitz JC. The effects of chemical modifications on the reconstitution, activity, and stability of clostridial ferredoxin. J Biol Chem 1970;245:4988–4994. 42. Gupta N, Bonomi F, Kurtz DM, Ravi N, Wang DL, Huynh BH. Recombinant Desulfovibrio vulgaris rubrerythrin–isolation and characterization of the diiron domain. Biochemistry 1995;34:3310–3318. 43. Iametti S, Uhlmann H, Ragg E, Sala N, Grinberg A, Beckert V, Bernhardt R, Bonomi F. Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states. Eur J Biochem 1998;251:673–681. 44. Wilson CJ, Apiyo D, Wittung-Stafshede P. Role of cofactors in metalloprotein folding. Q Rev Biophys 2004;37:285–314.

PROTEINS

9