- Email: [email protected]
ATP-induced noncooperative thermal unfolding of hen lysozyme
Biochemical and Biophysical Research Communications 397 (2010) 598–602
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ATP-induced noncooperative thermal unfolding of hen lysozyme Honglin Liu a, Peidong Yin a, Shengnan He a, Zhihu Sun a, Ye Tao b, Yan Huang b, Hao Zhuang b, Guobin Zhang a, Shiqiang Wei a,* a b
National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029, People’s Republic of China Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 14 May 2010 Available online 4 June 2010 Keywords: Hen egg white lysozyme Synchrotron radiation circular dichroism Secondary structure Noncooperative unfolding Partially unfolded intermediate
a b s t r a c t To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic lightscattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg2+–ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the b-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich a-helix and less b-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric b-sheet enriched intermediate. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The interaction between small molecules and proteins usually induces alterations in protein unfolding kinetics [1] (such as modifications in the melting temperature, enthalpy of unfolding, unfolding pathway, etc.) which has an important role in the amyloid aggregation of proteins [2]. Many studies have revealed that the process of amyloid aggregation could be affected by the presence of a significant quantity of charged polyelectrolytes [3,4]. In particular, ATP, a naturally occurring polyanion, dramatically increases the rate of amyloid aggregation of hen egg white lysozyme (HEWL) in elevated temperature and low pH [4–6], although it is considered to have no physiological role in the function of HEWL. HEWL is known as a highly positively charged protein at low pH. Its interaction with the negatively charged ATP compensates the electrostatic repulsions between positively charged protein monomers [4]. However, a different effect of the electrostatic neutralization may exist as it is shown that ionic liquids could significantly inhibit the amyloid aggregation of HEWL [7]. HEWL comprises an a-domain containing several helices and a b-domain comprised largely of b-strands (Fig. S1). The amyloidogenic region of HEWL mainly encompasses part of the b-domain and Helix C of the a-domain [8]. Some studies suggest that monomeric b-sheet enriched intermediate is required for amyloid aggregation [9,10], and ATP might bind to these amyloidogenic precursors [11]. Nevertheless,
* Corresponding author. Fax: +86 551 5141078. E-mail address: [email protected] (S.Q. Wei). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.005
no intermediate in amyloid aggregation of HEWL has been identified [7]. To date, the nature of the interactions and the degree of specificity involved in ATP-enhanced amyloidosis of HEWL are uncertain. Recently, Morshedi et al. suggested that a putative interaction site in HEWL for indole derivatives [12] is the binding site of ATP [5], in which three tryptophan residues including W62, W63, and W108 are involved. Earlier studies reveal that the position alteration of W62 weakens the hydrophobic forces which hold the protein in its native structure [13–15]. W62 and W63 may first unfold in the HEWL unfolding [16]. In addition, W62G mutation first induces the unfolding of the b-domain of HEWL at high temperatures, and then the unfolding process spreads into the a-domain through Helix C [14]. Hence, whether the presence of ATP alters the W62 conformation or the unfolding pathway of HEWL must be key factors for understanding the nature and significance of the interactions between HEWL and ATP. Here, synchrotron radiation circular dichroism (SRCD) and intrinsic tryptophan fluorescence are used to examine the conformational alterations of HEWL in the presence of ATP, Mg2+–ATP complex (MgATP), ADP, AMP, and MgCl2. SRCD and DSC are employed to characterize the thermal equilibrium unfolding of HEWL alone and in the presence of each additive. Moreover, dynamic light-scattering is used to probe the aggregation in the unfolding process. We for the first time observe an ATP-induced partially unfolded intermediate under the conditions of HEWL amyloid aggregation. The results imply that noncooperative unfolding of HEWL caused by ATP-induced perturbation of the residue W62, rather than the electrostatic neutralizing effect, is more critical for HEWL aggregation.
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
2. Materials and methods
599
equilibrated at 10.0 °C for 15 min to eliminate the effect of thermal history before the second heating process.
2.1. Materials 2.5. Dynamic light-scattering (DLS) HEWL, AMP, ADP, and ATP (sodium salts) were purchased (Sigma). The other chemicals used were of analytical grade. All solutions were prepared with Mill-Q water. A HEWL stock solution of 20 mg/ml was prepared, and was extensively dialyzed against the solvent (aqueous solution of HCl at pH 2.0). AMP, ADP, and ATP solutions of 100 mM and MgCl2 solution of 500 mM were prepared, and the pH was adjusted to 2.0 by addition of small amounts of 1 M HCl. MgCl2 (10-fold) was mixed with isopyknic ATP solution to form MgATP. These stock solutions were stored at 80 °C. The samples were prepared by dilution from these stock solutions, and were centrifuged (16,000 rpm, 10 min). All sample cells filled with solution were sealed to keep the pH constant during the experiments. The accurate determination of protein concentration was as described [17]. For simplicity, in the following text, the HEWL in the presence of AMP, ADP, ATP, and MgATP are denoted by amp-HEWL, adp-HEWL, atp-HEWL, and mgatp-HEWL, respectively. 2.2. Synchrotron radiation circular dichroism (SRCD) experiments The SRCD spectra were recorded from 25.0 to 70.0 °C on Beamline 4B8 in BSRF (Beijing, China) as described in [17]. All experiments were done in triplicate. The equilibrium time at each temperature was 5 min to ensure that three repeat scans exactly match each other. The same cell with a path length of 0.01 cm (Hellma) was used. The concentrations of HEWL and each additive used in samples were 0.14 and 1.40 mM, respectively. The protein was omitted from the control sample (baseline) of which the spectra at all temperatures were also recorded. The secondary structure contents were estimated by singular value decomposition (SVD) and the spectra set was deconvoluted using the convex constraint algorithm (CCA) [18]. DSSP was used to assign the secondary structure of HEWL in the crystal (PDB ID: 193L [19]) [20]. The SRCD performance, d, was characterized by the root-mean-square deviation (RMSD) between the DSSP assignment and the SRCD estimates [21]. The quality of the fit of the calculated data to the experimental data was characterized by the normalized RMSD (NRMSD), whose values of <0.1 mean that they are in close agreement [22].
DLS measurements were done from 25.0 to 60.0 °C with a DynaPro MSTC800 DLS instrument fitted with a 624.4 nm, 50 mW laser at a scattering angle of 90°. Fourteen micromolar of HEWL samples was prepared in the absence and presence of 140 lM each additive, respectively. All samples were filtered through a 100 nm membrane before the measurements. The hydrodynamic radius was analyzed using Dynamics V6.2 software. 3. Results and discussion 3.1. ATP-induced conformational alteration of the residue W62 In the ITF experiments, the emission maximum (EM) of 7 lM HEWL at pH 2.0 is around 338 nm. And the EM dramatically shifts to smaller wavelengths with the increasing of ATP concentration (Fig. 1A). However, ADP has much weaker effects, and no effect of AMP or Mg2+ is observed. Interestingly, the EM of 7 lM HEWL in the presence of 7 mM ATP gradually shifts back to 338 nm with the increasing of Mg2+ concentration (Fig. 1B); this should be because the negative charges of ATP phosphate groups which can chelate Mg2+ with very high affinity are neutralized. As a conclusion, the EM shifting to smaller wavelengths attributed to the movement of tryptophan residues to more compact, hydrophobic environments [23] is caused by the electrostatic effect of ATP phosphate groups and is strongly dependent on the density of negative charges. The secondary structure contents of HEWL determined from the SRCD spectra at 25 °C are in good agreement with DSSP assignment, as indicated by the factor d (Table 1). There is no apparent alteration in the SRCD signal of HEWL in the presence of MgATP, ADP, or AMP. However, the addition of ATP induces that the
2.3. Intrinsic tryptophan fluorescence (ITF) experiments The ITF spectra were monitored from 300 to 400 nm at 25 °C using a spectrofluorometer (Aminco-Bowman series 2) equipped with a temperature-controlled circulator bath. The excitation wavelength is 295 nm, and the excitation and emission bandwidths are both 4 nm. One hundred and fifty microliter portion of 7 lM HEWL was added to a cuvette with a path length of 5 mm in both the lateral and perpendicular directions. Each additive was then titrated into the HEWL solution in the cuvette. In the experiment of MgCl2 back-titration, the sample of 7 lM HEWL in the presence of 7 mM ATP was added to the same cuvette, and MgCl2 was then titrated. All baselines were measured in the same procedures. 2.4. Ultrasensitive differential scanning calorimetry (US-DSC) DSC measurements were done with a VP-DSC microcalorimeter (MicroCal). All samples were the same with that used in the SRCD experiments. The reference cell and the sample cell were prewashed by the control and the sample, respectively. Each sample and the control were equilibrated at 10.0 °C for 15 min before heating. The scanning rate was 1.0 °C/min. After the first heating process, the cells were cooled. Then, the samples were again
Fig. 1. The ITF and SRCD assays of HEWL in the absence and presence of different additives at 25 °C, pH 2.0. (A) The emission maximum shifts of the tryptophan fluorescence of HEWL produced by varying concentrations of ATP (squares), ADP (triangles), AMP (diamonds), and MgCl2 (crosses). (B) MgCl2 back-titration of atpHEWL. The inset shows the fluorescence spectra marked with the concentrations of MgCl2. (C) SRCD spectra of HEWL (solid line), mgatp-HEWL (dash line), and atpHEWL (dot line). The error bars represent one standard deviation of the measurements from three sequential scans of both spectra and baselines.
600
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
The ratios of DHcal between the second and the first heating process is an indicator of the degree of reversibility [25]. The melting curve of HEWL is more than 90% reproducible upon reheating (Table 2); neither ADP nor AMP visibly affects the reversibility. However, the reversibility of atp-HEWL unfolding is greatly decreased from 95% to 76%, indicating the multimolecular reactions or interchain interaction [25]. Fig. 2 shows the CCA analysis [18] of the SRCD spectra sets of HEWL and atp-HEWL determined as a function of temperature. When the spectra set of HEWL in Fig. 2A are deconvoluted into three basis spectra (Fig. 2C), two of the basis spectra are almost identical, showing that only two conformations are needed to fit all the data. The percentage of each basis curve contributing to the spectra is shown in Fig. 2E, where the fractions of the two identical curves corresponding to the native structure are added. Fig. 2E shows that HEWL unfolds with a T1/2 of about 54 °C. In contrast, when the spectra set of atp-HEWL in Fig. 2B are deconvoluted, at least three curves are needed to fit the data (Fig. 2D). The change of the curve corresponding to the partially unfolded intermediate reaches the largest fractional weight at 47 °C (Fig. 2F). The protein unfolding is also characterized by monitoring the change in ellipticities at two selected wavelengths (222 and 191 nm) as a function of temperature (Fig. 3). In the SRCD spectra, the negative peak at 222 nm assigned to the peptide n ? p* transition is mainly contributed by the a-helix structure, and the positive peak at 190 nm assigned to the peptide p ? p* transition is contributed by the a-helix and b-sheet structures [26]. As shown in Table 3, both the T1/2–222 values of HEWL and atpHEWL are about 54 °C, and the T1/2–191 of HEWL is also 54 °C. However, the T1/2–191 of atp-HEWL is significantly lowered to 51 °C. The data indicate that the b-sheet structure first unfolds in the equilibrium unfolding of atp-HEWL. SVD analysis [18] reveals that 35% of total b-sheet and only 6.5% of total a-helix of atp-HEWL unfold at 47 °C (Fig. S3). In contrast, the b-sheet content of HEWL does not decrease at 47 °C. Although analysis of the SRCD spectra provides worse estimates of b-sheet than
magnitudes of the SRCD peaks at 208 and 191 nm increase by 11.4% and by 8.5%, respectively (Fig. 1C). The calculated results show that the helical content of atp-HEWL increases by 2.3% and the turn content decreases by 2.5%. Such alterations correspond to 2–3 amino acids in HEWL molecule. The molecular of HEWL has six Trp residues (Fig. S1). Detailed studies already show that W62 is the largest contributor to HEWL emission at low pH, and other Trp residues all lay near Cys, Met, or protonated Glu, acting as efficient quenchers [24]. Moreover, W123, W111, W108, and W28 are all buried in hydrophobic environments; their conformations cannot be easily altered [16]. However, W62 and W63 are exposed and located in a turn structure which is arranged along one side of the active site in a hinge region between the a- and b-domains [14]. Hence, it should be the residue W62, and even W63, which fold into more compact, hydrophobic environments. Consequently, the turn is changed into a helical structure. As mentioned before, the position alteration of W62 will affect the unfolding process of HEWL [13–15]. 3.2. ATP-induced noncooperative unfolding of HEWL The thermodynamic parameters of HEWL unfolding provided by DSC are showed in Table 2. The DHcal-to-DHV ratio, n, is an indicator of the degree of cooperativity of unfolding [25]. In the first heating process, it is evident that the temperature dependence of the excess heat capacity (C exc p ) for HEWL is well approximated by the two-state model (Fig. S2A). The n value of HEWL is very close to one, indicating a simple transition between two discrete macroscopic states. The addition of AMP or ADP only slightly lowers the n values, and does not alter the Tm of HEWL unfolding which is at about 58 °C. However, the addition of ATP significantly lowers the Tm down to 55 °C and makes the n value significantly less than 1. The C exc p curve of atp-HEWL cannot be fitted well to a two-state model with one transition. A three-state model with two sequential transitions is used to fit the curve of atp-HEWL (Fig. S2C). The Tm of the first transition is 51 °C, and that of the second is 56 °C. Table 1 Secondary-structure contents determined by DSSP assignment and SRCD spectra. Protein HEWL HEWL atp-HEWL a
DSSP SRCD SRCD
a-Helix
b-Sheet
Turn
Unordered
Total
NRMSD
da
40.3 39.4 41.7
6.2 11.5 11.4
30.2 22.1 19.6
23.3 27.0 27.3
100 100 100
— 0.023 0.022
— 0.042 —
The factor d indicates good agreement between the DSSP assignment and the SRCD estimates.
Table 2 Calorimetric results on HEWL at pH 2.0 in the absence and presence of different additives. Additives g
– AMP ADP ATP MgATP
ATP a
h
Tma (°C)
DHcalb (kcal/mol)
DHVc (kcal/mol)
nd
v2/DOFe ((kcal/mol/°C)2)
Rf
57.7 58.1 57.5 55.2 56.4
96.9 96.5 89.5 80.3 92.3
99.7 101.4 96.2 90.9 100.3
0.97 0.95 0.93 0.88 0.92
0.039 0.051 0.078 0.106 0.043
0.92 0.92 0.91 0.76 0.94
Tm1
Tm2
DHcal1
DHcal2
DHV1
DHV2
n1
n2
v2/DOF
51.0
56.0
26.4
64.7
81.7
109.3
0.32
0.59
0.006
Melting temperature (Tm) derived from fitting to a two-state model. Error is ±0.2 °C. b The calorimetrically measured total enthalpy change (DHcal) obtained using the two-state model. Errors on DHcal values are ±1.0%. c The van‘t Hoff enthalpy (DHV) from applying the two-state model to calorimetric data. Errors on DHV values are ±1.5%. d The ratio of DHcal-to-DHV, for the two-state model. e The reduced v2 value used to evaluate the goodness of fitting. f The ratio of DHcal in the second heating process to that in the first heating process. g The solution of HEWL without any additive. In addition, no effect of MgCl2 on the Tm value of HEWL was observed. h Applying the three-state model to calorimetric data analysis. The parameters with the index ‘1’ are for the first transition and that with the index ‘2’ for the second transition.
601
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
Fig. 3. Apparent unfolded fraction of (A) HEWL, (B) adp-HEWL, (C) atp-HEWL, and (D) mgatp-HEWL as a function of temperature monitored by the changes of ellipticity at 222 nm (squares) and 191 nm (circles). The ellipticities at 25 and 70 °C were considered to represent the native and unfolded states, respectively, and were normalized to the range of 0–1. The solid lines represent theoretical curves resulting from the nonlinear least square fitting of the individual data sets. Fig. 2. (A,B) The SRCD spectra sets of HEWL and atp-HEWL at pH 2.0 for a temperature range of 25–70 °C. (C,D) The data in (A,B) deconvoluted into three basis curves using the CCA. (E,F) The fraction of each basis curve contributing to each spectrum at each temperature. The fractional weights of the two curves corresponding to the native protein were summed in (E) because they were almost identical.
Table 3 T1/2 valuesa determined by the least square fitting of the ellipticities at 191 and 222 nm as a function of temperature. Error is ±0.5 °C.
a-helix, the b-sheet changes here are big enough to support our conclusion. At 70 °C, the magnitude of the atp-HEWL spectrum at 222 nm is about 34% larger than that of the HEWL spectrum (Fig. 2A and B), indicating that atp-HEWL remains more a-helix structure than HEWL at high temperature. SVD analysis reveals that the a-helix content of atp-HEWL is 5.3% more, on average, than that of HEWL over the temperature range of 47–70 °C (Fig. S3). This conclusion is consistent also with the DSC data. The DHcal of atp-HEWL obtained using the two-state model can be compared to that of HEWL. There is a difference of 16.6 kcal/mol, or about 17%, in DHcal between atpHEWL and HEWL. This difference should be due to the incomplete unfolding of atp-HEWL at high temperature [27]. It should be noted that the T1/2 values determined by SRCD are all smaller than the Tm values determined by DSC, because SRCD reflects the features of secondary structures while DSC reflects the overall profile of heat capacity. Nevertheless, both techniques monitor the same process [28]. The combination of SRCD and DSC data demonstrates this view: In the unfolding process of atp-HEWL, the b-domain breaks up first. At about 50 °C, atp-HEWL unfolds into a partially unfolded intermediate with relatively rich a-helix and low b-sheet structures instead of a monomeric b-sheet enriched intermediate. 3.3. ATP-promoted aggregation of partially unfolded intermediate In the unfolding process of HEWL, the hydrodynamic radius varies from 1.5 to 3.0 nm (Fig. 4A), confirming previous literature data [29]. Actually, HEWL has four disulfide bonds distributed over the polypeptide chain, which limit the freedom of expansion of the polypeptide coil. However, in the unfolding process of atp-HEWL,
Additives
T1/2–191
T1/2–222
–b AMP ADP ATP MgATP
53.8 53.5 52.8 50.7 53.1
53.8 53.8 53.8 54.2 53.8
a
T1/2 is the temperature value at which the apparent unfolded fraction equals
0.5. b
The solution of HEWL without any additive. In addition, no effect of MgCl2 on the T1/2 value of HEWL was observed.
the aggregation begins to arise at 42 °C. Interestingly, the aggregation is not observed when the temperature is lower than 42 °C, and gradually breaks up and returns to monomers when the temperature is higher than 52 °C (Fig. 4C). This indicates that the partially unfolded intermediate has a much greater tendency to aggregate than the native and the unfolded states. Here is a question whether the electrostatic neutralizing effect of ATP dominates the aggregation of proteins. Although the negatively charged ADP and AMP can compensate the electrostatic repulsions between positively charged HEWL monomers, there is no aggregation of HEWL in the presence of ADP, AMP, and MgATP in the temperature range of 25–60 °C (Fig. 4). Correspondingly, ADP, AMP, and MgATP do not affect the cooperativity of HEWL unfolding. Hence, it is demonstrated that ATP-induced intermediate is more critical for protein aggregation, i.e., the ease of aggregating is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric b-sheet enriched intermediate. In support of this, two amyloidogenic human lysozyme variants have been shown to aggregate on heating resulted from the instability of the b-domain [2], and a peptide corresponding to the b-sheet region of HEWL has
602
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
Fig. 4. Temperature dependence of hydrodynamic radius of (A) HEWL, (B) adpHEWL, (C) atp-HEWL, and (D) mgatp-HEWL estimated via the DLS measurements. The temperature varied from 25 to 60 °C.
been found to form extensive interchain interactions [30]. Moreover, the fibrils formed from atp-HEWL are much longer, straighter, and more periodical (Fig. S4), indicating that the intermediate makes the self-assembly of fibrils more orderly and easier. In summary, the binding of ATP to HEWL makes the residue W62, and even W63, move into more hydrophobic environments. Compared to the effects of ADP, AMP, and MgATP, it is reasonable to infer that the much stronger electrostatic effect of ATP induces the alteration of secondary structures of HEWL. This alteration decreases the b-domain stability of HEWL in the thermal unfolding process, results in a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate at about 50 °C. This intermediate containing relatively rich a-helix and less b-sheet structure has a much greater tendency to aggregate than the native and the unfolded states. It is demonstrated that ATP-induced intermediate, rather than the electrostatic neutralizing effect, is more critical for protein aggregation. This study implies that monomeric b-sheet enriched intermediate does not represent a general step under the conditions of amyloid aggregation. We expect that more detailed studies of longer polyanions will lead to a better understanding of the role of polyelectrolytes underlying the amyloid aggregation of proteins. Acknowledgment This work was supported by Chinese National Natural Science Foundation (No. 10635060 and 10725522). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.005. References [1] M.S. Celej, C.G. Montich, G.D. Fidelio, Protein stability induced by ligand binding correlates with changes in protein flexibility, Protein Sci. 12 (2003) 1496–1506.
[2] D.R. Booth, M. Sunde, V. Bellotti, C.V. Robinson, W.L. Hutchinson, P.E. Fraser, P.N. Hawkins, C.M. Dobson, S.E. Radford, C.C. Blake, M.B. Pepys, Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis, Nature 385 (1997) 787–793. [3] C. Exley, ATP-promoted amyloidosis of an amyloid beta peptide, Neuroreport 8 (1997) 3411–3414. [4] M. Calamai, J.R. Kumita, J. Mifsud, C. Parrini, M. Ramazzotti, G. Ramponi, N. Taddei, F. Chiti, C.M. Dobson, Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes, Biochemistry 45 (2006) 12806–12815. [5] D. Morshedi, A. Ghasemi, M. Evini, A. Ebrahim-Hababi, M. Nemat-Gorgani, Dramatic enhancement of lysozyme fibrillogenesis by ATP, FEBS J. 275 (2008) 194. [6] L.N. Arnaudov, R. de Vries, Thermally induced fibrillar aggregation of hen egg white lysozyme, Biophys. J. 88 (2005) 515–526. [7] H.R. Kalhor, M. Kamizi, J. Akbari, A. Heydari, Inhibition of amyloid formation by ionic liquids: ionic liquids affecting intermediate oligomers, Biomacromolecules 10 (2009) 2468–2475. [8] E. Frare, P. Polverino De Laureto, J. Zurdo, C.M. Dobson, A. Fontana, A highly amyloidogenic region of hen lysozyme, J. Mol. Biol. 340 (2004) 1153–1165. [9] S. Srisailam, H.M. Wang, T.K.S. Kumar, D. Rajalingam, V. Sivaraja, H.S. Sheu, Y.C. Chang, C. Yu, Amyloid-like fibril formation in an all beta-barrel protein involves the formation of partially structured intermediate(s), J. Biol. Chem. 277 (2002) 19027–19036. [10] V.J. McParland, A.P. Kalverda, S.W. Homans, S.E. Radford, Structural properties of an amyloid precursor of beta(2)-microglobulin, Nat. Struct. Biol. 9 (2002) 326–331. [11] C. Exley, O.V. Korchazhkina, Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP), J. Inorg. Biochem. 84 (2001) 215– 224. [12] D. Morshedi, N. Rezaei-Ghaleh, A. Ebrahim-Habibi, S. Ahmadian, M. NematGorgani, Inhibition of amyloid fibrillation of lysozyme by indole derivatives – possible mechanism of action, FEBS J. 274 (2007) 6415–6425. [13] J. Klein-Seetharaman, M. Oikawa, S.B. Grimshaw, J. Wirmer, E. Duchardt, T. Ueda, T. Imoto, L.J. Smith, C.M. Dobson, H. Schwalbe, Long-range interactions within a nonnative protein, Science 295 (2002) 1719–1722. [14] M. Eleftheriou, R.S. Germain, A.K. Royyuru, R. Zhou, Thermal denaturing of mutant lysozyme with both the OPLSAA and the CHARMM force fields, J. Am. Chem. Soc. 128 (2006) 13388–13395. [15] T. Ohkuri, T. Imoto, T. Ueda, Effect of W62G mutation of hen lysozyme on the folding in vivo, Biochem. Biophys. Res. Commun. 338 (2005) 820–824. [16] D.V. Laurents, R.L. Baldwin, Characterization of the unfolding pathway of hen egg white lysozyme, Biochemistry 36 (1997) 1496–1504. [17] H.L. Liu, X.H. Peng, F. Zhao, G.B. Zhang, Y. Tao, Z.F. Luo, Y. Li, M.K. Teng, X. Li, S.Q. Wei, N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1, Biochem. Biophys. Res. Commun. 379 (2009) 1120–1125. [18] N.J. Greenfield, Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions, Nat. Protoc. 1 (2007) 2527–2535. [19] M.C. Vaney, S. Maignan, M. Ries-Kautt, A. Ducruix, High-resolution structure (1.33 Å) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission, Acta Crystallogr. D 52 (1996) 505–517. [20] C.S. Wolfgang Kabsch, Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, Biopolymers 22 (1983) 2577–2637. [21] N. Sreerama, S.Y. Venyaminov, R.W. Woody, Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy, Protein Sci. 8 (1999) 370–380. [22] S. Brahms, J. Brahms, Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism, J. Mol. Biol. 138 (1980) 149–178. [23] F.X. Schmid, Optical spectroscopy to characterize protein conformation and conformational changes, in: T.E. Creighton (Ed.), Protein Structure: A Practical Approach, IRL Press, Oxford, 1997, pp. 261–297. [24] J.M. Beechem, L. Brand, Time-resolved fluorescence of proteins, Annu. Rev. Biochem. 54 (1985) 43–71. [25] P.L. Privalov, Scanning microcalorimeters for studying macromolecules, Pure Appl. Chem. 52 (1980) 479–497. [26] R.W. Woody, Theory of circular dichroism of proteins, in: G.D. Fasman (Ed.), Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, pp. 25–67. [27] L. Mitra, N. Smolin, R. Ravindra, C. Royer, R. Winter, Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution – experiments and theoretical interpretation, Phys. Chem. Chem. Phys. 8 (2006) 1249–1265. [28] D.G. Pina, J. Gómez, P. England, C.T. Craescu, L. Johannes, V.L. Shnyrov, Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit, Biochimie 88 (2006) 1199–1207. [29] A. Esposito, L. Comez, S. Cinelli, F. Scarponi, G. Onori, Influence of glycerol on the structure and thermal stability of lysozyme: a dynamic light scattering and circular dichroism study, J. Phys. Chem. B 113 (2009) 16420–16424. [30] J.J. Yang, M. Pitkeathly, S.E. Radford, Far-UV circular-dichroism reveals a conformational switch in a peptide fragment from the beta-sheet of hen lysozyme, Biochemistry 33 (1994) 7345–7353.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ATP-induced noncooperative thermal unfolding of hen lysozyme Honglin Liu a, Peidong Yin a, Shengnan He a, Zhihu Sun a, Ye Tao b, Yan Huang b, Hao Zhuang b, Guobin Zhang a, Shiqiang Wei a,* a b
National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029, People’s Republic of China Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 14 May 2010 Available online 4 June 2010 Keywords: Hen egg white lysozyme Synchrotron radiation circular dichroism Secondary structure Noncooperative unfolding Partially unfolded intermediate
a b s t r a c t To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic lightscattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg2+–ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the b-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich a-helix and less b-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric b-sheet enriched intermediate. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The interaction between small molecules and proteins usually induces alterations in protein unfolding kinetics [1] (such as modifications in the melting temperature, enthalpy of unfolding, unfolding pathway, etc.) which has an important role in the amyloid aggregation of proteins [2]. Many studies have revealed that the process of amyloid aggregation could be affected by the presence of a significant quantity of charged polyelectrolytes [3,4]. In particular, ATP, a naturally occurring polyanion, dramatically increases the rate of amyloid aggregation of hen egg white lysozyme (HEWL) in elevated temperature and low pH [4–6], although it is considered to have no physiological role in the function of HEWL. HEWL is known as a highly positively charged protein at low pH. Its interaction with the negatively charged ATP compensates the electrostatic repulsions between positively charged protein monomers [4]. However, a different effect of the electrostatic neutralization may exist as it is shown that ionic liquids could significantly inhibit the amyloid aggregation of HEWL [7]. HEWL comprises an a-domain containing several helices and a b-domain comprised largely of b-strands (Fig. S1). The amyloidogenic region of HEWL mainly encompasses part of the b-domain and Helix C of the a-domain [8]. Some studies suggest that monomeric b-sheet enriched intermediate is required for amyloid aggregation [9,10], and ATP might bind to these amyloidogenic precursors [11]. Nevertheless,
* Corresponding author. Fax: +86 551 5141078. E-mail address: [email protected] (S.Q. Wei). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.005
no intermediate in amyloid aggregation of HEWL has been identified [7]. To date, the nature of the interactions and the degree of specificity involved in ATP-enhanced amyloidosis of HEWL are uncertain. Recently, Morshedi et al. suggested that a putative interaction site in HEWL for indole derivatives [12] is the binding site of ATP [5], in which three tryptophan residues including W62, W63, and W108 are involved. Earlier studies reveal that the position alteration of W62 weakens the hydrophobic forces which hold the protein in its native structure [13–15]. W62 and W63 may first unfold in the HEWL unfolding [16]. In addition, W62G mutation first induces the unfolding of the b-domain of HEWL at high temperatures, and then the unfolding process spreads into the a-domain through Helix C [14]. Hence, whether the presence of ATP alters the W62 conformation or the unfolding pathway of HEWL must be key factors for understanding the nature and significance of the interactions between HEWL and ATP. Here, synchrotron radiation circular dichroism (SRCD) and intrinsic tryptophan fluorescence are used to examine the conformational alterations of HEWL in the presence of ATP, Mg2+–ATP complex (MgATP), ADP, AMP, and MgCl2. SRCD and DSC are employed to characterize the thermal equilibrium unfolding of HEWL alone and in the presence of each additive. Moreover, dynamic light-scattering is used to probe the aggregation in the unfolding process. We for the first time observe an ATP-induced partially unfolded intermediate under the conditions of HEWL amyloid aggregation. The results imply that noncooperative unfolding of HEWL caused by ATP-induced perturbation of the residue W62, rather than the electrostatic neutralizing effect, is more critical for HEWL aggregation.
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
2. Materials and methods
599
equilibrated at 10.0 °C for 15 min to eliminate the effect of thermal history before the second heating process.
2.1. Materials 2.5. Dynamic light-scattering (DLS) HEWL, AMP, ADP, and ATP (sodium salts) were purchased (Sigma). The other chemicals used were of analytical grade. All solutions were prepared with Mill-Q water. A HEWL stock solution of 20 mg/ml was prepared, and was extensively dialyzed against the solvent (aqueous solution of HCl at pH 2.0). AMP, ADP, and ATP solutions of 100 mM and MgCl2 solution of 500 mM were prepared, and the pH was adjusted to 2.0 by addition of small amounts of 1 M HCl. MgCl2 (10-fold) was mixed with isopyknic ATP solution to form MgATP. These stock solutions were stored at 80 °C. The samples were prepared by dilution from these stock solutions, and were centrifuged (16,000 rpm, 10 min). All sample cells filled with solution were sealed to keep the pH constant during the experiments. The accurate determination of protein concentration was as described [17]. For simplicity, in the following text, the HEWL in the presence of AMP, ADP, ATP, and MgATP are denoted by amp-HEWL, adp-HEWL, atp-HEWL, and mgatp-HEWL, respectively. 2.2. Synchrotron radiation circular dichroism (SRCD) experiments The SRCD spectra were recorded from 25.0 to 70.0 °C on Beamline 4B8 in BSRF (Beijing, China) as described in [17]. All experiments were done in triplicate. The equilibrium time at each temperature was 5 min to ensure that three repeat scans exactly match each other. The same cell with a path length of 0.01 cm (Hellma) was used. The concentrations of HEWL and each additive used in samples were 0.14 and 1.40 mM, respectively. The protein was omitted from the control sample (baseline) of which the spectra at all temperatures were also recorded. The secondary structure contents were estimated by singular value decomposition (SVD) and the spectra set was deconvoluted using the convex constraint algorithm (CCA) [18]. DSSP was used to assign the secondary structure of HEWL in the crystal (PDB ID: 193L [19]) [20]. The SRCD performance, d, was characterized by the root-mean-square deviation (RMSD) between the DSSP assignment and the SRCD estimates [21]. The quality of the fit of the calculated data to the experimental data was characterized by the normalized RMSD (NRMSD), whose values of <0.1 mean that they are in close agreement [22].
DLS measurements were done from 25.0 to 60.0 °C with a DynaPro MSTC800 DLS instrument fitted with a 624.4 nm, 50 mW laser at a scattering angle of 90°. Fourteen micromolar of HEWL samples was prepared in the absence and presence of 140 lM each additive, respectively. All samples were filtered through a 100 nm membrane before the measurements. The hydrodynamic radius was analyzed using Dynamics V6.2 software. 3. Results and discussion 3.1. ATP-induced conformational alteration of the residue W62 In the ITF experiments, the emission maximum (EM) of 7 lM HEWL at pH 2.0 is around 338 nm. And the EM dramatically shifts to smaller wavelengths with the increasing of ATP concentration (Fig. 1A). However, ADP has much weaker effects, and no effect of AMP or Mg2+ is observed. Interestingly, the EM of 7 lM HEWL in the presence of 7 mM ATP gradually shifts back to 338 nm with the increasing of Mg2+ concentration (Fig. 1B); this should be because the negative charges of ATP phosphate groups which can chelate Mg2+ with very high affinity are neutralized. As a conclusion, the EM shifting to smaller wavelengths attributed to the movement of tryptophan residues to more compact, hydrophobic environments [23] is caused by the electrostatic effect of ATP phosphate groups and is strongly dependent on the density of negative charges. The secondary structure contents of HEWL determined from the SRCD spectra at 25 °C are in good agreement with DSSP assignment, as indicated by the factor d (Table 1). There is no apparent alteration in the SRCD signal of HEWL in the presence of MgATP, ADP, or AMP. However, the addition of ATP induces that the
2.3. Intrinsic tryptophan fluorescence (ITF) experiments The ITF spectra were monitored from 300 to 400 nm at 25 °C using a spectrofluorometer (Aminco-Bowman series 2) equipped with a temperature-controlled circulator bath. The excitation wavelength is 295 nm, and the excitation and emission bandwidths are both 4 nm. One hundred and fifty microliter portion of 7 lM HEWL was added to a cuvette with a path length of 5 mm in both the lateral and perpendicular directions. Each additive was then titrated into the HEWL solution in the cuvette. In the experiment of MgCl2 back-titration, the sample of 7 lM HEWL in the presence of 7 mM ATP was added to the same cuvette, and MgCl2 was then titrated. All baselines were measured in the same procedures. 2.4. Ultrasensitive differential scanning calorimetry (US-DSC) DSC measurements were done with a VP-DSC microcalorimeter (MicroCal). All samples were the same with that used in the SRCD experiments. The reference cell and the sample cell were prewashed by the control and the sample, respectively. Each sample and the control were equilibrated at 10.0 °C for 15 min before heating. The scanning rate was 1.0 °C/min. After the first heating process, the cells were cooled. Then, the samples were again
Fig. 1. The ITF and SRCD assays of HEWL in the absence and presence of different additives at 25 °C, pH 2.0. (A) The emission maximum shifts of the tryptophan fluorescence of HEWL produced by varying concentrations of ATP (squares), ADP (triangles), AMP (diamonds), and MgCl2 (crosses). (B) MgCl2 back-titration of atpHEWL. The inset shows the fluorescence spectra marked with the concentrations of MgCl2. (C) SRCD spectra of HEWL (solid line), mgatp-HEWL (dash line), and atpHEWL (dot line). The error bars represent one standard deviation of the measurements from three sequential scans of both spectra and baselines.
600
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
The ratios of DHcal between the second and the first heating process is an indicator of the degree of reversibility [25]. The melting curve of HEWL is more than 90% reproducible upon reheating (Table 2); neither ADP nor AMP visibly affects the reversibility. However, the reversibility of atp-HEWL unfolding is greatly decreased from 95% to 76%, indicating the multimolecular reactions or interchain interaction [25]. Fig. 2 shows the CCA analysis [18] of the SRCD spectra sets of HEWL and atp-HEWL determined as a function of temperature. When the spectra set of HEWL in Fig. 2A are deconvoluted into three basis spectra (Fig. 2C), two of the basis spectra are almost identical, showing that only two conformations are needed to fit all the data. The percentage of each basis curve contributing to the spectra is shown in Fig. 2E, where the fractions of the two identical curves corresponding to the native structure are added. Fig. 2E shows that HEWL unfolds with a T1/2 of about 54 °C. In contrast, when the spectra set of atp-HEWL in Fig. 2B are deconvoluted, at least three curves are needed to fit the data (Fig. 2D). The change of the curve corresponding to the partially unfolded intermediate reaches the largest fractional weight at 47 °C (Fig. 2F). The protein unfolding is also characterized by monitoring the change in ellipticities at two selected wavelengths (222 and 191 nm) as a function of temperature (Fig. 3). In the SRCD spectra, the negative peak at 222 nm assigned to the peptide n ? p* transition is mainly contributed by the a-helix structure, and the positive peak at 190 nm assigned to the peptide p ? p* transition is contributed by the a-helix and b-sheet structures [26]. As shown in Table 3, both the T1/2–222 values of HEWL and atpHEWL are about 54 °C, and the T1/2–191 of HEWL is also 54 °C. However, the T1/2–191 of atp-HEWL is significantly lowered to 51 °C. The data indicate that the b-sheet structure first unfolds in the equilibrium unfolding of atp-HEWL. SVD analysis [18] reveals that 35% of total b-sheet and only 6.5% of total a-helix of atp-HEWL unfold at 47 °C (Fig. S3). In contrast, the b-sheet content of HEWL does not decrease at 47 °C. Although analysis of the SRCD spectra provides worse estimates of b-sheet than
magnitudes of the SRCD peaks at 208 and 191 nm increase by 11.4% and by 8.5%, respectively (Fig. 1C). The calculated results show that the helical content of atp-HEWL increases by 2.3% and the turn content decreases by 2.5%. Such alterations correspond to 2–3 amino acids in HEWL molecule. The molecular of HEWL has six Trp residues (Fig. S1). Detailed studies already show that W62 is the largest contributor to HEWL emission at low pH, and other Trp residues all lay near Cys, Met, or protonated Glu, acting as efficient quenchers [24]. Moreover, W123, W111, W108, and W28 are all buried in hydrophobic environments; their conformations cannot be easily altered [16]. However, W62 and W63 are exposed and located in a turn structure which is arranged along one side of the active site in a hinge region between the a- and b-domains [14]. Hence, it should be the residue W62, and even W63, which fold into more compact, hydrophobic environments. Consequently, the turn is changed into a helical structure. As mentioned before, the position alteration of W62 will affect the unfolding process of HEWL [13–15]. 3.2. ATP-induced noncooperative unfolding of HEWL The thermodynamic parameters of HEWL unfolding provided by DSC are showed in Table 2. The DHcal-to-DHV ratio, n, is an indicator of the degree of cooperativity of unfolding [25]. In the first heating process, it is evident that the temperature dependence of the excess heat capacity (C exc p ) for HEWL is well approximated by the two-state model (Fig. S2A). The n value of HEWL is very close to one, indicating a simple transition between two discrete macroscopic states. The addition of AMP or ADP only slightly lowers the n values, and does not alter the Tm of HEWL unfolding which is at about 58 °C. However, the addition of ATP significantly lowers the Tm down to 55 °C and makes the n value significantly less than 1. The C exc p curve of atp-HEWL cannot be fitted well to a two-state model with one transition. A three-state model with two sequential transitions is used to fit the curve of atp-HEWL (Fig. S2C). The Tm of the first transition is 51 °C, and that of the second is 56 °C. Table 1 Secondary-structure contents determined by DSSP assignment and SRCD spectra. Protein HEWL HEWL atp-HEWL a
DSSP SRCD SRCD
a-Helix
b-Sheet
Turn
Unordered
Total
NRMSD
da
40.3 39.4 41.7
6.2 11.5 11.4
30.2 22.1 19.6
23.3 27.0 27.3
100 100 100
— 0.023 0.022
— 0.042 —
The factor d indicates good agreement between the DSSP assignment and the SRCD estimates.
Table 2 Calorimetric results on HEWL at pH 2.0 in the absence and presence of different additives. Additives g
– AMP ADP ATP MgATP
ATP a
h
Tma (°C)
DHcalb (kcal/mol)
DHVc (kcal/mol)
nd
v2/DOFe ((kcal/mol/°C)2)
Rf
57.7 58.1 57.5 55.2 56.4
96.9 96.5 89.5 80.3 92.3
99.7 101.4 96.2 90.9 100.3
0.97 0.95 0.93 0.88 0.92
0.039 0.051 0.078 0.106 0.043
0.92 0.92 0.91 0.76 0.94
Tm1
Tm2
DHcal1
DHcal2
DHV1
DHV2
n1
n2
v2/DOF
51.0
56.0
26.4
64.7
81.7
109.3
0.32
0.59
0.006
Melting temperature (Tm) derived from fitting to a two-state model. Error is ±0.2 °C. b The calorimetrically measured total enthalpy change (DHcal) obtained using the two-state model. Errors on DHcal values are ±1.0%. c The van‘t Hoff enthalpy (DHV) from applying the two-state model to calorimetric data. Errors on DHV values are ±1.5%. d The ratio of DHcal-to-DHV, for the two-state model. e The reduced v2 value used to evaluate the goodness of fitting. f The ratio of DHcal in the second heating process to that in the first heating process. g The solution of HEWL without any additive. In addition, no effect of MgCl2 on the Tm value of HEWL was observed. h Applying the three-state model to calorimetric data analysis. The parameters with the index ‘1’ are for the first transition and that with the index ‘2’ for the second transition.
601
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
Fig. 3. Apparent unfolded fraction of (A) HEWL, (B) adp-HEWL, (C) atp-HEWL, and (D) mgatp-HEWL as a function of temperature monitored by the changes of ellipticity at 222 nm (squares) and 191 nm (circles). The ellipticities at 25 and 70 °C were considered to represent the native and unfolded states, respectively, and were normalized to the range of 0–1. The solid lines represent theoretical curves resulting from the nonlinear least square fitting of the individual data sets. Fig. 2. (A,B) The SRCD spectra sets of HEWL and atp-HEWL at pH 2.0 for a temperature range of 25–70 °C. (C,D) The data in (A,B) deconvoluted into three basis curves using the CCA. (E,F) The fraction of each basis curve contributing to each spectrum at each temperature. The fractional weights of the two curves corresponding to the native protein were summed in (E) because they were almost identical.
Table 3 T1/2 valuesa determined by the least square fitting of the ellipticities at 191 and 222 nm as a function of temperature. Error is ±0.5 °C.
a-helix, the b-sheet changes here are big enough to support our conclusion. At 70 °C, the magnitude of the atp-HEWL spectrum at 222 nm is about 34% larger than that of the HEWL spectrum (Fig. 2A and B), indicating that atp-HEWL remains more a-helix structure than HEWL at high temperature. SVD analysis reveals that the a-helix content of atp-HEWL is 5.3% more, on average, than that of HEWL over the temperature range of 47–70 °C (Fig. S3). This conclusion is consistent also with the DSC data. The DHcal of atp-HEWL obtained using the two-state model can be compared to that of HEWL. There is a difference of 16.6 kcal/mol, or about 17%, in DHcal between atpHEWL and HEWL. This difference should be due to the incomplete unfolding of atp-HEWL at high temperature [27]. It should be noted that the T1/2 values determined by SRCD are all smaller than the Tm values determined by DSC, because SRCD reflects the features of secondary structures while DSC reflects the overall profile of heat capacity. Nevertheless, both techniques monitor the same process [28]. The combination of SRCD and DSC data demonstrates this view: In the unfolding process of atp-HEWL, the b-domain breaks up first. At about 50 °C, atp-HEWL unfolds into a partially unfolded intermediate with relatively rich a-helix and low b-sheet structures instead of a monomeric b-sheet enriched intermediate. 3.3. ATP-promoted aggregation of partially unfolded intermediate In the unfolding process of HEWL, the hydrodynamic radius varies from 1.5 to 3.0 nm (Fig. 4A), confirming previous literature data [29]. Actually, HEWL has four disulfide bonds distributed over the polypeptide chain, which limit the freedom of expansion of the polypeptide coil. However, in the unfolding process of atp-HEWL,
Additives
T1/2–191
T1/2–222
–b AMP ADP ATP MgATP
53.8 53.5 52.8 50.7 53.1
53.8 53.8 53.8 54.2 53.8
a
T1/2 is the temperature value at which the apparent unfolded fraction equals
0.5. b
The solution of HEWL without any additive. In addition, no effect of MgCl2 on the T1/2 value of HEWL was observed.
the aggregation begins to arise at 42 °C. Interestingly, the aggregation is not observed when the temperature is lower than 42 °C, and gradually breaks up and returns to monomers when the temperature is higher than 52 °C (Fig. 4C). This indicates that the partially unfolded intermediate has a much greater tendency to aggregate than the native and the unfolded states. Here is a question whether the electrostatic neutralizing effect of ATP dominates the aggregation of proteins. Although the negatively charged ADP and AMP can compensate the electrostatic repulsions between positively charged HEWL monomers, there is no aggregation of HEWL in the presence of ADP, AMP, and MgATP in the temperature range of 25–60 °C (Fig. 4). Correspondingly, ADP, AMP, and MgATP do not affect the cooperativity of HEWL unfolding. Hence, it is demonstrated that ATP-induced intermediate is more critical for protein aggregation, i.e., the ease of aggregating is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric b-sheet enriched intermediate. In support of this, two amyloidogenic human lysozyme variants have been shown to aggregate on heating resulted from the instability of the b-domain [2], and a peptide corresponding to the b-sheet region of HEWL has
602
H. Liu et al. / Biochemical and Biophysical Research Communications 397 (2010) 598–602
Fig. 4. Temperature dependence of hydrodynamic radius of (A) HEWL, (B) adpHEWL, (C) atp-HEWL, and (D) mgatp-HEWL estimated via the DLS measurements. The temperature varied from 25 to 60 °C.
been found to form extensive interchain interactions [30]. Moreover, the fibrils formed from atp-HEWL are much longer, straighter, and more periodical (Fig. S4), indicating that the intermediate makes the self-assembly of fibrils more orderly and easier. In summary, the binding of ATP to HEWL makes the residue W62, and even W63, move into more hydrophobic environments. Compared to the effects of ADP, AMP, and MgATP, it is reasonable to infer that the much stronger electrostatic effect of ATP induces the alteration of secondary structures of HEWL. This alteration decreases the b-domain stability of HEWL in the thermal unfolding process, results in a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate at about 50 °C. This intermediate containing relatively rich a-helix and less b-sheet structure has a much greater tendency to aggregate than the native and the unfolded states. It is demonstrated that ATP-induced intermediate, rather than the electrostatic neutralizing effect, is more critical for protein aggregation. This study implies that monomeric b-sheet enriched intermediate does not represent a general step under the conditions of amyloid aggregation. We expect that more detailed studies of longer polyanions will lead to a better understanding of the role of polyelectrolytes underlying the amyloid aggregation of proteins. Acknowledgment This work was supported by Chinese National Natural Science Foundation (No. 10635060 and 10725522). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.005. References [1] M.S. Celej, C.G. Montich, G.D. Fidelio, Protein stability induced by ligand binding correlates with changes in protein flexibility, Protein Sci. 12 (2003) 1496–1506.
[2] D.R. Booth, M. Sunde, V. Bellotti, C.V. Robinson, W.L. Hutchinson, P.E. Fraser, P.N. Hawkins, C.M. Dobson, S.E. Radford, C.C. Blake, M.B. Pepys, Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis, Nature 385 (1997) 787–793. [3] C. Exley, ATP-promoted amyloidosis of an amyloid beta peptide, Neuroreport 8 (1997) 3411–3414. [4] M. Calamai, J.R. Kumita, J. Mifsud, C. Parrini, M. Ramazzotti, G. Ramponi, N. Taddei, F. Chiti, C.M. Dobson, Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes, Biochemistry 45 (2006) 12806–12815. [5] D. Morshedi, A. Ghasemi, M. Evini, A. Ebrahim-Hababi, M. Nemat-Gorgani, Dramatic enhancement of lysozyme fibrillogenesis by ATP, FEBS J. 275 (2008) 194. [6] L.N. Arnaudov, R. de Vries, Thermally induced fibrillar aggregation of hen egg white lysozyme, Biophys. J. 88 (2005) 515–526. [7] H.R. Kalhor, M. Kamizi, J. Akbari, A. Heydari, Inhibition of amyloid formation by ionic liquids: ionic liquids affecting intermediate oligomers, Biomacromolecules 10 (2009) 2468–2475. [8] E. Frare, P. Polverino De Laureto, J. Zurdo, C.M. Dobson, A. Fontana, A highly amyloidogenic region of hen lysozyme, J. Mol. Biol. 340 (2004) 1153–1165. [9] S. Srisailam, H.M. Wang, T.K.S. Kumar, D. Rajalingam, V. Sivaraja, H.S. Sheu, Y.C. Chang, C. Yu, Amyloid-like fibril formation in an all beta-barrel protein involves the formation of partially structured intermediate(s), J. Biol. Chem. 277 (2002) 19027–19036. [10] V.J. McParland, A.P. Kalverda, S.W. Homans, S.E. Radford, Structural properties of an amyloid precursor of beta(2)-microglobulin, Nat. Struct. Biol. 9 (2002) 326–331. [11] C. Exley, O.V. Korchazhkina, Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP), J. Inorg. Biochem. 84 (2001) 215– 224. [12] D. Morshedi, N. Rezaei-Ghaleh, A. Ebrahim-Habibi, S. Ahmadian, M. NematGorgani, Inhibition of amyloid fibrillation of lysozyme by indole derivatives – possible mechanism of action, FEBS J. 274 (2007) 6415–6425. [13] J. Klein-Seetharaman, M. Oikawa, S.B. Grimshaw, J. Wirmer, E. Duchardt, T. Ueda, T. Imoto, L.J. Smith, C.M. Dobson, H. Schwalbe, Long-range interactions within a nonnative protein, Science 295 (2002) 1719–1722. [14] M. Eleftheriou, R.S. Germain, A.K. Royyuru, R. Zhou, Thermal denaturing of mutant lysozyme with both the OPLSAA and the CHARMM force fields, J. Am. Chem. Soc. 128 (2006) 13388–13395. [15] T. Ohkuri, T. Imoto, T. Ueda, Effect of W62G mutation of hen lysozyme on the folding in vivo, Biochem. Biophys. Res. Commun. 338 (2005) 820–824. [16] D.V. Laurents, R.L. Baldwin, Characterization of the unfolding pathway of hen egg white lysozyme, Biochemistry 36 (1997) 1496–1504. [17] H.L. Liu, X.H. Peng, F. Zhao, G.B. Zhang, Y. Tao, Z.F. Luo, Y. Li, M.K. Teng, X. Li, S.Q. Wei, N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1, Biochem. Biophys. Res. Commun. 379 (2009) 1120–1125. [18] N.J. Greenfield, Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions, Nat. Protoc. 1 (2007) 2527–2535. [19] M.C. Vaney, S. Maignan, M. Ries-Kautt, A. Ducruix, High-resolution structure (1.33 Å) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission, Acta Crystallogr. D 52 (1996) 505–517. [20] C.S. Wolfgang Kabsch, Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features, Biopolymers 22 (1983) 2577–2637. [21] N. Sreerama, S.Y. Venyaminov, R.W. Woody, Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy, Protein Sci. 8 (1999) 370–380. [22] S. Brahms, J. Brahms, Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism, J. Mol. Biol. 138 (1980) 149–178. [23] F.X. Schmid, Optical spectroscopy to characterize protein conformation and conformational changes, in: T.E. Creighton (Ed.), Protein Structure: A Practical Approach, IRL Press, Oxford, 1997, pp. 261–297. [24] J.M. Beechem, L. Brand, Time-resolved fluorescence of proteins, Annu. Rev. Biochem. 54 (1985) 43–71. [25] P.L. Privalov, Scanning microcalorimeters for studying macromolecules, Pure Appl. Chem. 52 (1980) 479–497. [26] R.W. Woody, Theory of circular dichroism of proteins, in: G.D. Fasman (Ed.), Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996, pp. 25–67. [27] L. Mitra, N. Smolin, R. Ravindra, C. Royer, R. Winter, Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution – experiments and theoretical interpretation, Phys. Chem. Chem. Phys. 8 (2006) 1249–1265. [28] D.G. Pina, J. Gómez, P. England, C.T. Craescu, L. Johannes, V.L. Shnyrov, Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit, Biochimie 88 (2006) 1199–1207. [29] A. Esposito, L. Comez, S. Cinelli, F. Scarponi, G. Onori, Influence of glycerol on the structure and thermal stability of lysozyme: a dynamic light scattering and circular dichroism study, J. Phys. Chem. B 113 (2009) 16420–16424. [30] J.J. Yang, M. Pitkeathly, S.E. Radford, Far-UV circular-dichroism reveals a conformational switch in a peptide fragment from the beta-sheet of hen lysozyme, Biochemistry 33 (1994) 7345–7353.