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Switch in the Aggregation Pathway of Bovine Serum Albumin Mediated by Electrostatic Interactions Shivnetra Saha and Shashank Deep* Department of Chemistry, Indian Institute of Technology, Delhi, Hauz-Khas, New Delhi 110016, India S Supporting Information *

ABSTRACT: A strong denaturant, guanidinium hydrochloride (GdnHCl), is shown to delay and alter the inherent aggregation pathway of bovine serum albumin (BSA) from a downhill polymerization to a nucleated polymerization. We hypothesize that such an alteration is closely connected to the conformational population of the protein, and ion-binding to such an ensemble. Hindered molecular collisions due to electrostatic repulsions in an ion-bound denatured ensemble increase the activation barrier for aggregation to such an extent that the growth, which was spontaneous in the absence of any cosolute, goes through an unfavorable nucleation phase. Our study shows that the behavior in GdnHCl is not unique to it, but occurs in a certain class of cosolutes those which are charged and bind to BSA. Variation in pH of the medium, which gives rise to extra charges on the protein backbone, also showed such repulsive effects, further confirming the involvement of electrostatic interactions. We have further shown that the coexistence of both an appropriate population and an appropriate cosolute is necessary. An absence of either of these prevents a switch in the pathway.

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ceases to exist.13 A reversal from downhill to nucleated polymerization though, to our knowledge, is not very well reported. A vast number of cosolutes have been shown to affect the lag phase or growth phase of aggregation.14−16 However, very few studies have been reported about the changing routes of aggregation in the presence of these cosolutes.6 We report here a case where a strong protein denaturant, guanidinium hydrochloride (GdnHCl), has been shown to transform the downhill aggregation pathway of BSA to a nucleated one. Our study shows that the behavior observed in GdnHCl is not unique to it, but occurs in a certain class of cosolutes, viz., charged species that have the ability to bind to BSA.

INTRODUCTION Nucleation-dependent polymerizations are often characterized by the onset of aggregation after a certain lag.1−3 The existence of a lag phase is suggestive of an energy barrier that needs to be overcome, before a protein can start forming aggregates.4−8 Some proteins, however, immediately surrender to aggregation, without any lag phase, when subjected to appropriate conditions.9−12 Such proteins are said to follow a downhill pathway and encounter no energy barrier before aggregating. What is peculiar about the pathways is the fact that they have no correlation with the secondary structure of proteins. Of two different helical proteins, one can undergo nucleated polymerization with a well-defined lag phase (insulin),2 while the other can aggregate immediately without any delay (bovine serum albumin (BSA)).10 On the other hand, both a helical protein (BSA) and a β-sheet-rich protein (transthyretin) can undergo instant polymerization without any lag.11 In spite of the extensive studies done in the context of molecular selfassembly, it is still not very clear as to why some proteins aggregate without a nucleus and some do not. In this report, we show that a protein which intrinsically aggregates downhill can be made to follow a nucleated aggregation pathway by manipulating its conformational population and the net charge on the protein. A nucleated pathway can be transformed to a downhill pathway above a certain concentration of the protein, known as the super critical concentration, beyond which the lag phase © 2014 American Chemical Society

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MATERIALS AND METHODS Chemicals. BSA, fraction V, and GdnHCl were purchased from Sisco Research Laboratories, Mumbai, India. Fluorescent probes Thioflavin T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS) and 9-(2,2-Dicyanovinyl) julolidine (DCVJ) were purchased from Sigma-Aldrich. Glycerol and histidine were purchased from Sisco Research Laboratories; valine and arginine were purchased from Spectrochem Pvt. Ltd., Mumbai, India; dextrose and ethanol were purchased from Merck; alpha Received: March 11, 2014 Revised: July 11, 2014 Published: July 15, 2014 9155

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kept at 450 and 485 nm, respectively, and ThT fluorescence was recorded for 800 s. Dynamic Light Scattering. Aggregated samples of BSA (20 μM) at 343 K (in presence and absence of cosolutes) were drawn out at regular time intervals and quenched to 5 μM by dilution using 10 mM Tris buffer, pH 7.5. The size of the species in the different aliquots was then measured using Zetasizer Nano, Malvern instrument for dynamic light scattering (DLS), at a scattering angle of 90°. All the measurements were performed at 298 K. At longer aggregation times, the size could not be measured accurately due to formation of larger aggregates. Zeta Potential. Zeta potential of 5 μM BSA in the presence of varying concentrations of cosolutes was measured in a cuvette equipped with a folded capillary tube and gold electrodes in a Zetasizer Nano, Malvern instrument. The concentrations of the cosolutes that have been used in the aggregation experiments (0.8 M) are too high for the measurement of zeta potential due to an increased charge. Hence, much lower concentrations were used to see the effect of changing concentration of cosolutes on the protein. Size Exclusion Chromatography. Twenty micromolar aggregated samples of BSA (in the presence and absence of cosolutes) at 343 K were taken out at the required time points and then diluted to 15 μM using 10 mM Tris, pH 7.5. The samples were filtered using a 0.22 μm syringe filter to remove any higher or insoluble aggregates, to prevent chocking of the column. The sample was then analyzed using size exclusion chromatography using Superdex 200 increase 10/300 GL column in a GE Akta purifier FPLC system using a loop of 1 mL and an injection volume of 800 μL. Samples were eluted isocratically, and the mobile phase was 20 mM Tris, 100 mM NaCl. The absorbance from the samples was recorded at 215 and 280 nm. Transmission Electron Microscopy. Five microliters of the aggregated solution of BSA was placed on a copper grid and then stained with uranyl acetate solution. This was allowed to dry for half an hour and then viewed with a JEOL 2100F transmission electron microscope. Data Analysis. The kinetic traces of the evolution of ThT fluorescence intensity and Rayleigh scattering intensity was fitted to the following multiexponential equation:

cyclodextrin and sorbitol were purchased from Sigma-Aldrich. Tris was purchased from Merck. Stock Solutions. Stock solution of BSA was prepared at pH 7.5 in 10 mM Tris-HCl, and the concentration was determined using a molar extinction coefficient of 43 824 M−1cm−1 at 280 nm, on a Cary 100 Bio UV−visible spectrophotometer. Stock solutions of ANS and ThT were made in Milli Q water, purified from the Millipore system, and concentration was determined using a molar extinction coefficient of 5000 M−1cm−1 at 350 nm and 35 000 M−1cm−1 at 412 nm for the two dyes, respectively. Stock solution of DCVJ was prepared in ethanol, and the concentration was determined using a molar extinction coefficient of 65 900 M−1cm−1 at 453 nm. Stock solutions of the cosolutes were prepared in 10 mM Tris-HCl, and pH was adjusted to 7.5. Aggregation at pH 6.8 or 8.5 was also carried out in 10 mM Tris-HCl, and cosolutes required for aggregation at these pH values were prepared in the respective buffers. Aggregation at pH 1.6 or 12 was carried out in 0.1 M NaCl− HCl and 0.1 M KCl−NaOH, respectively. All the steady state fluorescence measurements were done in a Varian Cary Eclipse Fluorescence Spectrophotometer. Aggregation Kinetics. To prepare the aggregated samples, 20 μM BSA, alone and in the presence of cosolutes, was incubated at 343 K. The kinetics of aggregation was monitored both in situ and ex situ. The obtained parameters from the two methods were nearly the same and within the limits of experimental error. For the ex situ experiments, aliquots were withdrawn at different time intervals and diluted four times in cold buffer (10 mM Tris-HCl, pH 7.5) to quench the aggregation. Rayleigh scattering intensity of the aggregates was measured at 600 nm (λex and λem = 600 nm). For the extrinsic fluorescence studies, ThT and ANS were added before making the measurements. ThT was excited at 450 nm, and the emission was collected at 482 nm. ANS was excited at 350 nm, and the emission was collected at 473 nm, while DCVJ was excited at 470 nm, and the emission was collected at 505 nm. Tryptophan fluorescence was monitored only in situ by exciting the protein at 295 nm and collecting the emission at 350 nm. For in situ measurements, the dye was added to the working solution at the beginning and the change in the fluorescence intensity was monitored with time in a Varian Cary Eclipse Fluorescence Spectrophotometer. The Rayleigh scattering was also monitored similarly, in the absence of any dye. Aggregation at pH 12 was performed only ex situ, due to the instability of ThT in alkaline medium. For monitoring the changes in secondary structure with time, aliquots were diluted 5 times in cold buffer, and the CD spectra were recorded from 260 to 200 nm on an Aviv, Model 420 SF Circular Dichroism Spectrometer. Seeds were prepared by aggregating 50 μM BSA in the absence of any cosolute. Freshly prepared seeds were sonicated and added to a fresh solution of 20 μM monomeric BSA, in the presence of 0.8 M GdnHCl, with a final seed concentration of 20 μM. Stopped-Flow Fluorescence. Stopped-flow fluorescence was measured in BioLogic Stopped-Flow Spectrometer system. The instrument was kept at 323 K using the water bath provided with the system. ThT, dissolved in 10 mM Tris, pH 7.5, was heated in a separate water bath at 348 K. BSA and the heated buffer (at 348 K) was injected just before starting the experiment. The mixing ratio from the two syringes was kept at 7:2, with a final volume of 1018 μL in the cuvette FC-08. The dead time of mixing was 2.0 ms. Excitation and emission was

F = F ∞ + ΔF exp( − (kt )n )

(1)

where F is the observed signal at any time t, F∞ is the final intensity, ΔF is the difference between the initial and final signal, k is the rate of aggregation, and n is any number. The number n is a signature of the cooperativity of aggregation. The higher the value of n, the higher is the cooperativity and longer will be the lag phase. The length of the lag phase was calculated using eq 2, where y0 is the initial intensity, yf is the final intensity, 1/b is the apparent rate constant for the growth of aggregates, and x0 is the time at which the aggregation is half complete. The lag phase is given by (x0 − 2b).2,8,17 y = y0 +

A 1 + exp( −(x − x0)/b)

(2)

It should be noted that although eqs 1 and 2 have been used robustly in literature, they are entirely empirical in nature, and they do not provide any information about the actual underlying molecular events. A more detailed analysis is presented in the text. 9156

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enhance the aggregation of proteins.9,15 However, the addition of a strong denaturant, GdnHCl, during the aggregation of BSA, led to a very unusual result. Addition of 0.8 M GdnHCl was seen to induce a distinct lag phase in the aggregation of BSA, implying that it is providing the protein with an energy barrier to aggregation, despite being a denaturant. The aggregation profile, as monitored by both ThT fluorescence and Rayleigh scattering, resulted in a typical sigmoidal curve (Figure 1A,B), as found for a large number of proteins that aggregate via a nucleated pathway. To avoid the insensitivity of these techniques in detecting oligomers of sizes below a certain threshold value,19,20 the aggregation was also monitored by DLS. Aggregation, when monitored by DLS, also did not show any remarkable change in size of the particle in the initial stages of aggregation (Supporting Figure 1C). However, the width of the distribution at longer incubation times became broader, indicating the increasing heterogeneity of the solution as the aggregation progressed. The total time taken for aggregation in 0.8 M GdnHCl was also higher than that in its absence. However, the longer aggregation time and the initial inhibition are deceptivethe reason being a significantly higher Rayleigh scattering intensity in the presence of GdnHCl than in its absence (Figure 1B). The Rayleigh scattering intensity in the presence of 0.8 M GdnHCl was also significantly higher than the ThT fluorescence intensity (Figure 2A), which probably indicates a bias toward the formation of a certain aggregate morphology. While the aggregates of BSA in buffer alone developed a prominent fluorescence at 425 nm (λex = 355 nm), such an intrinsic fluorescence was missing in the native protein, or when BSA was aggregated in 0.8 M GdnHCl (Figure 2B). Recent reports21,22 say that such an intrinsic fluorescence develops due to energy states that become available when the protein aggregates into a fibrillar structure. The loss of fluorescence from the aggregates in the presence of 0.8 M GdnHCl points toward a change in the aggregate morphology. This is further supported by the TEM images (Figure 2C,D), where aggregates in the presence of 0.8 M GdnHCl showed lumpy structures, confirming the selective growth of amorphous aggregates. The observed lag phase could be shortened by increasing the concentration of the protein. However, concentration dependence of the aggregation of BSA in the presence of 0.8 M GdnHCl was high (Supporting Figure 1D), unlike the case in its absence, hinting toward a possible change in the mechanism of aggregation. The lag phase in the presence of 0.8 M GdnHCl could be shortened upon the addition of seeds (Figure 3). This was an important observation, since the aggregation of BSA alone remained unaffected by seeding.10 In order to eliminate the possibility of an undetectable short lag phase in buffer alone, the aggregation of BSA was also monitored using stopped-flow fluorescence, in a separate experimental set up. The dead time of mixing was 2.0 ms and the kinetic profile still had no lag phase (Supporting Figure 2A,B). Besides, stabilizing cosolutes, which can increase the lag time of aggregation and delay the process, only decreased the growth rate of aggregation of BSA, without the induction of any lag phase. Other commonly known cosolutes affected only the rate of the process, suggesting that the association mechanism, on a broader scale, still remained the same (Supporting Figure 2C). The temporal evolution of the secondary structure of BSA, during its aggregation in the presence of 0.8 M GdnHCl, when monitored by the change in ellipticity at 222 nm, showed an

RESULTS AND DISCUSSION The aggregation of BSA, in the presence or absence of cosolutes was measured by monitoring the ThT fluorescence intensity with time. The kinetic profile of the aggregation of BSA alone, at pH 7.5, 343 K shows a concave profile, without any lag phase (Figure 1A).10,18 Under this condition, BSA

Figure 1. Comparison of the kinetics of the ThT fluorescence (A) and Rayleigh scattering intensity (B) of 20 μM BSA at 343 K, pH 7.5, in the presence and absence of 0.8 M GdnHCl. Graphs in the inset show the same at short times. For a better comparison of the difference in the extent of aggregation, the figures have been normalized from 1, where 1 corresponds to the lowest intensity.

forms worm-like fibrillar structures, as already reported.10 A similar concave profile was seen when the aggregation was monitored by Rayleigh scattering at 600 nm (Figure 1B). The Rayleigh scattering intensity, as a function of time, is thought to have contribution from all the different species of aggregates, ThT-positive, or otherwise. The aggregation remains unaffected by seeding (data not shown).10 Increasing concentrations of protein increased the rate of aggregation linearly. This is evident from the log−log plot of the rate of aggregation versus the concentration, which had a slope of 0.986 (Supporting Figure 1A). DLS measurements have also shown an immediate increase in the size of the species from 7.031 to 17.38 nm upon the initiation of aggregation (Supporting Figure 1B). Aggregation in globular proteins occurs under conditions that can destabilize the native state of the protein.7 In sync with this, addition of destabilizing cosolutes has been shown to 9157

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Figure 2. (A) Graph showing the relatively high increase in Rayleigh scattering during the aggregation of BSA in 0.8 M GdnHCl (pH 7.5, 343 K), suggesting predominantly amorphous aggregation. (B) Intrinsic fluorescence of BSA fibril in pH 7.5, 343 K, without cosolute (black) and the disappearance of the fluorescence in the presence of 0.8 M GdnHCl, pH 7.5, 343 K (blue). (C) TEM images of BSA fibrils. (D) TEM images of BSA aggregated in the presence of 0.8 M GdnHCl (pH 7.5, 343 K).

in the hydrophobic patches, along with the tryptophan residues getting exposed to a more solvent accessible state (Supporting Figure 3). Thus, the kinetics of these conformational changes still proceed without a lag even in the presence of GdnHCl (similar to that observed in its absence), despite there being a prominent lag in kinetics of aggregation. This leads us to conclude that while GdnHCl does not affect the intramolecular conformational changes during the course of aggregation, it does affect the intermolecular association during this process. Such behavior contrasts that of another protein, insulin, where both the conformational changes and the aggregation proceed concomitantly via a distinct lag phase.23 Now that we have established that there is a significant change in how BSA aggregates in the absence and presence of 0.8 M GdnHCl, it is worth investigating how the monomers are getting incorporated into the aggregates during the course of the reaction. Monomeric BSA in the absence of any additive was eluted at 13.45 mL, with a monomer peak area of 443.41 mAU mL, in size exclusion chromatography (Figure 4A). Upon aggregation at 343 K in buffer alone, the monomer peak area decreased, along with the emergence of a new peak at 9.06 mL due to the formation of new soluble oligomers (Figure 4B). The temporal evolution of the size exclusion profiles had a continuous increase in the oligomer peak area along with a

Figure 3. (A) Effect of addition of preformed seeds in the aggregation of BSA at 343 K in 0.8 M GdnHCl.

immediate loss of the helicity, without any lag (Supporting Figure 3, inset). A time scan of the tryptophan and ANS fluorescence showed a concomitant decrease in the fluorescence intensity, indicating that the distortion of the secondary structure during aggregation is also accompanied by a decrease 9158

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Figure 4. SEC profiles of the aggregation of BSA in the absence and presence of GdnHCl. (A) Change in monomer peak of BSA with increasing time during aggregation at 343 K in buffer alone. (B) Change in oligomer peak of BSA with increasing time during aggregation at 343 K in buffer alone. Oligomers are formed from the initial times. (C) Change in the monomer and oligomer content (peak area) of BSA with time during aggregation at 343 K in buffer alone. Monomer loss occurs in sync with oligomer formation. (D) Change in monomer peak of BSA with increasing time during aggregation at 343 K in the presence of 0.8 M GdnHCl. (E) Change in the oligomer peak of BSA with increasing time during aggregation at 343 K in the presence of 0.8 M GdnHCl. No oligomers were seen initially. (F) Change in the monomer and oligomer content of BSA with time during aggregation at 343 K in 0.8 M GdnHCl.

the virtue of diffusion alone. It is only when a sufficient fraction of the denatured population (C) is attained, for example, at elevated temperatures, that aggregation begins, and proceeds immediately without any lag. Such a process, limited by monomer conformational change, does not respond to preformed seeds. A primary outline of the underlying events thus predicts the depletion of monomers (N) from the solution, occurring in sync with the formation of soluble oligomers (O) via conformationally changed monomers (C). The exponential decay of N, and a corresponding exponential growth of O (via C) with a linear dependence on time, all of which are observed for the aggregation of BSA in buffer alone, can be represented as N(t) = N(0)[1 − exp(−kNCt)].24 On the basis of these observations, along with a linear dependence of the rate on the monomer concentration (Supporting Figure 1A), it can be confirmed that BSA aggregates via a downhill pathway. Downhill pathways are those in which the protein can aggregate without any energy barrier, owing to which aggregation can commence immediately. It is possible that such an aggregation will depend on the rate at which

concomitant reduction in the monomer peak area. The peak for the oligomers also shifted to lower elution volumes with increasing time, indicating an increase in both the number and the size of the aggregates. The simultaneous loss in the monomer confirmed that they were indeed getting incorporated into the oligomers, and that the change in the oligomer peak was not due to oligomers coalescing with each other. With further increase in time, the amount of soluble oligomers reached a maximum, after which they began to decrease (Figure 4C) as a result of the conversion of the soluble oligomers into higher insoluble aggregates. The loss of monomers from the solution and the growth of soluble oligomers were exponential in nature (like a first order process, Figure 4C), similar to that observed for the aggregation monitored by ThT fluorescence or Rayleigh scattering. The rate of growth of aggregates increased linearly with increasing concentration of the protein (Supporting Figure 1A). Also, as already discussed, the growth of aggregates is accompanied by the loss of the protein conformation, as seen by CD, ANS fluorescence and tryptophan fluorescence. Monomers (N), as present in solution, do not have any propensity to aggregate by 9159

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Figure 5. Existence of secondary pathways during the aggregation of BSA at 343 K in the presence of 0.8 M GdnHCl, as seen from a good fit to the relation Δ = A(cosh Bt − 1).

monomers (N) encounter each other. This will be limited by diffusion, and will depict quadratic time dependence during aggregation.13,24,25 However, our observations rule out this possibility, since BSA, as prepared in solution, does not aggregate; a conformational change is indispensable before aggregation. This categorizes BSA in that class of downhill polymerizations that are limited by conformational changes, as suggested by Powers and Ferrone.24 During the aggregation of BSA at 343 K in the presence of GdnHCl, the depletion of monomers from the solution in the initial times, as observed through size exclusion chromatography, was quite negligible, as compared to the aggregation in buffer alone (Figure 4D). This is not unexpected in the regime of lag phase in an aggregating system. However, with increasing time, the kinetics of monomer loss became much more rapid than in the early times (Figure 4F, inset). Besides, no additional peak at lower elution volumes was detected initially (unlike the case of BSA alone), confirming that GdnHCl prevents the formation of new oligomers, which are otherwise favorable in its absence (Figure 4E). It is only after a certain lag that a new peak for oligomers appears at a lower elution volume. As the aggregation proceeds, the soluble oligomers increase both in number and size, similar to that observed in BSA alone. In this case also, the amount of soluble oligomers first reached a maximum, and then started decreasing upon formation of larger insoluble aggregates (Figure 4F). Since monomers were getting depleted, although insignificantly, the absence of any new oligomer in the initial stages shows that the rate of depletion of the monomers was much faster than the rate of formation of the oligomers. This is consistent with a process where the reactants (monomers) are combining to form a higher order species (oligomers). Such a phase was absent for the aggregation of BSA in buffer alone, again suggesting a probable alteration in the mechanism of aggregation.

A shift of the aggregation profile to one with a distinct lag phase hints toward a prominent shift in the association mechanism of BSA, and this is also evident from the kinetics of monomer loss in solution. The lag phase observed in sigmoidal aggregation profiles of proteins has often been assumed to be the time required for the formation of an unfavorable nucleus in a nucleated polymerization. This, however, need not always be the case, as suggested by Ferrone26 and Cohen et al.27 Lag phases can exist even without a nucleus, or both nucleation and growth can be operative in the initial flat phase of the reaction, or most nuclei can be formed after the lag.4,26 For example, Ferrone has explained that a flat lag phase may also be a result of downhill polymerization with secondary pathways.26 This, however, does not hold for our system, as we shall see shortly. Analysis of the kinetic profiles of aggregation has long been used to derive insights into the mechanism of aggregation.28,29 The time course of the aggregation of BSA in the presence of 0.8 M GdnHCl showed an early exponential growth, which was in good agreement with Δ= A(cosh Bt −1) for the early stages (∼15−20%) of the reaction (Figure 5). Such an observation, often reflected by an abrupt growth in the kinetic curve, is indicative of the existence of secondary pathways in the system, wherein existing aggregates aid in the formation of newer aggregates. These pathways may or may not be monomer dependent. The existence of secondary pathways was first reported by Ferrone et al., in order to explain the abrupt growth during the aggregation of sickle cell hemoglobin.30 Secondary pathways are an inherent property of many proteins.30−33 They are, however, not intrinsic in BSA, as seen from the kinetic profile (Figure 1). Our study shows that secondary pathways can be introduced by modulating the conditions of aggregation. In the alternate, aggregates can also be created from the monomers alone, a phenomenon commonly referred to as 9160

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primary nucleation. Depending on the protein, aggregation may occur via primary or secondary pathways alone, or both. It must be realized that secondary pathways cannot be operative unless some aggregates are formed or are already present in the solution, since such pathways cannot be initiated without the presence of aggregates. BSA, in the presence of 0.8 M GdnHCl, can aggregate even without the addition of preformed seeds, pointing to the fact that primary pathways alone can create aggregates required for the initiation of secondary pathways. A close examination of the seeded aggregation of BSA in the presence of 0.8 M GdnHCl (Figure 3) shows that the reaction is complete even before the growth of detectable aggregates have started in the unseeded reaction. The creation of these new aggregates in a time scale where unseeded reaction does not create enough aggregates is a reflection of the fact that the preformed seeds play a significant role in creating new aggregates. This observation, in addition to the early exponential growth, establishes the importance of both primary and secondary pathways during the aggregation of BSA in 0.8 M GdnHCl. In another representative case, where seeds are a prerequisite for the formation of aggregates, primary pathways have a very little role to play.34,35 Various secondary pathways include fragmentation, heterogeneous nucleation and branching. A plethora of studies have shown that primary and secondary pathways during the course of aggregation are controlled by two parameters, λ and κ.27,36,37 If the rate constant for the formation of the primary nucleus be kn and that of elongation be k+, then λ is given by λ = (2k + knm(0)nc)1/2, where nc is size of the critical nucleus/the order of the reaction,4,27,36 and m(0) is the initial concentration of the monomer. If secondary pathways exist, and k− (or k2) be the rate constants for monomer-independent fragmentation (or monomer-dependent heterogeneous nucleation), then κ is given by κ = (2k+k−m(0)n2+1)1/2 (or κ = (2k+k2m(0)n2+1)1/2), where n2 is the size of the secondary nucleus/reaction order for the secondary processes. With fragmentation as a secondary pathway, n2 will have a value of 0. k+, kn, k−, and k2 are the microscopic rate constants of aggregation, and are independent of the concentration of the protein.4,27 The existence of secondary pathway during aggregation in the presence of 0.8 M GdnHCl has been confirmed (Figure 5). An individual estimate of the primary and the secondary rate constants can be obtained from the fits of early time behavior of the reaction using the relation38 M(t ) = m(0)

λ2 (cosh(κt ) − 1) κ2

Figure 6. Determination of the primary and the secondary nucleus size from the kinetic parameters λ and κ. The slope obtained for the monomer concentration dependence of λ is 1.526, and that of κ is 1.501.

We now identify the secondary pathway operative in the system. It is to be noted that for a system where both primary and secondary pathways exist, deconvolution of the two can be complex. A preliminary idea about the prevalent secondary pathway in the system can be obtained from the scaling of kinetic parameters with the concentration of monomers. Such a scaling relation can be represented as τlag ∼ m(0)γ. γ equals (nc/ 2) and ((n2 + 1)/2), depending upon whether the primary or secondary pathway is predominant.4,27,34,36,40 The scaling exponent for the aggregation of BSA in 0.8 M GdnHCl was determined from the lag times of aggregation (determined from eq 2) and yielded a value of 1.74 (Supporting Figure 1C). A similar scaling was obtained using the half times of aggregation. The observed values of γ eliminates fragmentation as the secondary pathway in our system, since fragmentation would lead γ to be nearly equal to 0.5 (n2 = 0). A log−log plot of κ versus the monomer concentration gives us a value of 2 for the secondary nucleus, confirming that the secondary pathway is not fragmentation, but is heterogeneous nucleation (Figure 6). The prominent features in the aggregation of BSA in the presence of 0.8 M GdnHCl, viz., the presence of a lag phase during aggregation, response to seeding, and a higher order dependence of aggregation on the concentration of protein, all of which are in contrast to that of the aggregation in absence of GdnHCl, provides us with a mechanistic basis on which the two cases may be isolated. All the features just listed ascertain that the mechanism has shifted to a nucleated polymerization in 0.8 M GdnHCl, from a downhill polymerization in its absence. The induction of lag phase by 0.8 M GdnHCl was not observed in another well-known denaturant, urea (Supporting Figure 4A). Concentrations of urea up to 3.5 M did not impart any lag at the temperature of aggregation (343 K). This might seem striking in the first place, since both GdnHCl and urea denature the protein, and have a similar starting population at the temperature of aggregation (Supporting Figure 5). However, urea is a neutral denaturant, while GdnHCl is charged and is involved in electrostatic interactions with the charged residues of the protein.41,42 To see if the unusual behavior in GdnHCl was an effect of charge alone, the unipositive guanidinium cations and the chloride anions were mimicked with NaCl. However, concentrations of up to 2 M NaCl did not show any lag phase at 343 K, and in fact stabilized

(3)

As suggested by Ferrone,26 the reaction order of the primary and the secondary processes can be obtained from a linear fit of the logarithmic values of κ or λ versus the logarithm of the monomer concentration, the former being obtained by fitting the first 20% of the data to eq 3. This, in our case, gives us a value of nucleus size, nc ∼ 3 for the primary process, from the slope (= nc/2) of the log−log plot (Figure 6). Such a value of nc confirms the presence of a nucleus in the aggregation of BSA in 0.8 M GdnHCl and eliminates the possibility of it being a downhill aggregation with secondary pathways, which would also result in a long lag phase. It is also important to mention that the kinetic profiles in the presence of 0.8 M GdnHCl was not in agreement with a tn dependence39 (which again results in a lag phase), eliminating the possibility of an irreversible cascade reaction. 9161

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BSA toward aggregation (Supporting Figure 4B). Mimicking the combined electrostatic and denaturing effect of GdnHCl with 0.8 M urea and 0.8 M NaCl together, did not impart any lag phase either (Supporting Figure 4C). The contrasting observation in GdnHCl and NaCl is not inexplicable. High concentrations of NaCl have been suggested to make the tertiary structure of this protein more compact.43 The stabilization of BSA by sodium ions occurs by the phenomenon of preferential hydration,44 that is, the preferential exclusion of the salt from the surface of the protein. This preferential hydration of BSA drives its unfolding equilibrium more toward the native state, leading to lesser aggregation. GdnHCl on the other hand, binds to the protein and unfolds it. The unfolding of the protein exposes further sites onto which more GdnHCl can bind.41,42 The binding of GdnHCl to the protein increases the charge on it. This creates new repulsive forces among the unfolded monomers, owing to which, the monomers cannot interact with each other to form the aggregates. We believe that the hindered interactions due to enhanced intermolecular repulsions, generate a high activation barrier, and introduce the energetically unfavorable lag phase that is observed in the presence of 0.8 M GdnHCl. Constant exposure to high temperature provides the required energy to overcome the activation barrier, following which, the growth phase is observed. It is worth noting that urea also binds to BSA,45 but does not induce a lag phase due to the lack of charge, eliminating any possibility of intermolecular repulsions. If molecular repulsions are truly responsible for the lag phase, then other fluorescent probes that can sense and respond to a change in particle size through association should not show a significant change in the fluorescence in this period. The molecular rotor DCVJ, is one such probe, which strongly fluoresces upon restricted molecular rotation. Lindgren et al. have shown that this dye binds to the early species like the molten globule and other oligomers during the aggregation of transthyretin.46 In our case, when the aggregation of BSA in the presence of GdnHCl was monitored by DCVJ fluorescence, a significant lag phase was observed, indicating a lack of oligomerization, and hence confirming the intermolecular repulsions (Supporting Figure 6A). On the other hand, DCVJ showed an immediate response to aggregation in absence of GdnHCl (Supporting Figure 6B), thus distinguishing the phenomena in the two cases. If our speculation about the intermolecular repulsion is true, then other ions known to bind to BSA and destabilize it, should also impart a lag phase to the aggregation kinetics. And indeed, when the aggregation was carried in the presence of 0.8 M MgCl2, or 0.8 M CaCl2, both of which bind to BSA,44 a lag phase was observed (Supporting Figure 7). Both of them were found to induce a lag phase and selectively promote the amorphous aggregation, similar to GdnHCl. A lag was also seen in SDS, which again is charged and binds to BSA.47−49 The conformational changes still proceeded without any lag phase in the presence of these ions. Increasing concentrations of positively charged ions like GdnHCl and CaCl2 increased the zeta potential of BSA from −9.01 mV in buffer to −5.54 mV in the presence of GdnHCl (Figure 7) and to −2.82 mV in the presence of CaCl2 (data not shown). Higher zeta potential results from higher electrostatic repulsions. Such repulsions are expected to increase with temperature. Increasing temperature also unfolds the protein, leading to higher binding of the ions and hence higher repulsions, thus preventing the coalescence of monomers with each other. Increasing concentrations of

Figure 7. Change in the zeta potential of BSA with increasing concentration of GdnHCl.

GdnHCl or CaCl2 also resulted in increased electrophoretic mobility (Supporting Figure 8), indicating that migrating protein molecules were attaining higher charges due to binding of the cosolutes. These additional charges, bringing about an increased repulsion among the protein moleules, prevent oligomer formation during the process of aggregation. Interestingly, concentrations lower than 0.8 M GdnHCl did not show any lag phasethe curves were similar to those observed in absence of any cosolute (Supporting Figure 9). Studying the aggregation in higher concentrations of GdnHCl at the temperature of aggregation (343 K) for the entire time course was not feasible in the experimental set up used (although a lag phase was observed for the early times). However, higher concentrations of the destabilizing ions are expected to result in greater repulsions among the molecules, and this in turn should result in a longer lag phase. This is what was observed when the concentration of the binding cosolute (CaCl2 and SDS) was varied (Supporting Figure 10A,B). Lowering the concentration of these binding ions leads to a shortening, and ultimately, a disappearance of the lag phase. An analogous trend can be expected for increasing concentrations of GdnHCl since it differs in behavior in only the concentration at which a lag phase is induced. Other properties like shortening of the lag phase upon addition of preformed seeds or the observation of a lag phase when the aggregation was probed by DCVJ were also found to hold true for CaCl2, confirming that our hypothesis can be extended beyond GdnHCl (Supporting Figures 6A and 10C). The disappearance of the lag phase at lower concentrations of GdnHCl suggested a possible role of the conformational population of the protein, since the concentration of GdnHCl would affect the fraction of the unfolded species at a particular temperature. The higher the concentration, the higher this fraction would be. The aggregation was thus carried out in the absence of GdnHCl, at higher temperatures of 353 and 363 K (where the ensemble is expected to be fully denatured, as evident from the thermal profiles, Supporting Figure 5) to exclusively investigate the role of denatured states in the aggregation pathway. No lag phase was observed for both ordered and amorphous aggregation, but the relative increase in the Rayleigh scattering intensity from 353 to 363 K was much higher as compared to that in the ThT fluorescence intensity, which remained similar (Supporting Figure 11). This suggests that an unfolded ensemble is more prone to forming 9162

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amorphous aggregates, probably because it reduces the chances of specific interactions required to form the patterned fibrils. This behavior is reminiscent of the enhanced amorphous aggregation seen in the presence of 0.8 M GdnHCl, indicating that the conformational population is one of the factors responsible for this unusual phenomenon. To further support our hypothesis, the aggregation was studied in the presence of binding ions at 343 K at different pH conditions. If repulsion between positive ion-bound proteins has an important role to play in imparting a lag time, an increase in the positive charge on the protein, by lowering the pH, is expected to increase the lag time, whereas a decrease in the positive charge by increasing the pH should reduce the lag time. In the presence of 0.8 M Ca2+, lowering of pH to 6.8 drastically increased the lag time as compared to that in pH 7.5, whereas increasing the pH to 8.5 led to a reduction in the lag time (Figure 8). Also, we expect the reverse when negatively Figure 9. Relation between the starting population and the aggregation pathway of BSA in presence of 0.8 M GdnHCl. When the starting population is (a), 330 K, the aggregation is downhill, without any lag phase, whereas the aggregation is nucleated (with a lag phase) when the starting population is (b), 343 K.

appearance of a lag phase (Figure 9). The ions must be bound to a denatured population. A general scheme demonstrating our hypothesis for the switch in the aggregation pathway of BSA is given in Scheme 1. Scheme 1. Change in the Aggregation Pathway of BSA from Downhill Polymerization to Nucleated Polymerization

Figure 8. Effect of pH on the aggregation of BSA in the presence of Ca2+ ions at 343 K.

charged ions are involved. In the presence of SDS, a decrease in pH to 6.8 decreased the lag time, while it increased the lag time at pH 8.5 (data not shown). Lag time was also observed at pH 1.6 and pH 12 (where the protein will be highly positively charged and negatively charged, respectively), in the absence of any binding ions, reconfirming the role of electrostatic interactions in altering the pathway of aggregation (Supporting Figure 12). ThT-positive aggregates at such high and low pH values were negligible. At such extreme values of pH, the protein will be far away from its native conformation that exists at pH 7.5.50,51 In spite of this, they show similar aggregation behavior. This indicates that it is the charge on the protein, and not its conformation, that plays the predominant role in inducing the lag phase. Aggregation of BSA at 330 K in 0.8 M GdnHCl did not show any lag phase. The kinetic profile was similar to that in the absence of any additives. A close examination of the thermal profile shows that the population is not fully denatured at this temperature (Figure 9). These observations, viz., (i) the absence of a lag phase at concentrations of GdnHCl lower than 0.8 M, (ii) the absence of a lag phase at a lower temperature (330 K) in 0.8 M GdnHCl, and (iii) the presence of a lag phase at a higher temperature (343 K) in 0.8 M GdnHCl, establish the importance of the conformational population. The binding of ions is a necessary but not a sufficient condition for the

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SUMMARY AND CONCLUSION Partial denaturation of the native state of the protein is one of the prerequisites for the formation of aggregates,7 which, in this study, has been attained by thermally denaturing the protein at 343 K. Addition of GdnHCl at this temperature increases the fraction of the denatured population (Figure 9). Although denatured populations are required for aggregation to occur, a large population of denatured states has been shown to hinder aggregation in proteins, due to prevention of noncovalent interactions.9,15 The unusual emergence of a distinct lag phase in the aggregation of BSA in 0.8 M GdnHCl appears to be, at the outset, a reflection of this phenomenon, in that a fully 9163

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cosolutes. Aggregation kinetics of BSA at 343 K monitored by DCVJ. Aggregation of BSA in CaCl2, MgCl2 and SDS. Electrophoretic mobility of BSA in the presence of GdnHCl. Aggregation of BSA at lower concentrations of GdnHCl. Relative change of ThT positive aggregates and amorphous aggregates. Aggregation kinetics of BSA at high and low pH. Change in the secondary structure of BSA at different temperatures. This material is available free of charge via the Internet at http://pubs.acs.org.

denatured population initially resists aggregation. Once this resistance is overcome, BSA aggregates to a much larger extent in GdnHCl, contrary to what might be expected from a fully denatured population. A fully denatured population at the same temperature, in the presence of urea, on the other hand, does not show a lag phase, indicating that the initial resistance observed in GdnHCl is not simply a consequence of hindered noncovalent interactions. Our results suggest that the lag phase emerges as a consequence of intermolecular repulsions among the protein molecules. The length of the lag phase can be modified by manipulating the charges on the protein residues, brought about by a change in the pH or by changing the concentration of the binding ions. Aggregation of proteins, even to this date, remains an incompletely answered question. Protein aggregation holds a vital status in both industry and pathology, but at the same time, expounding the facts of protein aggregation is rendered challenging due to its noncorrelation with the amino acid sequence.52,53 Given the universality of this problem, we chose to study the aggregation of BSA and have shown that the intrinsic pathway of its aggregation can be changed by altering the conformational population and the environment of the protein. This is also accompanied by a change in the aggregate morphology. Amorphous or ordered aggregation is a consequence of the close balance between the hydrophobic interaction and hydrogen bonding. While amyloid fibril formations are dominated by hydrogen bonding interactions, increasing hydrophobic interactions result in amorphous aggregates.54,55 Cosolutes, which are charged and can bind to the denatured population, or environments that can increase the net charge on the protein, both of which give rise to electrostatic repulsion among the monomers, can switch the aggregation pathway of BSA from downhill to a nucleated one, with an increase in the extent of amorphous aggregation. Such conditions also seem to perturb the balance between the hydrophobic interactions and hydrogen bonding interactions, leading to a change in the nature of aggregates. Downhill polymerizations proceed via a series of irreversible steps, do not require the formation of a nucleus and encounter no energy barrier in the aggregation pathway. In such a scenario, monomers are the most unstable species in the aggregation pathway and all other oligomeric species are more stable than the native form. Downhill polymerization might therefore transpire as a setback to preventing aggregation. The formation of a nucleus in a nucleated polymerization involves equilibrium between the monomers and the nucleus, and is not energetically favored. In light of this, the ability to switch the mechanism of aggregation of proteins to a nucleated pathway, under physiologically relevant conditions, might serve as an important step toward formulating a general approach to hinder aggregation. We believe this study will prove useful in this regard, and help in elucidating the mechanistic nuances of the phenomena of protein aggregation.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.D. thanks the Council of Scientific and Industrial Research (CSIR), India, for the generous funding to carry out the above research. S.S. thanks CSIR, Government of India, for the fellowship. We thank Dr. Pramit Kumar Chowdhury, Department of Chemistry, IIT-Delhi, for his helpful discussions and invaluable suggestions towards improving this study. We are thankful to Dr. Gajender Saini and Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, for providing the TEM facility. We thank DeitY for providing us with the DLS facility. We thank Dr. Suman Kundu, Department of Biochemistry, University of Delhi, South Campus, for providing us with the stopped-flow fluorescence spectrometer, and Sheetal Uppal, his Ph.D student, for helping in carrying out the stopped-flow measurements.

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ABBREVIATIONS GdnHCl, guanidinium hydrochloride; ThT, thioflavin T; ANS, anilino-1-naphthalenesulfonic acid; DCVJ, 9-(2,2-dicyanovinyl) julolidine; TEM, transmission electron microscopy; SEC, size exclusion chromatography; DLS, dynamic light scattering

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ASSOCIATED CONTENT

S Supporting Information *

Dependence of the rate constant/lag times on the concentration of protein. DLS in the absence and presence of GdnHCl. Aggregation monitored by stopped-flow fluorescence. Kinetic profiles of the aggregation of BSA in the presence of different cosolutes. Kinetics of the conformational changes of BSA at 343 K. Aggregation of BSA in the presence of urea and NaCl. Thermal denaturation of BSA in the presence of 9164

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