Eur Biophys J (2014) 43:1–9 DOI 10.1007/s00249-013-0934-9
ORIGINAL PAPER
Effects of environmental factors on MSP21–25 aggregation indicate the roles of hydrophobic and electrostatic interactions in the aggregation process Xuecheng Zhang • Yuanqiu Dong • Jigang Yu Xiaoming Tu
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Received: 11 June 2013 / Revised: 28 September 2013 / Accepted: 15 October 2013 / Published online: 23 October 2013 Ó European Biophysical Societies’ Association 2013
Abstract Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the merozoite surface of Plasmodium falciparum, is recognized to be important for the parasite’s invasion into the host cell and is thus a promising malaria vaccine candidate. However, mediated mainly by its conserved N-terminal 25 residues (MSP21–25), MSP2 readily forms amyloid fibril-like aggregates under physiological conditions in vitro, which impairs its potential as a vaccine component. In addition, there is evidence that MSP2 exists in aggregated forms on the merozoite surface in vivo. To elucidate the aggregation mechanism of MSP21–25 and thereby understand the behavior of MSP2 in vivo and find ways to avoid the aggregation of relevant vaccine in vitro, we investigated the effects of agitation, pH, salts, 1-anilinonaphthalene-8-sulfonic acid (ANS), trimethylamine N-oxide dihydrate (TMAO), urea, and submicellar sodium dodecyl sulfate (SDS) on the aggregation kinetics of MSP21–25 using thioflavin T (ThT) fluorescence. The results showed that MSP21–25 aggregation was Electronic supplementary material The online version of this article (doi:10.1007/s00249-013-0934-9) contains supplementary material, which is available to authorized users. X. Zhang (&) Y. Dong J. Yu School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei 230601, Anhui, People’s Republic of China e-mail: [email protected] X. Zhang Y. Dong J. Yu Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 111 Jiulong Road, Hefei, Anhui 230601, People’s Republic of China X. Tu School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of China
accelerated by agitation, while repressed by acidic pHs. The salts promoted the aggregation in an anion naturedependent pattern. Hydrophobic surface-binding agent ANS and detergent urea repressed MSP21–25 aggregation, in contrast to hydrophobic interaction strengthener TMAO, which enhanced the aggregation. Notably, sub-micellar SDS, contrary to its micellar form, promoted MSP21–25 aggregation significantly. Our data indicated that hydrophobic interactions are the predominant driving force of the nucleation of MSP21–25 aggregation, while the elongation is controlled mainly by electrostatic interactions. A kinetic model of MSP21–25 aggregation and its implication were also discussed. Keywords Amyloid fibril Environmental factor Intrinsically disordered protein Merozoite surface protein 2 Protein aggregation Thioflavin T Abbreviations ANS 1-Anilinonaphthalene-8-sulfonic acid CD Circular dichroism MSP21–25 Conserved N-terminal 25 residues merozoite surface protein 2 SDS Sodium dodecylsulfate TEM Transmission electronic microscopy TFE Trifluoroethanol ThT Thioflavin T TMAO Trimethylamine N-oxide dehydrate
of
Introduction Malaria is one of the most serious infectious diseases in the world, infecting hundreds of millions of people and killing nearly a million of them annually, so the development of
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vaccines against it is a major global health priority (Snow et al. 2005). One of the most abundant proteins on the surface of malaria Plasmodium falciparum merozoites, Merozoite surface protein 2 (MSP2), has shown significant potential as a malaria vaccine component (Anders et al. 2010; Genton et al. 2003; McCarthy et al. 2011). However, recent studies have shown that full-length MSP2 exists in oligomeric forms on the merozoite surface in vivo and is prone to form amyloid fibril-like aggregates in vitro (Adda et al. 2009). The aggregation of MSP2 may impair its production, safety, and particularly the efficacy as a vaccine component, as the monomer and aggregates may have different antigenic and immunogenic properties. The amyloidogenic propensity of full-length MSP2 is mainly contributed by the conserved N-terminal 25 residues of MSP2 (MSP21–25), which constitute the structural core of the fibrils (Adda et al. 2009). This is supported by the fact that isolated MSP21–25 forms fibrils similar to those formed by full-length MSP2 (Low et al. 2007; Yang et al. 2007). In addition, MSP21–25 offers the full-length protein the ability to interact with the membrane (Zhang et al. 2008), which in return inhibits the aggregation of the peptide (Zhang et al. 2012). It is appreciated that studies on isolated amyloidogenic peptides may provide fundamental insights into the behavior of the intact proteins. Thus, we focus on isolated MSP21–25 to better understand the properties of full-length MSP2 and help develop a relevant malaria vaccine. To find ways to avoid or control the formation of amyloid fibrils, it is essential to understand the factors that affect the aggregation kinetics, which normally display a sigmoidal profile including a lag (nucleation) phase followed by a growth (elongation) phase and a final stationary (equilibrium) phase. Environmental factors modulate aggregation by affecting the production of aggregationprone monomers, which associate with each other to form nuclei in the lag phase or join into the oligomers/protofibrils in the growth phase (Straub and Thirumalai 2010, 2011). From the structural viewpoint, amyloid fibril formation requires either partial unfolding of native proteins (Fink 1998) or partial folding of intrinsically disordered proteins (IDPs) (Thirumalai et al. 2003) to populate the aggregation-prone states. Our previous study on MSP21–25 showed that it is intrinsically disordered, and low concentrations of TFE (trifluoroethanol) enhanced its aggregation by inducing a helical structure (Zhang et al. 2012), supporting that the partially folded state is a prerequisite for the amyloid fibril formation of IDPs. However, high concentrations of TFE in contrast repressed MSP21–25 aggregation (Zhang et al. 2012). Hydrophobic as well as electrostatic interactions are known to play critical roles in the amyloid fibril formation by proteins and peptides (Auer et al. 2008; Straub and Thirumalai 2011; Tarus et al. 2008). Thus, the varying effects of TFE on MSP21–25 aggregation
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may be explained by the interplay between hydrophobic and electrostatic interactions, both of which could be influenced by TFE. One way to understand in depth the roles of the two types of interactions in MSP21–25 aggregation is to investigate the effects of different environmental factors that influence either hydrophobic or electrostatic interactions, or both, on the aggregation. In the present study, we used ThT fluorescence as the probe of the aggregates formed by MSP21–25 to investigate the effects of agitation, pH, salt, ANS, urea, TMAO, and sub-micellar SDS on the aggregation kinetics. To exclude the contribution by the secondary structural changes in the peptide itself, CD spectra were also measured for these under various conditions. Our results indicated that the nucleation of MSP21–25 is driven predominantly by hydrophobic interactions, while the elongation of the aggregates is controlled mainly by electrostatic interactions.
Materials and methods Materials ThT and ANS were purchased from Sigma-Aldrich (Shanghai). Other reagents were all of analytic or higher purity. MSP21–25 (IKNESKYSNTFINNAYNMSIRRSMA, 98 % pure) was purchased from GL Biochem Ltd. (Shanghai) and used without further purification. Peptide stock solutions were prepared by dissolving the peptide in MilliQ water and incubating the solution at 80 °C for 10 min, followed by centrifugation at 13,000 rpm for 10 min and filtration by a 0.02-lm filter to remove preformed aggregates. The exact concentrations of peptide in the final stock solutions were determined by absorption at 280 nm using an extinction coefficient of 2,980 M-1 cm-1, which was calculated from the amino acid composition using the ProtParam program in ExPasy (http://www. expasy.ch/tools/#primary). ThT and ANS fluorescence Thioflavin T (ThT) has been widely used for the detection of amyloid fibrils, as it can bind to the cross-b structure of amyloid fibrils, with the excitation maximum (ex) and emission maximum (em) shifting from *385/445 nm of the free form to *450/480 nm of the bound form (Naiki et al. 1990). A stock solution of 500 lM ThT was prepared in MilliQ water and stored at 4 °C protected from light to prevent quenching. For ThT fluorescence assays, 200 ll solutions containing 10 lM ThT and *30 lM (*0.1 mg ml-1) MSP21–25 were incubated in 96-microwell black polystyrene plates with flat bottoms (Costar, Corning Incorp.) and measured every 2 min. Three
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replicates corresponding to three wells were measured for each sample to allow for well-to-well variation. A SpectraMax M5 multidetection reader (Molecular Devices Ltd.) was used to read the ThT fluorescence with excitation at 443 nm and emission at 484 nm. Both the excitation and the emission slits were maintained at 2 nm. The samples were shaken for 5 s before the first read and 3 s before every interval read. All measurements were carried out at 20 °C. The samples for ANS fluorescence kinetics measurements were prepared in the same way as for ThT assays, except that ThT was replaced with ANS and the assays were performed with ex/em of 360/480 nm. The ANS fluorescence emission spectrum was scanned from 420 to 600 nm with a 360-nm excitation wavelength. Transmission electronic microscopy Samples for TEM were prepared as for the ThT fluorescence assay except that they contained no ThT; 10 lL of each sample was deposited on a carbon-coated grid, excess material was removed by blotting, and samples were negatively stained twice with 10 ll of a 2 % uranyl acetate solution (w/v). The grids were air dried and viewed on a Tecnai F20 transmission electron microscope (FEI).
Fig. 1 The aggregation kinetics of MSP21–25 in PBS with and without agitation monitored by ThT fluorescence
Compared with the stationary samples, the agitated ones aggregated much faster, with the lag phase significantly reduced in an interval-dependent pattern (Fig. 1). Therefore, to make MSP21–25 aggregation suitably rapid for convenient analysis, the kinetics assays in the present study were all performed with agitation (shaking 3 s every 2 min) unless stated otherwise.
Circular dichroism MSP21–25 aggregation was pH dependent Circular dichroism measurements were carried out on a Jasco Model J-810 spectropolarimeter. Spectra were measured in the range of 190–250 nm at a protein concentration of 30 lM (0.1 mg ml-1) using a 0.1-cm path-length cell. All CD measurements were performed at room temperature (20 °C), and each spectrum was the average of three scans.
Results Agitation accelerated MSP21–25 aggregation Followed with ThT fluorescence, MSP21–25 incubated in PBS in the absence of agitation displayed a typical sigmoidal profile with an evident lag phase, a growth phase, and a stationary phase (Fig. 1), signifying a nucleationelongation process. To seek a condition under which MSP21–25 aggregation was accelerated to be completed within a suitable duration, the ThT fluorescence kinetics of samples under the same condition as the above but with agitation (shaking 5 s every 2 or 10 min) were tested. As expected, both the samples with and without agitation formed fibril-like aggregates, with the former leading to shorter fibrils than the latter (Online resource Fig S1).
In a previous study (Zhang et al. 2012), we noted that MSP21–25 aggregation was inhibited at pH 3.4, which was adopted to investigate the NMR structure of the peptide. In the present study, MSP21–25 aggregation at more pH values was monitored. To exclude the effects derived from buffer composition, all the solutions were prepared by 10 mM phosphate buffer at neutral pH titrated with H3PO4 or NaOH. The result showed that MSP21–25 aggregation was inhibited by acidic pHs of 3.4 and 5, whereas, at alkaline pHs of 9.4 and 11.4 MSP21–25 aggregated readily, with similar lag phases but faster growth rates compared with at physiological pHs (Fig. 2). At pH 3.4, changing the buffer from phosphate to acetate had no influence on MSP21–25 aggregation (Fig. 2). MSP21–25 aggregated a little faster in PBS at pH 7.3 than in 10 mM NaPi at pH 7.2, indicating that additional NaCl had a slight enhancement effect on the aggregation. Minor differences between the CD spectra of MSP21–25 under the various pH conditions (Online resource Fig. S2) implied a limited impact of pH on the secondary structure. Therefore, the enhancement effect of high pH on MSP21–25 aggregation might be exerted dominantly by decreased intermolecular electrostatic repulsion rather than through an intramolecular one, which would affect the structure in the peptide itself.
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Fig. 2 The aggregation kinetics of MSP21–25 under different pH conditions monitored by ThT fluorescence
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Fig. 4 The aggregation kinetics of MSP21–25 in 10 mM HOAc with the sulfates of sodium, magnesium, and zinc, respectively, monitored by ThT fluorescence
(100 mM) of the sulfates of sodium, magnesium, and zinc aggregated with undetectable lag phases and similar growth rates (Fig. 4), confirming the minor role of cations in the aggregation-promoting effect, while CD spectra of MSP21–25 exhibited nearly superimposing curves in the presence of the various salts (Online resource Fig. S4), excluding the contribution by the secondary structure change. ANS repressed MSP21–25 aggregation
Fig. 3 The aggregation kinetics of MSP21–25 in 10 mM HOAc with different sodium salts, monitored by ThT fluorescence
Salts promoted MSP21–25 aggregation in an anion nature-dependent manner As described above, at physiological pH, additional NaCl had little influence on MSP21–25 aggregation. However, the situation altered at acidic pHs. As shown in Fig. 3, MSP21–25 aggregation in 10 mM HOAc was promoted by the addition of NaCl in a concentration-dependent manner. A similar result was obtained for the addition of NaH2PO4, NaAc, and Na2SO4, with the promotion extents for both the lag phase and the elongation phase being different from those caused by the same concentration of NaCl (Fig. 3). In addition, adding NaH2PO4 or Na2SO4 to PBS also enhanced MSP21–25 aggregation, with the lag phase being reduced (Online resource Fig S3). These results suggested that the salt effect is primarily an anion-induced one. MSP21–25 in 10 mM HOAc with the same concentration
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ANS can bind to the hydrophobic clusters exposed on the molecular surface and emits fluorescence maximally at a wavelength shorter than that of free ANS (so called ‘‘blueshift’’); thus, it is widely used to detect the existence of partially folded states of proteins (Strickland and Mercola 1976). The ANS fluorescence spectrum of MSP21–25 in PBS showed a maximum emission wavelength at about 470 nm, strongly blue-shifted compared to that of free ANS, 520 nm (Fig. 5a), signifying the presence of a partially folded state. Presumably, ANS would hinder the aggregation of MSP21–25 by blocking hydrophobic associations such as those between the residues F11I12, which were demonstrated to be critical for the aggregation, particularly the nucleation stage of the peptide (Yang et al. 2010). Actually, in the presence of ANS, MSP21–25 aggregation in PBS was repressed in a concentrationdependent manner, with the lag phase extended to being unobservable within the measurement period at high ANS concentration (Fig. 5b). To test the abundance of accessible hydrophobic clusters of the components present along the aggregation pathway, ANS fluorescence at 480 nm was monitored throughout the MSP21–25 aggregation process. The result showed that ANS fluorescence, like ThT
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Fig. 5 a The fluorescence emission spectra of free and MSP21–25bound ANS in PBS, b the aggregation kinetics of MSP21–25 in PBS with different concentrations of ANS monitored by ThT fluorescence,
and c the aggregation kinetics monitored by ANS fluorescence at ex/ em of 360/480 nm
fluorescence, showed a typical sigmoidal profile when aggregation was progressing (Fig. 5c). This indicated that MSP21–25 aggregation would produce a more hydrophobic surface, which might be a characteristics of the aggregates.
kosmotropic (order-making) force, a power similar to hydrophobic force in essence (Baskakov and Bolen 1998; Wang and Bolen 1997). Therefore, contrary to urea, TMAO enhanced the aggregation of MSP21–25 when the lag phase decreased, also in a concentration-dependent manner (Fig. 6b). However, neither of the effects of urea and TMAO was exerted via structural change, demonstrated by the similar CD spectra of the peptide in the absence and presence of the two cosolvents (Online resource Fig. S5).
Urea and TMAO had opposite effects on MSP21–25 aggregation Urea is a good solvent and frequently used to denature protein by disrupting hydrophobic interactions. In addition, urea could also break the hydrogen bond by interfering with the electrostatic interactions (Klimov et al. 2004; Tobi et al. 2003). Therrefore, as expected, urea repressed MSP21–25 aggregation in PBS with both the lag phase and the growth phase elongated in a concentration-dependent manner (Fig. 6a). By contrast, TMAO is a naturally occurring osmolyte that stabilizes proteins in vivo under some stress conditions; it could induce unfolded protein to fold by
Sub-micellar SDS promoted MSP21–25 aggregation In a previous study, we used micellar SDS as a membrane mimetic to investigate the aggregation of MSP21–25 under a membranous environment, and the result showed that SDS micelles induced a helical structure and inhibited the aggregation (Zhang et al. 2012). However, in the present study, the effect of SDS was reversed when the
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Fig. 6 The aggregation kinetics monitored by ThT fluorescence for MSP21–25 in PBS with different concentrations of a urea and b TMAO Fig. 7 a The aggregation kinetics monitored by ThT fluorescence, and b the far UV CD spectra for MSP21–25 in the absence and presence of submicellar SDS
concentration decreased below the critical micellar concentration. As shown in Fig. 7a, addition of 1 mM SDS to PBS-enhanced MSP21–25 aggregation significantly, with the lag phase and part of the elongation phase being undetectable. Moreover, addition of 1 mM SDS in 10 mM HOAc diminished the inhibition effect of the acidic pH and promoted MSP21–25 aggregation to a degree comparable to that in PBS. SDS at concentrations between 0.1 and 1 mM caused similar results (data not shown). Intriguingly, submicellar concentrations of another frequently used phospholipid detergent, dodecylphosphocholine (DPC), had no influence on MSP21–25 aggregation in either PBS or in 10 mM HOAc (data not shown), implying the importance of the negatively charged head group of SDS. The
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CD spectrum of MSP21–25 in PBS with 1 mM SDS displayed a deep trough near 217 nm (Fig. 7b), indicating a structural change that facilitated the cross-b structure formation and might provide an additional contribution to the intensive aggregation enhancement effect.
Discussion Interplay between hydrophobic and electronic interactions in MSP21–25 aggregation MSP21–25 aggregation in vitro was shown to be a nucleation-elongation process (Zhang et al. 2012) and has been
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used to explain the assembly kinetics of amyloid-like fibrils composed of prions (Eigen 1996; Lundberg et al. 1997), Ab (Teplow 1998), and TTR (Kelly et al. 1997). The aggregation of all amyloidogenic proteins may share an essential mechanism, which could be attributed to the interplay between hydrophobic and electrostatic interactions (Auer et al. 2008; Straub and Thirumalai 2011; Tarus et al. 2008). It is assumed that the self-organization of the protein and the stability of the amyloid fibril arise by maximizing the number of hydrophobic and favorable electrostatic interactions, especially hydrogen bonding (Tarus et al. 2006). According to the assumption and based on our previous results that MSP21–25 aggregated with increasing lag phases and growth rates with rising TFE concentration (Zhang et al. 2012), we speculated that MSP21–25 aggregation is modulated by both hydrophobic and electrostatic interactions, with the nucleus formation driven predominantly by the former and the aggregate elongation controlled mainly by the latter. That is, any environmental factor facilitating intermolecular hydrophobic interactions, e.g., low concentrations of TFE, could promote nucleation, while those conditions favoring electrostatic interactions, e.g., high concentrations of TFE, could enhance elongation. The results obtained in the present study provide further evidence for this speculation. MSP21–25 is rich in basic residues (two Lys and two Arg) while deficient in acidic ones (only one Glu), leading to an alkaline theoretical pI of 9.99 (calculated by ProtParam program in ExPasy, http://www.expasy.ch/tools/ #primary). Therefore, it is reasonable that at alkaline pHs MSP21–25 aggregated readily, as it could easily self-associate to form nuclei because of weak inter-molecular electrostatic repulsion. On the other hand, the growth of MSP21–25 aggregation at alkaline pHs was faster than at neutral pHs, indicating the contribution of electrostatic interactions to the elongation phase. Salts have been shown to affect protein aggregation by either an electrostatic effect (Pedersen et al. 2006; Raman et al. 2005) or hydrophobic effect (Munishkina et al. 2004; Yeh et al. 2010), or both (Jain and Udgaonkar 2010; Klement et al. 2007). As inter- or intra-molecular electrostatic interactions could be shielded by ions with opposite charge, addition of salts at pH 3.4 would screen the charge repulsion and promote self-association between MSP21–25 peptides with the lag phase reduced in an anion naturedependent manner. The aggregation-promoting power of anions thus should be listed in the order of ionic strength and binding affinity: SO42-*HPO42- [ Ac-*Cl-. However, though the electrostatic effect of the salts was consistent with the growth rate difference, it alone could not explain the big difference between their nucleationpromoting effects, implying a contribution by other forces. Actually, anions could promote aggregation by acting as a
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kosmotrope (order-maker) by strengthening hydrophobic interactions in the order of Hofmeister series as follows: SO42- [ HPO42-*Ac- [ Cl- (Hofmeister 1888). This additional force could explain the greater lag phasereducing effect of acetate than chloride. Furthermore, addition of Na2SO4 or Na2HPO4 to 10 mM HOAc caused MSP21–25 to nucleate much faster than it did at alkaline pHs, stressing the predominant role of hydrophobic interactions in the nucleation. In addition, adding the salts to PBS also enhanced MSP21–25 aggregation with the lag phase reduced, providing further evidence for the role of hydrophobic interactions in the aggregate nucleation. The role of hydrophobic interactions in the nucleation phase of MSP21–25 aggregation is also supported by the effect of TMAO, which shortened the lag phase significantly to even undetectable levels. Addition of ANS elongating the lag phase provides further evidence for the essential role of hydrophobic interactions. However, ANS fluorescence, like the ThT signal, increasing along with the MSP21–25 aggregation process indicates that hydrophobic interactions are sparsely involved in the growth phase. The significance of hydrophobic interactions for MSP21–25 aggregation is also evidenced by the fact that the sample suffering agitation aggregated much faster than that staying stationary. Agitation would produce more water-air interface, which is hydrophobic and therefore favors hydrophobic interactions (Sluzky et al. 1992; Nielsen et al. 2001), and thus could promote the nucleation process. The roles of hydrophobic and electrostatic interactions in the lag phase of MSP21–25 aggregation have been demonstrated by the effect of TFE on the aggregation. In our previous study, we showed that low concentrations of TFE enhanced MSP21–25 aggregation by inducing a partially folded state, whereas high concentrations repressed the aggregation (Zhang et al. 2012). As we know, TFE denatures proteins by weakening hydrophobic interactions and induces secondary structures by enhancing electrostatic interactions, particularly hydrogen bonding, in a concentration-dependent manner (Albert and Hamilton 1995; Liu et al. 2004; Luo and Baldwin 1997; Thomas and Dill 1993; Yang et al. 1993). Therefore, the effect of high concentrations of TFE on MSP21–25 aggregation could be explained, besides in the structural aspect as described previously (Zhang et al. 2012), also by the interplay between increased electrostatic and decreased hydrophobic interactions: with TFE concentration increasing, the hydrophobic interaction-destabilizing power grows to repress the aggregation by hampering the nucleation, while strengthened hydrogen bonding enhances the growth rate. Like TFE, sub-micellar SDS might influence the aggregation of MSP21–25 by affecting both electrostatic and hydrophobic interactions via its polar head group and non-polar tail (Naiki et al. 2005; Wang et al. 1996; Yonath
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et al. 1977). The inter-molecular electrostatic interactions between the negatively charged sulfate group of SDS and the positive charges on the MSP21–25 surface would locate the aliphatic tail close to the peptide, strongly facilitating the peptide monomer self-associating to form nuclei or attaching to the aggregate end through hydrophobic interactions. Thus, SDS promoted MSP21–25 aggregation to a much higher extent than TMAO and the salts, with both the nucleation and elongation phases shortened. As for urea, it is believed to coat hydrophobic interactions by forming hydrogen bonds with backbone amides of proteins (Klimov et al. 2004; Tobi et al. 2003). Therefore, it is reasonable that urea elongated both the lag and growth phases of MSP21–25 aggregation. Implication for the MSP2 aggregation mechanism and relevant aggregation-preventing methods According to the current results, we suggest that the aggregation of MSP21–25 is a nucleation-elongation process, in which the nucleation is driven predominantly by hydrophobic interactions and the elongation is controlled mainly by electrostatic interactions. At physiological pHs, slightly charged and partially folded MSP21–25 self-associates to form nuclei via intermolecular hydrophobic interactions. Then, the nuclei transform to oligomers/protofibrils via conformational change from a helical to bstructure, which is stabilized by hydrogen bonding. The formation of the oligomers/protofibrils is a rate-limiting step, followed by relatively downhill growth. The growth phase may progress via a ‘‘dock and lock’’ mechanism (Straub and Thirumalai 2011), in which monomeric peptide attaches to the oligomers/protofibrils by a hydrophobic force-driven ‘‘docking’’ process, and the newly joined monomer transforms to a b-structure, which is stabilized by electrostatic hydrogen bonding and clusters of hydrophobic groups on surface, to finish the ‘‘locking’’ stage. The above model may provide clues for developing methods to prevent MSP21–25 and full-length MSP2 from aggregating in vitro; factors that could hinder the intermolecular association by hydrophobic interactions would inhibit the aggregation by hampering the nucleation, and these could weaken or eliminate electrostatic hydrogen bonding, which would inhibit the aggregation by hampering the elongation. Acknowledgments This work was supported by grants from the Educational Commission of Anhui Province of China (Grant no. KJ2010A024), Scientific Research Foundation for Returned Scholars, Ministry of Education of China, and the National Natural Science Foundation of China (Grant no. 30900228). Ethical standards We declare that all the experiments in the present work comply with the current laws of the PR China.
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The authors declare that they have no conflict
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ORIGINAL PAPER
Effects of environmental factors on MSP21–25 aggregation indicate the roles of hydrophobic and electrostatic interactions in the aggregation process Xuecheng Zhang • Yuanqiu Dong • Jigang Yu Xiaoming Tu
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Received: 11 June 2013 / Revised: 28 September 2013 / Accepted: 15 October 2013 / Published online: 23 October 2013 Ó European Biophysical Societies’ Association 2013
Abstract Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the merozoite surface of Plasmodium falciparum, is recognized to be important for the parasite’s invasion into the host cell and is thus a promising malaria vaccine candidate. However, mediated mainly by its conserved N-terminal 25 residues (MSP21–25), MSP2 readily forms amyloid fibril-like aggregates under physiological conditions in vitro, which impairs its potential as a vaccine component. In addition, there is evidence that MSP2 exists in aggregated forms on the merozoite surface in vivo. To elucidate the aggregation mechanism of MSP21–25 and thereby understand the behavior of MSP2 in vivo and find ways to avoid the aggregation of relevant vaccine in vitro, we investigated the effects of agitation, pH, salts, 1-anilinonaphthalene-8-sulfonic acid (ANS), trimethylamine N-oxide dihydrate (TMAO), urea, and submicellar sodium dodecyl sulfate (SDS) on the aggregation kinetics of MSP21–25 using thioflavin T (ThT) fluorescence. The results showed that MSP21–25 aggregation was Electronic supplementary material The online version of this article (doi:10.1007/s00249-013-0934-9) contains supplementary material, which is available to authorized users. X. Zhang (&) Y. Dong J. Yu School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei 230601, Anhui, People’s Republic of China e-mail: [email protected] X. Zhang Y. Dong J. Yu Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 111 Jiulong Road, Hefei, Anhui 230601, People’s Republic of China X. Tu School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of China
accelerated by agitation, while repressed by acidic pHs. The salts promoted the aggregation in an anion naturedependent pattern. Hydrophobic surface-binding agent ANS and detergent urea repressed MSP21–25 aggregation, in contrast to hydrophobic interaction strengthener TMAO, which enhanced the aggregation. Notably, sub-micellar SDS, contrary to its micellar form, promoted MSP21–25 aggregation significantly. Our data indicated that hydrophobic interactions are the predominant driving force of the nucleation of MSP21–25 aggregation, while the elongation is controlled mainly by electrostatic interactions. A kinetic model of MSP21–25 aggregation and its implication were also discussed. Keywords Amyloid fibril Environmental factor Intrinsically disordered protein Merozoite surface protein 2 Protein aggregation Thioflavin T Abbreviations ANS 1-Anilinonaphthalene-8-sulfonic acid CD Circular dichroism MSP21–25 Conserved N-terminal 25 residues merozoite surface protein 2 SDS Sodium dodecylsulfate TEM Transmission electronic microscopy TFE Trifluoroethanol ThT Thioflavin T TMAO Trimethylamine N-oxide dehydrate
of
Introduction Malaria is one of the most serious infectious diseases in the world, infecting hundreds of millions of people and killing nearly a million of them annually, so the development of
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vaccines against it is a major global health priority (Snow et al. 2005). One of the most abundant proteins on the surface of malaria Plasmodium falciparum merozoites, Merozoite surface protein 2 (MSP2), has shown significant potential as a malaria vaccine component (Anders et al. 2010; Genton et al. 2003; McCarthy et al. 2011). However, recent studies have shown that full-length MSP2 exists in oligomeric forms on the merozoite surface in vivo and is prone to form amyloid fibril-like aggregates in vitro (Adda et al. 2009). The aggregation of MSP2 may impair its production, safety, and particularly the efficacy as a vaccine component, as the monomer and aggregates may have different antigenic and immunogenic properties. The amyloidogenic propensity of full-length MSP2 is mainly contributed by the conserved N-terminal 25 residues of MSP2 (MSP21–25), which constitute the structural core of the fibrils (Adda et al. 2009). This is supported by the fact that isolated MSP21–25 forms fibrils similar to those formed by full-length MSP2 (Low et al. 2007; Yang et al. 2007). In addition, MSP21–25 offers the full-length protein the ability to interact with the membrane (Zhang et al. 2008), which in return inhibits the aggregation of the peptide (Zhang et al. 2012). It is appreciated that studies on isolated amyloidogenic peptides may provide fundamental insights into the behavior of the intact proteins. Thus, we focus on isolated MSP21–25 to better understand the properties of full-length MSP2 and help develop a relevant malaria vaccine. To find ways to avoid or control the formation of amyloid fibrils, it is essential to understand the factors that affect the aggregation kinetics, which normally display a sigmoidal profile including a lag (nucleation) phase followed by a growth (elongation) phase and a final stationary (equilibrium) phase. Environmental factors modulate aggregation by affecting the production of aggregationprone monomers, which associate with each other to form nuclei in the lag phase or join into the oligomers/protofibrils in the growth phase (Straub and Thirumalai 2010, 2011). From the structural viewpoint, amyloid fibril formation requires either partial unfolding of native proteins (Fink 1998) or partial folding of intrinsically disordered proteins (IDPs) (Thirumalai et al. 2003) to populate the aggregation-prone states. Our previous study on MSP21–25 showed that it is intrinsically disordered, and low concentrations of TFE (trifluoroethanol) enhanced its aggregation by inducing a helical structure (Zhang et al. 2012), supporting that the partially folded state is a prerequisite for the amyloid fibril formation of IDPs. However, high concentrations of TFE in contrast repressed MSP21–25 aggregation (Zhang et al. 2012). Hydrophobic as well as electrostatic interactions are known to play critical roles in the amyloid fibril formation by proteins and peptides (Auer et al. 2008; Straub and Thirumalai 2011; Tarus et al. 2008). Thus, the varying effects of TFE on MSP21–25 aggregation
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may be explained by the interplay between hydrophobic and electrostatic interactions, both of which could be influenced by TFE. One way to understand in depth the roles of the two types of interactions in MSP21–25 aggregation is to investigate the effects of different environmental factors that influence either hydrophobic or electrostatic interactions, or both, on the aggregation. In the present study, we used ThT fluorescence as the probe of the aggregates formed by MSP21–25 to investigate the effects of agitation, pH, salt, ANS, urea, TMAO, and sub-micellar SDS on the aggregation kinetics. To exclude the contribution by the secondary structural changes in the peptide itself, CD spectra were also measured for these under various conditions. Our results indicated that the nucleation of MSP21–25 is driven predominantly by hydrophobic interactions, while the elongation of the aggregates is controlled mainly by electrostatic interactions.
Materials and methods Materials ThT and ANS were purchased from Sigma-Aldrich (Shanghai). Other reagents were all of analytic or higher purity. MSP21–25 (IKNESKYSNTFINNAYNMSIRRSMA, 98 % pure) was purchased from GL Biochem Ltd. (Shanghai) and used without further purification. Peptide stock solutions were prepared by dissolving the peptide in MilliQ water and incubating the solution at 80 °C for 10 min, followed by centrifugation at 13,000 rpm for 10 min and filtration by a 0.02-lm filter to remove preformed aggregates. The exact concentrations of peptide in the final stock solutions were determined by absorption at 280 nm using an extinction coefficient of 2,980 M-1 cm-1, which was calculated from the amino acid composition using the ProtParam program in ExPasy (http://www. expasy.ch/tools/#primary). ThT and ANS fluorescence Thioflavin T (ThT) has been widely used for the detection of amyloid fibrils, as it can bind to the cross-b structure of amyloid fibrils, with the excitation maximum (ex) and emission maximum (em) shifting from *385/445 nm of the free form to *450/480 nm of the bound form (Naiki et al. 1990). A stock solution of 500 lM ThT was prepared in MilliQ water and stored at 4 °C protected from light to prevent quenching. For ThT fluorescence assays, 200 ll solutions containing 10 lM ThT and *30 lM (*0.1 mg ml-1) MSP21–25 were incubated in 96-microwell black polystyrene plates with flat bottoms (Costar, Corning Incorp.) and measured every 2 min. Three
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replicates corresponding to three wells were measured for each sample to allow for well-to-well variation. A SpectraMax M5 multidetection reader (Molecular Devices Ltd.) was used to read the ThT fluorescence with excitation at 443 nm and emission at 484 nm. Both the excitation and the emission slits were maintained at 2 nm. The samples were shaken for 5 s before the first read and 3 s before every interval read. All measurements were carried out at 20 °C. The samples for ANS fluorescence kinetics measurements were prepared in the same way as for ThT assays, except that ThT was replaced with ANS and the assays were performed with ex/em of 360/480 nm. The ANS fluorescence emission spectrum was scanned from 420 to 600 nm with a 360-nm excitation wavelength. Transmission electronic microscopy Samples for TEM were prepared as for the ThT fluorescence assay except that they contained no ThT; 10 lL of each sample was deposited on a carbon-coated grid, excess material was removed by blotting, and samples were negatively stained twice with 10 ll of a 2 % uranyl acetate solution (w/v). The grids were air dried and viewed on a Tecnai F20 transmission electron microscope (FEI).
Fig. 1 The aggregation kinetics of MSP21–25 in PBS with and without agitation monitored by ThT fluorescence
Compared with the stationary samples, the agitated ones aggregated much faster, with the lag phase significantly reduced in an interval-dependent pattern (Fig. 1). Therefore, to make MSP21–25 aggregation suitably rapid for convenient analysis, the kinetics assays in the present study were all performed with agitation (shaking 3 s every 2 min) unless stated otherwise.
Circular dichroism MSP21–25 aggregation was pH dependent Circular dichroism measurements were carried out on a Jasco Model J-810 spectropolarimeter. Spectra were measured in the range of 190–250 nm at a protein concentration of 30 lM (0.1 mg ml-1) using a 0.1-cm path-length cell. All CD measurements were performed at room temperature (20 °C), and each spectrum was the average of three scans.
Results Agitation accelerated MSP21–25 aggregation Followed with ThT fluorescence, MSP21–25 incubated in PBS in the absence of agitation displayed a typical sigmoidal profile with an evident lag phase, a growth phase, and a stationary phase (Fig. 1), signifying a nucleationelongation process. To seek a condition under which MSP21–25 aggregation was accelerated to be completed within a suitable duration, the ThT fluorescence kinetics of samples under the same condition as the above but with agitation (shaking 5 s every 2 or 10 min) were tested. As expected, both the samples with and without agitation formed fibril-like aggregates, with the former leading to shorter fibrils than the latter (Online resource Fig S1).
In a previous study (Zhang et al. 2012), we noted that MSP21–25 aggregation was inhibited at pH 3.4, which was adopted to investigate the NMR structure of the peptide. In the present study, MSP21–25 aggregation at more pH values was monitored. To exclude the effects derived from buffer composition, all the solutions were prepared by 10 mM phosphate buffer at neutral pH titrated with H3PO4 or NaOH. The result showed that MSP21–25 aggregation was inhibited by acidic pHs of 3.4 and 5, whereas, at alkaline pHs of 9.4 and 11.4 MSP21–25 aggregated readily, with similar lag phases but faster growth rates compared with at physiological pHs (Fig. 2). At pH 3.4, changing the buffer from phosphate to acetate had no influence on MSP21–25 aggregation (Fig. 2). MSP21–25 aggregated a little faster in PBS at pH 7.3 than in 10 mM NaPi at pH 7.2, indicating that additional NaCl had a slight enhancement effect on the aggregation. Minor differences between the CD spectra of MSP21–25 under the various pH conditions (Online resource Fig. S2) implied a limited impact of pH on the secondary structure. Therefore, the enhancement effect of high pH on MSP21–25 aggregation might be exerted dominantly by decreased intermolecular electrostatic repulsion rather than through an intramolecular one, which would affect the structure in the peptide itself.
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Fig. 2 The aggregation kinetics of MSP21–25 under different pH conditions monitored by ThT fluorescence
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Fig. 4 The aggregation kinetics of MSP21–25 in 10 mM HOAc with the sulfates of sodium, magnesium, and zinc, respectively, monitored by ThT fluorescence
(100 mM) of the sulfates of sodium, magnesium, and zinc aggregated with undetectable lag phases and similar growth rates (Fig. 4), confirming the minor role of cations in the aggregation-promoting effect, while CD spectra of MSP21–25 exhibited nearly superimposing curves in the presence of the various salts (Online resource Fig. S4), excluding the contribution by the secondary structure change. ANS repressed MSP21–25 aggregation
Fig. 3 The aggregation kinetics of MSP21–25 in 10 mM HOAc with different sodium salts, monitored by ThT fluorescence
Salts promoted MSP21–25 aggregation in an anion nature-dependent manner As described above, at physiological pH, additional NaCl had little influence on MSP21–25 aggregation. However, the situation altered at acidic pHs. As shown in Fig. 3, MSP21–25 aggregation in 10 mM HOAc was promoted by the addition of NaCl in a concentration-dependent manner. A similar result was obtained for the addition of NaH2PO4, NaAc, and Na2SO4, with the promotion extents for both the lag phase and the elongation phase being different from those caused by the same concentration of NaCl (Fig. 3). In addition, adding NaH2PO4 or Na2SO4 to PBS also enhanced MSP21–25 aggregation, with the lag phase being reduced (Online resource Fig S3). These results suggested that the salt effect is primarily an anion-induced one. MSP21–25 in 10 mM HOAc with the same concentration
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ANS can bind to the hydrophobic clusters exposed on the molecular surface and emits fluorescence maximally at a wavelength shorter than that of free ANS (so called ‘‘blueshift’’); thus, it is widely used to detect the existence of partially folded states of proteins (Strickland and Mercola 1976). The ANS fluorescence spectrum of MSP21–25 in PBS showed a maximum emission wavelength at about 470 nm, strongly blue-shifted compared to that of free ANS, 520 nm (Fig. 5a), signifying the presence of a partially folded state. Presumably, ANS would hinder the aggregation of MSP21–25 by blocking hydrophobic associations such as those between the residues F11I12, which were demonstrated to be critical for the aggregation, particularly the nucleation stage of the peptide (Yang et al. 2010). Actually, in the presence of ANS, MSP21–25 aggregation in PBS was repressed in a concentrationdependent manner, with the lag phase extended to being unobservable within the measurement period at high ANS concentration (Fig. 5b). To test the abundance of accessible hydrophobic clusters of the components present along the aggregation pathway, ANS fluorescence at 480 nm was monitored throughout the MSP21–25 aggregation process. The result showed that ANS fluorescence, like ThT
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Fig. 5 a The fluorescence emission spectra of free and MSP21–25bound ANS in PBS, b the aggregation kinetics of MSP21–25 in PBS with different concentrations of ANS monitored by ThT fluorescence,
and c the aggregation kinetics monitored by ANS fluorescence at ex/ em of 360/480 nm
fluorescence, showed a typical sigmoidal profile when aggregation was progressing (Fig. 5c). This indicated that MSP21–25 aggregation would produce a more hydrophobic surface, which might be a characteristics of the aggregates.
kosmotropic (order-making) force, a power similar to hydrophobic force in essence (Baskakov and Bolen 1998; Wang and Bolen 1997). Therefore, contrary to urea, TMAO enhanced the aggregation of MSP21–25 when the lag phase decreased, also in a concentration-dependent manner (Fig. 6b). However, neither of the effects of urea and TMAO was exerted via structural change, demonstrated by the similar CD spectra of the peptide in the absence and presence of the two cosolvents (Online resource Fig. S5).
Urea and TMAO had opposite effects on MSP21–25 aggregation Urea is a good solvent and frequently used to denature protein by disrupting hydrophobic interactions. In addition, urea could also break the hydrogen bond by interfering with the electrostatic interactions (Klimov et al. 2004; Tobi et al. 2003). Therrefore, as expected, urea repressed MSP21–25 aggregation in PBS with both the lag phase and the growth phase elongated in a concentration-dependent manner (Fig. 6a). By contrast, TMAO is a naturally occurring osmolyte that stabilizes proteins in vivo under some stress conditions; it could induce unfolded protein to fold by
Sub-micellar SDS promoted MSP21–25 aggregation In a previous study, we used micellar SDS as a membrane mimetic to investigate the aggregation of MSP21–25 under a membranous environment, and the result showed that SDS micelles induced a helical structure and inhibited the aggregation (Zhang et al. 2012). However, in the present study, the effect of SDS was reversed when the
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Fig. 6 The aggregation kinetics monitored by ThT fluorescence for MSP21–25 in PBS with different concentrations of a urea and b TMAO Fig. 7 a The aggregation kinetics monitored by ThT fluorescence, and b the far UV CD spectra for MSP21–25 in the absence and presence of submicellar SDS
concentration decreased below the critical micellar concentration. As shown in Fig. 7a, addition of 1 mM SDS to PBS-enhanced MSP21–25 aggregation significantly, with the lag phase and part of the elongation phase being undetectable. Moreover, addition of 1 mM SDS in 10 mM HOAc diminished the inhibition effect of the acidic pH and promoted MSP21–25 aggregation to a degree comparable to that in PBS. SDS at concentrations between 0.1 and 1 mM caused similar results (data not shown). Intriguingly, submicellar concentrations of another frequently used phospholipid detergent, dodecylphosphocholine (DPC), had no influence on MSP21–25 aggregation in either PBS or in 10 mM HOAc (data not shown), implying the importance of the negatively charged head group of SDS. The
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CD spectrum of MSP21–25 in PBS with 1 mM SDS displayed a deep trough near 217 nm (Fig. 7b), indicating a structural change that facilitated the cross-b structure formation and might provide an additional contribution to the intensive aggregation enhancement effect.
Discussion Interplay between hydrophobic and electronic interactions in MSP21–25 aggregation MSP21–25 aggregation in vitro was shown to be a nucleation-elongation process (Zhang et al. 2012) and has been
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used to explain the assembly kinetics of amyloid-like fibrils composed of prions (Eigen 1996; Lundberg et al. 1997), Ab (Teplow 1998), and TTR (Kelly et al. 1997). The aggregation of all amyloidogenic proteins may share an essential mechanism, which could be attributed to the interplay between hydrophobic and electrostatic interactions (Auer et al. 2008; Straub and Thirumalai 2011; Tarus et al. 2008). It is assumed that the self-organization of the protein and the stability of the amyloid fibril arise by maximizing the number of hydrophobic and favorable electrostatic interactions, especially hydrogen bonding (Tarus et al. 2006). According to the assumption and based on our previous results that MSP21–25 aggregated with increasing lag phases and growth rates with rising TFE concentration (Zhang et al. 2012), we speculated that MSP21–25 aggregation is modulated by both hydrophobic and electrostatic interactions, with the nucleus formation driven predominantly by the former and the aggregate elongation controlled mainly by the latter. That is, any environmental factor facilitating intermolecular hydrophobic interactions, e.g., low concentrations of TFE, could promote nucleation, while those conditions favoring electrostatic interactions, e.g., high concentrations of TFE, could enhance elongation. The results obtained in the present study provide further evidence for this speculation. MSP21–25 is rich in basic residues (two Lys and two Arg) while deficient in acidic ones (only one Glu), leading to an alkaline theoretical pI of 9.99 (calculated by ProtParam program in ExPasy, http://www.expasy.ch/tools/ #primary). Therefore, it is reasonable that at alkaline pHs MSP21–25 aggregated readily, as it could easily self-associate to form nuclei because of weak inter-molecular electrostatic repulsion. On the other hand, the growth of MSP21–25 aggregation at alkaline pHs was faster than at neutral pHs, indicating the contribution of electrostatic interactions to the elongation phase. Salts have been shown to affect protein aggregation by either an electrostatic effect (Pedersen et al. 2006; Raman et al. 2005) or hydrophobic effect (Munishkina et al. 2004; Yeh et al. 2010), or both (Jain and Udgaonkar 2010; Klement et al. 2007). As inter- or intra-molecular electrostatic interactions could be shielded by ions with opposite charge, addition of salts at pH 3.4 would screen the charge repulsion and promote self-association between MSP21–25 peptides with the lag phase reduced in an anion naturedependent manner. The aggregation-promoting power of anions thus should be listed in the order of ionic strength and binding affinity: SO42-*HPO42- [ Ac-*Cl-. However, though the electrostatic effect of the salts was consistent with the growth rate difference, it alone could not explain the big difference between their nucleationpromoting effects, implying a contribution by other forces. Actually, anions could promote aggregation by acting as a
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kosmotrope (order-maker) by strengthening hydrophobic interactions in the order of Hofmeister series as follows: SO42- [ HPO42-*Ac- [ Cl- (Hofmeister 1888). This additional force could explain the greater lag phasereducing effect of acetate than chloride. Furthermore, addition of Na2SO4 or Na2HPO4 to 10 mM HOAc caused MSP21–25 to nucleate much faster than it did at alkaline pHs, stressing the predominant role of hydrophobic interactions in the nucleation. In addition, adding the salts to PBS also enhanced MSP21–25 aggregation with the lag phase reduced, providing further evidence for the role of hydrophobic interactions in the aggregate nucleation. The role of hydrophobic interactions in the nucleation phase of MSP21–25 aggregation is also supported by the effect of TMAO, which shortened the lag phase significantly to even undetectable levels. Addition of ANS elongating the lag phase provides further evidence for the essential role of hydrophobic interactions. However, ANS fluorescence, like the ThT signal, increasing along with the MSP21–25 aggregation process indicates that hydrophobic interactions are sparsely involved in the growth phase. The significance of hydrophobic interactions for MSP21–25 aggregation is also evidenced by the fact that the sample suffering agitation aggregated much faster than that staying stationary. Agitation would produce more water-air interface, which is hydrophobic and therefore favors hydrophobic interactions (Sluzky et al. 1992; Nielsen et al. 2001), and thus could promote the nucleation process. The roles of hydrophobic and electrostatic interactions in the lag phase of MSP21–25 aggregation have been demonstrated by the effect of TFE on the aggregation. In our previous study, we showed that low concentrations of TFE enhanced MSP21–25 aggregation by inducing a partially folded state, whereas high concentrations repressed the aggregation (Zhang et al. 2012). As we know, TFE denatures proteins by weakening hydrophobic interactions and induces secondary structures by enhancing electrostatic interactions, particularly hydrogen bonding, in a concentration-dependent manner (Albert and Hamilton 1995; Liu et al. 2004; Luo and Baldwin 1997; Thomas and Dill 1993; Yang et al. 1993). Therefore, the effect of high concentrations of TFE on MSP21–25 aggregation could be explained, besides in the structural aspect as described previously (Zhang et al. 2012), also by the interplay between increased electrostatic and decreased hydrophobic interactions: with TFE concentration increasing, the hydrophobic interaction-destabilizing power grows to repress the aggregation by hampering the nucleation, while strengthened hydrogen bonding enhances the growth rate. Like TFE, sub-micellar SDS might influence the aggregation of MSP21–25 by affecting both electrostatic and hydrophobic interactions via its polar head group and non-polar tail (Naiki et al. 2005; Wang et al. 1996; Yonath
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et al. 1977). The inter-molecular electrostatic interactions between the negatively charged sulfate group of SDS and the positive charges on the MSP21–25 surface would locate the aliphatic tail close to the peptide, strongly facilitating the peptide monomer self-associating to form nuclei or attaching to the aggregate end through hydrophobic interactions. Thus, SDS promoted MSP21–25 aggregation to a much higher extent than TMAO and the salts, with both the nucleation and elongation phases shortened. As for urea, it is believed to coat hydrophobic interactions by forming hydrogen bonds with backbone amides of proteins (Klimov et al. 2004; Tobi et al. 2003). Therefore, it is reasonable that urea elongated both the lag and growth phases of MSP21–25 aggregation. Implication for the MSP2 aggregation mechanism and relevant aggregation-preventing methods According to the current results, we suggest that the aggregation of MSP21–25 is a nucleation-elongation process, in which the nucleation is driven predominantly by hydrophobic interactions and the elongation is controlled mainly by electrostatic interactions. At physiological pHs, slightly charged and partially folded MSP21–25 self-associates to form nuclei via intermolecular hydrophobic interactions. Then, the nuclei transform to oligomers/protofibrils via conformational change from a helical to bstructure, which is stabilized by hydrogen bonding. The formation of the oligomers/protofibrils is a rate-limiting step, followed by relatively downhill growth. The growth phase may progress via a ‘‘dock and lock’’ mechanism (Straub and Thirumalai 2011), in which monomeric peptide attaches to the oligomers/protofibrils by a hydrophobic force-driven ‘‘docking’’ process, and the newly joined monomer transforms to a b-structure, which is stabilized by electrostatic hydrogen bonding and clusters of hydrophobic groups on surface, to finish the ‘‘locking’’ stage. The above model may provide clues for developing methods to prevent MSP21–25 and full-length MSP2 from aggregating in vitro; factors that could hinder the intermolecular association by hydrophobic interactions would inhibit the aggregation by hampering the nucleation, and these could weaken or eliminate electrostatic hydrogen bonding, which would inhibit the aggregation by hampering the elongation. Acknowledgments This work was supported by grants from the Educational Commission of Anhui Province of China (Grant no. KJ2010A024), Scientific Research Foundation for Returned Scholars, Ministry of Education of China, and the National Natural Science Foundation of China (Grant no. 30900228). Ethical standards We declare that all the experiments in the present work comply with the current laws of the PR China.
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Eur Biophys J (2014) 43:1–9 Conflict of interest of interest.
The authors declare that they have no conflict
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