Mechanistic Insights into the Stabilization of srcSH3 by PEGylation

Article pubs.acs.org/Langmuir

Mechanistic Insights into the Stabilization of srcSH3 by PEGylation Wei Meng, Xinlu Guo, Meng Qin, Hai Pan, Yi Cao,* and Wei Wang* National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu 210093, People’s Republic of China S Supporting Information *

ABSTRACT: Protein PEGylation (attaching PEG chains to proteins) has been widely used in pharmaceuticals and nanotechnology. Although it is widely known that PEGylation can increase the thermodynamic stability of proteins, the underlying mechanism remains elusive. In this Article, we studied the effect of PEGylation on the thermodynamic and kinetic stability of a protein, SH3. We show that the thermodynamic stability of SH3 is enhanced upon PEGylation, mainly due to the slowing of the unfolding rate. Moreover, PEGylation can decrease the solvent-accessible surface area of SH3, leading to an increase of the mvalue (the change in free energy with respect to denaturant concentration, which is a measure of the transition cooperativity between corresponding states). Such an effect also causes an enhancement of the thermodynamic stability. We quantitatively measured how the physical properties of PEG, such as the molecular weight and the number of PEGylation sites, affect the stabilization effect. We found that the stabilization effect is largely dependent on the number of PEGylation sites but only has a weak correlation with the molecular weight of the attached PEG. These experimental findings inspire us to derive a physical model based on excluded volume effect, which can satisfactorily describe all experimental observations. This model allows quantitatively calculating the free energy change upon PEGylation based on the change of water excluded zone on the protein surface. Although it is still unknown whether such a mechanism can be extended to other proteins, our work represents a key step toward the understanding of the nature of protein stabilization upon PEGylation.

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INTRODUCTION Proteins have been increasingly used as novel drugs for the treatment of human diseases.1,2 However, different from small molecule drugs, the stability of proteins is marginal and prone to aggregate or degrade after administration, which greatly impedes the application of protein-based drugs.3−5 To achieve optimum activities of protein-based drugs, many strategies have been developed to circumvent these drawbacks, which include using polymeric micelles as protein delivery system and direct conjugation of hydrophilic polymers to proteins.6,7 Poly(ethylene glycol) (PEG) is one of the most widely used macromolecular modifiers for protein conjugation.8−15 Thanks to its high water solubility, covalently linking PEG or PEGylation to protein can effectively increase the solubility of proteins and therefore prevent aggregation. Moreover, PEG can also increase the thermodynamic stability of proteins and retard the proteolysis. The history of PEGylation can be traced back to the later 1970s.16,17 Abuchowski and co-workers showed that PEG-conjugated proteins as drugs are significantly more effective than the unmodified ones.17 Since then, PEGylation has become a versatile strategy to improve the efficacy of protein drugs and is widely used in pharmaceutical research.18−33 Besides extensive pharmaceutical application, PEGylation is also widely used in surface chemistry and nanotechnology.34−36 PEG linkers have been developed as an efficient strategy to link proteins to nanoparticle surface, which could potentially © 2012 American Chemical Society

prevent the adsorption of proteins to nanoparticles and avoid protein unfolding and aggregation. Especially, in the recently emerging single-molecule studies, PEG is widely used to fix biomacromolecules, mainly proteins, to various surface or manipulation devices (e.g., cantilever tip of atomic force microscope).37−41Therefore, it is important to address whether such PEG-based tethering could lead to the alteration of the stability and function of proteins. Despite the broad applications of protein PEGylation, the effects of PEGylation on the chemical and physical properties of proteins are less understood. Most studies so far are focused on the consequence of PEGylation on the activity of proteins. At the more fundamental level, the studies of PEGylation on the thermodynamic stability are far behind.20,23,27,30,31,42 Although there are two studies showed that PEGylation could increase the thermodynamic stability of proteins in a length- and position-dependent fashion due to the change of the surface coverage of PEG molecules,43,44 these studies mainly focused on the effects of PEGylation on the thermodynamic stability, and no kinetic studies were provided. A quantitative model that describes the PEGylation effect is lacking. Received: August 28, 2012 Revised: October 24, 2012 Published: October 30, 2012 16133

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PAGE using a Mini-PROTEAN 4-gel electrophoresis system (BioRad). PEGylated SH3 samples were mixed with SDS sample buffer (10% glycerol, 62.5 mM Tris-HCl, pH 6.8 containing 2% SDS, 5% βmercaptoethanol, and 0.01 mg mL−1 bromophenol blue). Next, these samples were boiled at 100 °C for 3 min to completely denature the proteins before being loaded onto the SDS polyacrylamide gels. Depending on the molecular weight of the PEGylated SH3, we used either 13% or 16% gels to achieve optimal separation. The electrophoresis was running at 150 V for 120−150 min. Gels were stained in a solution containing 0.1% Coomassie brilliant blue and destained in a solution containing 10% acetic acid and 50% methanol. Circular Dichroism. CD spectra were measured using a JASCO J815 CD spectropolarimeter (Jasco, Japan). The concentration of protein samples was 0.17 mg mL−1, and the buffer was PB buffer (50 mM). The data were recorded from 250 to 200 nm at room temperature using a quartz cell with 0.1 mm path length. The data reported in this Article were averaged from at least five scans to improve the signal-to-noise ratio, and the contribution from buffer was subtracted. Results were expressed as mean residue ellipticity (θMRE), calculated according to eq 1, where θobs is the observed ellipticity (in deg), d is the path length (in cm), C is the concentration of protein samples (M), and n is the total number of amino acids in the protein.

Herein, we studied the effect of PEGylation on both the thermodynamic and the kinetic stability of a SH3 domain (srcSH3). SH3 comprises two three-stranded β-sheets that packed orthogonally against each other and has been extensively used as a model protein for protein folding studies.45−49 SH3 folds through a simple two-state pathway without any detectable folding intermediate states.45 Although SH3 is not therapeutically relevant, it makes up a very large family (over 400 members) and widely exists in a variety of proteins as an important domain. SH3 contains two lysine residues at positions 19 and 20. The amino groups from these two lysine residues and the N-terminus of SH3 can be PEGylated with aldehyde-terminated PEG (mPEG-ALD).50,51 Our kinetic studies indicate that both folding and unfolding of SH3 are slowed upon PEGylation. However, the slowing of the unfolding rate is more significant than the folding rate. Therefore, the overall thermodynamic stability of SH3 upon PEGylation is increased. Moreover, PEGylation reduces the solvent-accessible surface area of SH3, leading to an increase of the m-value. Such effect also accounts for the increase of the thermodynamic stability. We further propose a physical model based on the excluded volume effect, which can adequately explain all our experimental findings. On the basis of this model, the effect of PEG length and number of PEGylation sites on the increase of thermodynamic stability can be predicted.

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θMRE = (100θobs)/[dC(n − 1)]

(1)

Equilibrium Denaturation Measurements. Equilibrium denaturation was performed to determine ΔG and m-value. Equilibrium fluorescence data were collected on a JASCO FP6500 spectrofluorometer. Excitation was set at 280 nm, and emission was recorded from 300 to 400 nm. Samples for denaturation titrations were prepared by mixing desired amounts of protein sample with guanidine hydrochloride (GdnCl) stock solution in PB buffer to achieve a series of specified GdnCl concentrations. Temperature was maintained at 295 K. All samples were centrifuged before measurements. Denaturation curves were analyzed using an equilibrium two-state model:

MATERIALS AND METHODS

Chemicals. Aldehyde-terminated linear PEG (mPEG-ALD) with molecular weights of 5000 and 10 000 (>95% pure) were purchased from Kaizheng Biotech Co. (Beijing, China). TEV protease was purchased from NEB (Ipswich, MA). All other chemicals are of reagent or molecular biology grade. Protein Expression and Purification. The chicken srcSH3 gene was directly purchased from Genscript (Nanjing, China) and subcloned into pQE80L vector between BamHI and KpnI restriction sites. There is a 6× His-tag and a TEV cleavage site at the N-terminus of SH3. The protein was expressed in the BL21 pLysS strain of Escherichia coli. Each clone harboring a corresponding plasmid was grown in 2.5% LB medium containing 100 mg L−1 ampicillin at 37 °C under vigorous stirring (225 rpm), and induced with 1 mM isopropyl1-β-D-thiogalactoside (IPTG) when its optical density (OD) at 600 nm reached ∼0.6−0.8. Protein expression continued for 4 h at 37 °C. The cells were harvested by centrifugation and resuspended in precooled lysis buffer (20 mM Na3PO4, 500 mM NaCl, pH 7.4)), further lysed by 100 μM lysozyme, and sonicated on ice for 8 min. Next, the cell lysate was centrifuged at 12 000 rpm for 30 min at 4 °C. The protein was purified using Ni2+-NTA protein resin (GE healthcare, Shanghai) and eluted in elution buffer (20 mM Na3PO4, 500 mM NaCl, 250 mM imidazole, pH 7.4). Typically, 50−100 mg of protein was obtained from 1 L of LB medium. The purified protein was dialyzed against a buffer containing 50 mM NaH2PO4 (PB buffer, pH 5) for all subsequent experiments. PEGylation and Purification. PEGylation was done by mixing desired ratios of SH3 (1.5 mg mL−1) and mPEG-ALD at room temperature in PB buffer, pH 5, in the presence of 50 mM sodium cyanoborohydride (NaBH3CN), and under a stirring speed of 100 rad min−1. The reaction was carried out in a 50 mL falcon centrifuge tube. Approximately 30 mg or 20 mg of srcSH3 was used for mono- or multi-PEGylation each time. The reaction proceeded for 24 h and was purified using a gel filtration column (Superdex 75 10/300 GL) equipped in an Ä KTA FPLC system (GE Healthcare). The flow rate was set as 0.6 mL min−1. Before the purification, the column was equilibrated with 2 column volume (CV) of PB buffer (50 mM, pH 5). In a typical run, ∼0.4−0.8 mg of protein samples was injected, and the UV absorbance at 280 nm was used to monitor the separation. The purified PEGylated SH3 samples were further confirmed using SDS-

F = (αN + βN[denaturant] + αD + βD[denaturant]) exp(m([denaturant] − [denaturant]50% )/RT ) /(1 + exp[m([denaturant] − [denaturant]50% )/RT )

(2)

where αN+βN[denaturant] and αD+βD[denaturant] are the baselines for the native state and the unfolded state, respectively, R is gas constant, T is temperature, [denaturant]50% is the transition middle point, F is the observed spectroscopic signal, and m is a constant of proportionality and reflects the change in solvent-accessible surface area between the unfolded and folded states. Typically, m-value is also a measure of folding cooperativity of proteins. Stopped-Flow Folding/Unfolding Kinetics. Kinetics of folding and unfolding was followed by stopped-flow fluorescence (Biologic SFM300, France). Native protein stock was added to PB buffer or GdnCl stock to a final protein concentration of 3−5 μM to initiate unfolding or folding at 295 K. At each GdnCl concentration, five experiments were performed sequentially, and the kinetic traces were averaged. The kinetic traces were monitored by fluorescence emission at 320 nm and excitation at 280 nm. Folding and unfolding rates were determined from single-exponential fits to the kinetic traces using Biologic software. The chevron plot was fitted by the following equation:

ln(kobs) = ln(k fH2O exp(− mf [denaturant]/RT ) + k uH2O exp(m u[denaturant]/RT ))

(3)

where kobs is the observed rate constant, kH2O and kH2O are the folding f u and unfolding constants in the absence of denaturant, respectively, mf and mu are the folding and unfolding m-values, respectively, R is gas constant, and T is temperature. 16134

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RESULTS PEGylation of SH3. There are three sites in SH3 that can be functionalized with mPEG-ALD. They are the N-terminal amino group and the other two amino groups from lysine residues (Lys19 and Lys20), respectively (Figure 1). Because

multifunctionalized SH3 (mPEG-ALD:SH3 = 20:1) are shown in Figure 2a. For the PEG molecular weights of 5000 and 10 000 Da, the conjugation rates are similar. Moreover, in both cases, the monoconjugated and triconjugated SH3 can be well separated due to their different molecular weights using a size-exclusion column. The separated PEGylated SH3 samples were further confirmed by SDS-PAGE before further biophysical characterization (Figure 2b). It is worth mentioning that in the SDS-PAGE experiment, proteins are denatured by SDS and heat treated (100 °C for 3 min). This process may make the denatured PEGylated proteins prone to aggregate, leading to higher molecular weight oligomers shown in the gel. However, these high molecular weight aggregates are not found in FPLC traces, indicating that the PEGylated proteins do not aggregate in their native conditions. We then checked the PEGylated position for monoconjugated SH3. Because there is a TEV digestive sequence between the His-tag and the Nterminal of SH3 (see the Supporting Information for the detailed protein sequence), TEV digestion can remove the Nterminal conjugated PEG. Enzymatic digestion confirmed that the PEG tag in the monofunctionalized SH3 sample was indeed attached to the N-terminal amine (Figure 2c). Because there are three possible attaching sites for bifunctionalized SH3, the bifunctionalized SH3 samples were a mixture and were not used in this study. The monoconjugated SH3 of PEG molecular weights of 5000 and 10 000 Da are named as SH3-PEG5k and SH3-PEG10k, respectively. Accordingly, the triconjugated SH3 of PEG molecular weights of 5000 and 10 000 Da are named as SH3-(PEG5k)3 and SH3-(PEG10k)3, respectively. Effect of PEGylation on the Structure of SH3. We first characterize the structure of PEGylated SH3 using far-UV circular dichroism. Because β strands in SH3 are short and twisted (Figure 1), the CD spectrum of SH3 is atypical for βsheet proteins.52 As shown in Figure 3, unmodified SH3 shows a weak positive peak at ∼236 nm, a minor negative peak at ∼228 nm, and a major negative peak at ∼202 nm. PEGylation causes a slight shift of all three peaks to longer wavelength and reduces the intensity of the major peak at ∼202 nm by less than 20%. More PEGylation sites and longer PEG chains can lead to more dramatic structural alteration to SH3. However, the overall structural change of SH3 upon PEGylation is minute, suggesting that there are no strong interactions between PEG and SH3.

Figure 1. The native structure of srcSH3. The three PEGylation sites are highlighted in yellow.

the pKa of the N- terminal amino group is generally lower than that of the lysine ε-amino group, the N-terminal amino group has much higher reactivity than the side chain of lysine residues at a low pH. Therefore, it is possible to preferentially functionalize the N-terminal amino group at a low pH. Moreover, increasing the ratio of PEG could get more amino groups functionalized. We screened the reaction conditions to optimize the ratio of PEG to SH3 to obtain desired PEGylated products (Figure S1). Low mPEG-ALD to SH3 ratio could ensure that the vast majority of products were monoconjugated proteins at pH 5. Excess mPEG-ALD gradually converted monoconjugated products to multiconjugated ones. However, even at an mPEG-ALD to SH3 ratio of 30, only less than 40% of SH3 are triconjugated. This is probably because the two lysine residues in SH3 are in proximity. To balance the yield and conjugation efficiency, we used the molar ratio of mPEGALD to SH3 of 1:1 for N-terminal conjugation and 20:1 for three-site conjugation (Figure S1). The gel-filtration traces of monofunctionalized SH3 (mPEG-ALD:SH3 = 1:1) and

Figure 2. Isolation and purification of mono- and multi-PEGylated SH3. (a) Size-exclusion fast performance liquid chromatography (FPLC) traces of PEGylated-SH3. (b)The purity of the FPLC purified PEGylated SH3 is confirmed by SDS-PAGE (13%). (c) The mono-PEGylated SH3 was confirmed to be linked to the N-terminus of SH3. Because a TEV digestive site is introduced to the N-terminal of SH3, the monoconjugated PEG can be cleaved by TEV protease digestion leading to a decrease of the molecular weight of PEGylated SH3 as indicated in the SDS-PAGE gel (16%). 16135

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(PEG10k)3, PEGylation increases the thermodynamic stability by ∼0.93 kcal mol−1, ∼30% of that of unmodified SH3. The stabilization effect upon PEGylation is also confirmed by thermal denaturation experiments (Figure S2). The m-value of SH3 also increases from 1.41 to 1.58 kcal mol−1 M−1 upon three-site PEGylation, indicating an increase of the solventaccessible surface area (SASA) of SH3. The detailed fitting parameters are summarized in Table 1. This result is a bit surprising, as we might expect that attaching PEG to a protein surface could slightly block the attachment site from solvent and decrease the SASA. The origin of the increase of m-value will be discussed later in this Article. Effect of PEGylation on the Folding/Unfolding Kinetics of SH3. Subsequently, we study how PEGylation affects the folding and unfolding kinetics of unmodified and PEGylated SH3 using a stopped-flow apparatus. The tryptophan residues buried inside the hydrophobic core of SH3 will be exposed to polar solvent upon unfolding, resulting in a decrease of fluorescence intensity at 335−345 nm. Therefore, the change of the fluorescence signal can be used to monitor the folding and unfolding reactions. Representative folding and unfolding profiles are shown in Figure 5a and b, respectively. It is clear that both folding and unfolding of unmodified and PEGylated SH3 show single exponential kinetics and can be described using a two-state model. Therefore, PEGylation does not presumably change the folding and unfolding pathways. For N-terminal PEGylated SH3, the folding and unfolding kinetics are only slightly retarded. However, for triconjugated SH3, both folding and unfolding kinetics are significantly slowed. The folding and unfolding kinetics of unmodified SH3 and PEGylated SH3 are summarized in Figure 5c. Fitting chevron plots yields kinetic parameters for the free energy landscape of the folding reaction of unmodified SH3 and PEGylated SH3 (Table 1). Similar to thermodynamic stability, the kinetics of PEGylated SH3 is also independent of the molecular weight of the attached PEG (Figure 5). Interestingly, although both folding and unfolding of SH3 are slowed upon PEGylation, the underlying mechanism is different. For SH3-(PEG5k)3, the slowing of folding is mainly due to the increase of folding m-value (mf) from 0.92 to 0.98 kcal mol−1 M−1 without changing of the folding rate (kfH2O). However, the slowing of unfolding is mainly due to the decrease of unfolding rate (kuH2O) from 0.14 to 0.06 s−1 without changing the unfolding m-value (mu).

Figure 3. PEGylation has no significant effects on the structure of SH3. Far-UV CD spectra of unmodified and PEGylated SH3 in 50 mM phosphate buffer (pH 5.0) at 295 K.

Effect of PEGylation on the Thermodynamic Stability of SH3. We use chemical denaturation to study the thermodynamic stability of SH3 upon PEGylation. As shown in Figure 4, the GdnCl denaturation curves of both unmodified

Figure 4. Thermodynamic studies of PEGylated SH3. Representative GdnCl denaturation curves of unmodified and PEGylated SH3 at 295 K. The data are fitted to an equilibrium two-state model (eq 2), and the fitting parameters are given in Table 1.

and PEGylated SH3 can be adequately fitted using a simple two-state model. The thermodynamic stability is ∼3.14 kcal mol−1, and the m-value is ∼1.41 kcal mol−1 M−1 for unmodified SH3. For the N-terminal conjugated SH3, both the thermodynamic stability and the m-value are unaltered upon PEGylation. Furthermore, increasing the molecular weight of PEG from 5000 to 10 000 Da does not lead to additional increase in the thermodynamic stability. However, for SH3-

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DISCUSSION Folding Free Energy Landscape of PEGylated SH3. Having measured the effects of PEGylation on the thermodynamics and kinetics of the SH3, we are able to obtain a detailed picture of such effects from the free energy landscape. As shown in Figure 6, the major changes of the free energy

Table 1. Thermodynamic and Kinetic Stability of the Unmodified and PEGylated SH3a ΔG (kcal mol−1) SH3 SH3-PEG5k SH3-(PEG5k)3 SH3-PEG10k SH3(PEG10k)3 a

3.14 3.22 3.99 2.99 4.07

± ± ± ± ±

0.15 0.17 0.24 0.14 0.20

m-value (kcal mol−1 M−1) 1.41 1.42 1.52 1.42 1.58

± ± ± ± ±

0.06 0.07 0.09 0.06 0.07

ΔGN−U (kcal mol−1) 3.39 3.32 3.81 3.33 3.77

± ± ± ± ±

0.04 0.03 0.04 0.04 0.02

ΔGN−TS (kcal mol−1) 18.62 18.59 19.12 18.67 19.10

± ± ± ± ±

0.07 0.05 0.09 0.03 0.04

ΔGU−TS (kcal mol−1) 15.22 15.27 15.31 15.34 15.33

± ± ± ± ±

0.05 0.02 0.06 0.02 0.03

mu (kcal mol−1 M−1) 0.46 0.44 0.45 0.45 0.44

± ± ± ± ±

0.02 0.01 0.01 0.02 0.01

mf (kcal mol−1 M−1) 0.92 0.92 0.98 0.90 0.98

± ± ± ± ±

0.01 0.01 0.02 0.02 0.01

N, TS, and U represent native state, transition state, and unfolded state, respectively. 16136

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Figure 6. The effect of PEGylation on the free energy landscape for the folding and unfolding of SH3. ΔGN−TS and ΔGU−TS represent the free energy barriers for unfolding and folding, respectively. ΔGN−U is the thermodynamic stability. mu and mf are the unfolding and folding m-values, respectively.

which leads to an increase of m-value upon PEGylation. Similarly, because the transition state is highly native like, the ΔSASA between the unfolded state and the transition state increases upon PEGylation, while the ΔSASA between the transition state and folded state does not change too much. This accounts for an increased mf and an unchanged mu. Second, upon PEGylation, the thermodynamic stability of SH3 increases by ∼0.93 kcal mol−1. Kinetically, such enhancement of stability is mainly contributed from the decrease of the unfolding rate constant. How does PEGylation stabilize SH3? It was shown that the stabilization of a protein in a PEG solution can be explained by the steric exclusion of water molecules from protein surface due to the large volume occupied by PEG molecules. For the PEGylated SH3, although the overall PEG concentration in solution is low ( ns/nw. Besides, dμs = d[kBT ln(as)], where as is the solute activity and kB is the Boltzmann constant, or dμs = d[kBT ln(γCs)], where γ is the activity coefficient and Cs is the concentration of PEG. The perturbation of the chemical potential over molar concentration of PEG, (∂μs/∂ms)T,P,mM, is positive.59 Thus, dGM is sure to be negative, which means a protein is stabilized by linking with PEG. Although both ΔGN(PEG)−N and ΔGU(PEG)−U are negative, the thermodynamic stability of a protein is enhanced only if ΔGN(PEG)−N < ΔGU(PEG)−U. From eq 8, how much a protein is stabilized upon PEGylation is correlated with the number of PEG molecules and water molecules around the protein and in the bathing solution. To quantitatively measure Ns and Nw, we adopt a model proposed by Bhat and Timasheff (Figure 7b).59 Ns is obviously proportional to the volume of PEG occupied around protein surface (VPEG), and Nw is proportional to the steric exclusion volume of PEG or the preferential hydration volume of the effective shell, Ve, surrounding the protein.59

Figure 7. The effect of PEGylation on the thermodynamic and kinetic stability of proteins can be explained by the excluding volume effect. (a) Thermodynamic free energy cycle analysis of a protein upon PEGylation. N and U stand for the native and the unfolded states of the protein, respectively. PEG stands for PEGylated protein. (b) Schematic representation of the steric exclusion model of the folded and unfolded protein. Rp and Re are the effective radii of the protein and the exclusion, respectively. The exclusion volume Ve (between dashed blue line and solid blue line) includes the volume of the cosolute PEG (gray ellipse) and the water. Upon unfolding, both the volume of the protein and the exclusion expand, while the volume of PEG remains almost the same.

Ve =

∑ ni dμi i

(5)

where μi is the chemical potential of component i. For a solution of PEGylated protein, we consider three components of the solution: macromolecule, M (protein); water, w; and solute, s (PEG). At constant pressure, p, and constant temperature, T, the change in macromolecular chemical potential or free energy is linked to the changes in the chemical potentials of solute and water: dμM = −Nw dμw − Ns dμs

(6)

in which Nw and Ns are molecules of water and solute per macromolecule. This solution is in equilibrium with a reference solution containing nw and ns molecules of water and solutes. n w dμw + ns dμs = 0

(7)

Thus, the change in macromolecular free energy can be described by the extent that the ratio Ns/Nw in the vicinity of the macromolecule differs from their number ratio ns/nw in the bathing solution. ⎛ ⎛ n ⎞ n /n ⎞ dGM = −⎜Ns − Nw s ⎟ dμs = −Ns⎜1 − s w ⎟ dμs nw ⎠ Ns/Nw ⎠ ⎝ ⎝

(9)

where NA is Avogadro’s number, Rp is the radius of the protein, and Re is the radius of exclusion. Ve increases with the increasing of Rp and Re. The volume of the protein expands upon unfolding, which leads to larger Ve for the unfolded state than the native state. Therefore, Ns/Nw for the native state is larger than that for the unfolded state. Considering that ns/nw for the bathing solution is almost the same for the native and the unfolded states, according to eq 8, ΔGN(PEG)−N < ΔGU(PEG)−U. These derivations clearly explain why the thermodynamic stability of a protein increases upon PEGylation. Experimental Observations Can Be Adequately Described Using This Physical Model. First, our measurement of the change of thermodynamic stability of SH3 upon PEGylation is consistent with the model derived above. Because Ns is proportional to the number of PEG molecules attached to a protein, the more PEG chains there are, the greater is the thermodynamic stability of a protein, according to eq 8. Moreover, because both Ns and Ns/Nw increase, the changes of dGM upon attaching n PEG chains will be greater than n × dGM. This is consistent with our experimental observation on PEGylated SH3. However, the effect of the molecular weight of PEG on thermodynamic stability is more complicated. Because Re can be approximated as the radius of gyration (Rg) of PEG and is proportional to the square root of its molecular weight, Ve increases with the increase of the molecular weight of PEG in the same way as VPEG. On the basis of eqs 8 and 9, the molecular weight of PEG affects the thermodynamic stability of the PEGylated protein in a nonlinear fashion depending on the size of the protein. In our experiments, the molecular weight effect is not pronounced, which is also consistent with the experimental results from others.43,44 Second, the effect of PEGylation on the folding and unfolding kinetics can also be explained using this physical model. The effect of PEGylation on the folding and unfolding kinetics depends on how PEGylation affects the transition structure. According to previous ϕ-value analysis, the three PEGylated sites (N-terminal amino group, K19, and K20) are all in the unstructured region of the transition state,45−49 similar

PEGylation at its native and unfolded states. Therefore, to obtain the effect of PEGylation on the change of thermodynamic stability of a protein, we need to calculate the effect for both the native state and the unfolded state. We first derive how PEGylation affects the free energy of a protein. In a solution, the change in temperature T, hydrostatic pressure p, and chemical potentials μi is constrained by the Gibbs−Duhem equation.57 0 = − S dT + v dp −

4π NA[(R p + R e)3 − R p3] × 10−24 3

(8) 16138

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to those in the unfolded state. Thus, the stabilization effect for the transition state upon PEGylation, ΔGT(PEG)−T, is similar to ΔGU(PEG)−U. Accordingly, kf should not decrease significantly, while ku should slow. This is indeed the case for the folding and unfolding kinetics of SH3 upon PEGylation. Similarly, if the PEGylation sites were in the structured region of a protein, we would expect that PEGylation speeds the folding rate and does not change the unfolding rate. However, it is worth noting that the change of m-value upon PEGylation is also correlated with the change of folding and unfolding rates. Prediction of the change of folding and unfolding rates could be more complicated.

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CONCLUSIONS In summary, in this work, we have successfully engineered and purified PEGylated SH3 and quantitatively measured the effect of PEGylation on the thermodynamic and kinetic stability. We show that thermodynamic stability of SH3 is enhanced upon PEGylation. The stabilization effect is dependent on the number of attached PEG chains but shows a weak correlation with the size of PEG. The kinetic data showed that the stabilization effect mainly results from the slowing of the unfolding rate. Moreover, both the overall m-value and mf increase upon PEGylation due to the decrease of solventaccessible surface area of SH3. Starting from fundamental physical chemistry theory, we derive a physical model that can satisfactorily account for the experimentally observed changes of thermodynamic and kinetic properties of SH3 upon PEGylation. Hence, this study provides many insights toward the understanding of the mechanism for the stabilization of proteins by PEGylation. Nevertheless, it is still unknown whether this mechanism can be applied to other proteins whose structure is distinct from SH3 and how the PEGylation position affects the stability. Addressing these questions will be our next endeavor. ASSOCIATED CONTENT

S Supporting Information *

SDS PAGE of PEGylated SH3 at different PEG:SH3 ratios, sequence of SH3 used in this study, and thermal denaturation of PEGylated SH3. This material is available free of charge via the Internet at http://pubs.acs.org.

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GdnCl = guanidine hydrochloride SASA = solvent-accessible surface area

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.C.); [email protected] (W.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China under grant nos. 10834002, 31170813, and 11074115, the program for New Century Excellent Talents in University, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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ABBREVIATIONS CD = circular dichroism PB = phosphate buffer PEG = poly(ethylene glycol) mPEG-ALD = aldehyde-terminated linear PEG 16139

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