Concentrations Of Trifluoroethanol Research Articles

1P-037 βラクトグロブリンとsrc SH3ドメイン蛋白質が高濃度EGOHとTFEの中で取るαヘリックスの多い状態は,コンパクトでも完全変性状態でもない(蛋白質-物性(安定性,折れたたみなど),第47回日本生物物理学会年会)

1P-037 βラクトグロブリンとsrc SH3ドメイン蛋白質が高濃度EGOHとTFEの中で取るαヘリックスの多い状態は,コンパクトでも完全変性状態でもない(蛋白質-物性(安定性,折れたたみなど),第47回日本生物物理学会年会)

Activity and Structural Changes of Euphorbia characias Peroxidase in the Presence of Trifluoroethanol

Activity assays, conformational changes and transitional switches between secondary structures of a peroxidase from Euphorbia characias were studied in the presence of trifluoroethanol and in the presence or absence of calcium ions. The addition of trifluoroethanol up to 10-20% first induced a drastic decrease of alpha-helix content followed by an increase of tryptophan fluorescence emission intensity, a progressive re-induction of the formation of alpha-helical elements concomitant with loss of enzyme activity. In the presence of calcium ions, the fluorescence of the enzyme almost remained unchanged in the trifluoroethanol concentration range 5-20%. Further increase in trifluoroethanol concentration led to a protein structure characterized by a progressive re-induction of alpha-helical elements, a remarkable increase of the tryptophan fluorescence and a loss of enzyme activity. These results indicate that calcium ions in Euphorbia peroxidase play an essential role in maintaining the hydrophobic interactions on the protein structure preserving enzymatic activity.

Amyloidogenic potential of α‐chymotrypsin in different conformational states

Amyloid fibril formation is widely believed to be a generic property of polypeptide chains. In the present study, alpha-chymotrypsin, a well-known serine protease has been driven toward these structures by the use of two different conditions involving (I) high temperature, pH 2.5, and (II) low concentration of trifluoroethanol (TFE), pH 2.5. A variety of experimental methods, including fluorescence emission, dynamic quenching, steady-state fluorescence anisotropy, far-UV circular dichroism, nuclear magnetic resonance spectroscopy, and dynamic light scattering were employed to characterize the conformational states of alpha-chymotrypsin that precede formation of amyloid fibrils. The structure formed under Condition I was an unfolded monomer, whereas an alpha-helical rich oligomer was induced in Condition II. Both the amyloid aggregation-prone species manifested a higher solvent exposure of hydrophobic and aromatic residues compared with the native state. Upon incubation of the protein in these conditions for 48 h, amyloid-like fibrils were formed with diameters of about 10-12 nm. In contrast, at neutral pH and low concentration of TFE, a significant degree of amorphous aggregation was observed, suggesting that charge neutralization of acidic residues in the amyloid core region has a positive influence on amyloid fibril formation. In summary, results presented in this communication suggest that amyloid fibrils of alpha-chymotrypsin may be obtained from a variety of structurally distinct conformational ensembles highlighting the critical importance of protein evolution mechanisms related to prevention of protein misfolding.

Structural Changes and Aggregation Process of Cu/Containing Amine Oxidase in the Presence of 2,2,2-Trifluoroethanol

Conformational and structural changes of lentil seedlings amine oxidase (LSAO) were studied in the presence of trifluoroethanol (TFE) by spectroscopic and analytical techniques. At TFE concentrations up to 5%, the induction of a structural transition from beta-sheet to alpha-helix and up to 10% TFE a structural transition from alpha-helix to beta-sheet as well as inactivation of the enzyme are observed. At TFE concentrations between 10-35%, LSAO proves to be prone to aggregation and beyond 35% TFE leads to a non-native protein structure with a high alpha-helix content. The obtained results revealed that the aggregation of LSAO is strongly linked to the nature of secondary structures.

The Complex Structure Transition of Humanin Peptides by Sodium Dodecylsulfate and Trifluoroethanol

We have examined the structure of two Humanin (HN) analog peptides, HNG and AGA-(C8R)HNG17, in the presence of sodium dodecylsulfate (SDS) and trifluoroethanol (TFE) using CD and sedimentation velocity. Both HNG and AGA-(C8R)HNG17 underwent complex conformational changes with increasing concentrations of SDS and TFE, in contrast to general trend of increasing alpha-helix with their concentration. To our surprise, both peptides appear to converge into a similar structure in SDS and TFE at higher concentrations; e.g., above 0.05 % SDS or 30-40 % TFE. Sedimentation velocity analysis showed extensive aggregation of HNG at 0.1 mg/ml in PBS in the absence of SDS, but a highly homogeneous solution in 0.1 % SDS, indicating formation of a uniform structure by SDS. These two peptides also formed an intermediate structure both in SDS and TFE at lower concentrations, which appeared to be associated with extensive aggregation. It is interesting that the structure changes of these peptides occur well below the critical micelle concentration of SDS, suggesting that conformational changes are mediated through molecular, not micellar, interactions with SDS.

Conformational Analysis of Dynorphin A (1–13) Using Hydrogen—Deuterium Exchange and Tandem Mass Spectrometry

Trifluoroethanol (TFE)-induced conformational changes in dynorphin A (1-13) were investigated using charge-state distribution (CSD) and hydrogen-deuterium exchange (HDX), combined with electrospray ionization (ESI) mass spectrometry (MS). Individual amino acids involved in secondary structural elements were identified by collision-induced dissociation-tandem mass spectrometry (MS/MS). It was observed that dynorphin A (1-13) largely exists in an unfolded conformation and a folded structure in increasing concentrations of TFE. In 50% TFE, it forms an alpha-helix that encompasses residues 1-9 and remains flexible from residues 10 to 13.

Characterization of the structure and dynamics of mastoparan‐X during folding in aqueous TFE by CD and NMR spectroscopy

Mastoparan-X, a 14-residue peptide found in wasp venom, does not adopt a well-defined structure in water, but it folds into an alpha-helix upon addition of trifluoroethanol (TFE). At low levels of TFE, the peptide is partially folded, passing through intermediate stages of folding as the amount of TFE is increased. These partially folded states have been characterized by CD and NMR spectroscopy, and methods to estimate the helical content from CD, chemical shift, and nuclear overhauser effect (NOE) data are compared. Variation in the sign and intensity of NOE cross-peaks is observed in different regions of the peptide, indicative of greater mobility of the sidechains compared to the backbone of the peptide. Furthermore, variation in the sidechain mobility is observed, both between sidechains of different amino acids and within the sidechain of a given amino acid. By monitoring chemical shifts and NOE intensities as the TFE concentration is increased, the initiation site for helix formation could be identified. Furthermore, details of the peptide structure and dynamics during the folding process were elucidated.

The bioactive conformation of glucose‐dependent insulinotropic polypeptide by NMR and CD spectroscopy

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal incretin hormone, which modulates physiological insulin secretion. Because of its glucose-sensitive insulinotropic activity, there has been a considerable interest in utilizing the hormone as a potential treatment for type 2 diabetes. Structural parameters obtained from NMR spectroscopy combined with molecular modeling techniques play a vital role in the design of new therapeutic drugs. Therefore, to understand the structural requirements for the biological activity of GIP, the solution structure of GIP was investigated by circular dichroism (CD) followed by proton nuclear magnetic resonance (NMR) spectroscopy. CD studies showed an increase in the helical character of the peptide with increasing concentration of trifluoroethanol (TFE) up to 50%. Therefore, the solution structure of GIP in 50% TFE was determined. It was found that there was an alpha-helix between residues 6 and 29, which tends to extend further up to residue 36. The implications of the C-terminal extended helical segment in the inhibitory properties of GIP on gastric acid secretion are discussed. It is shown that the adoption by GIP of an alpha-helical secondary structure is a requirement for its biological activity. Knowledge of the solution structure of GIP will help in the understanding of how the peptide interacts with its receptor and aids in the design of new therapeutic agents useful for the treatment of diabetes.

Detection of the equilibrium folding intermediate of β‐lactoglobulin in the presence of trifluoroethanol by mass spectrometry

Nano-electrospray ionization mass spectrometry (nano-ESI-MS) was used to monitor the effect of trifluoroethanol (TFE) on the conformational properties of beta-lactoglobulin (BLG). TFE stabilizes protein secondary structure, particularly alpha-helices. However, it also acts as a denaturant above critical concentrations. In the case of BLG, TFE at low concentrations is known to induce formation of an equilibrium intermediate that contains non-native helical structure. Such an intermediate is thought to form also under physiological conditions, playing a role in BLG folding in vivo by preventing aggregation. This well-characterized system was chosen in order to test species distributions obtained by nano-ESI-MS. BLG spectra at increasing concentrations of TFE at pH 2 indicate transient accumulation of a conformer whose charge-state distribution (CSD) falls between that of the native and that of the denatured protein, indicating that the TFE-induced, partially folded form can be selectively monitored by this technique. The condition of its maximum accumulation corresponds to 16% TFE, in excellent agreement with results from solution experiments. In contrast, titrations with methanol or acetonitrile (ACN) reveal apparent two-state transitions from native to fully unfolded BLG. At 10% TFE, the protein appears to be still fully folded at room temperature but, if unfolding is elicited by the combination with other denaturing agents, e.g. heat or low concentrations of ACN, it proceeds via formation of the intermediate. Thus, TFE can also induce formation of the BLG intermediate in synergism with generic denaturing agents. This study indicates good agreement between ESI-MS and other biophysical methods monitoring protein conformational transitions in the presence of TFE.

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all α-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

Two peptide fragments G55–I72 and K97–A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.

Effect of Organic Solvents on the Molten Globule State of Procerain: β-Sheet to α-Helix Switchover in Presence of Trifluoroethanol

The effect of methanol and trifluoroethanol (TFE) on the structure and folding of molten globule state of procerain, a cysteine protease from Calotropis procera, was studied by circular dichroism spectroscopy. The magnitude of ellipticity at 215 nm, as a measure of beta-sheet content, is dependent on the concentration of the TFE. Interestingly, a switch over from the beta-sheet structure of the molten globule state to alpha-helix was observed at 60% TFE and the ellipticity at 222 nm increased as a function of TFE concentration beyond this critical TFE concentration. Temperature induced unfolding of the molten globule state of procerain in 10% methanol showed stabilization of alpha-rich domain with concomitant destabilization of beta-rich domain. Using higher concentration of methanol (20-40 %) had no stabilizing effect on the alpha-rich domain however, the beta-rich domain was destabilized, indicating that the stability of the domains were not interdependent and that a low concentration of methanol induced stabilization in alpha-rich domain.

2DCOS and MCR-ALS as a combined tool of analysis of β-lactoglobulin CD spectra

Since the biological activity of a protein depends on its conformation, therefore a full characterization of a protein involves an understanding of its three-dimensional structure. Any technique involved in determination of a protein structure is aimed at a detailed characterization of measured signal in context of the most common conformational motifs. From among different spectroscopic techniques employed to study a protein structure in solution and processes involved in protein unfolding far-UV circular dichroism spectroscopy is most commonly used. Although CD could give reasonable estimates of the secondary structure content of a protein, raw CD spectra are difficult to interpret due to a strong overlapping of broad bands having significantly different ellipticity that moreover are of opposite sign. For improving resolution of the feature-less CD spectra some resolution-enhancement techniques should be applied. Such methods are generally used for interpretation of the FTIR spectra of protein, but are scarcely applied to the CD spectra. Here, the analysis of the far-UV CD spectra of β-lactoglobulin (BLG) measured as a function of temperature and concentration of trifluoroethanol (TFE) has been performed by the two-dimensional correlation spectroscopy (2DCOS) and multivariate curve resolution-alternating least-squares (MCR-ALS) techniques. On this example a comparison of results obtained by means of the two methods are carried out.

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.

NMR Investigation of the Dihydrogen‐Bonding and Proton‐Transfer Equilibria between the Hydrido Carbonyl Anion [HRe2(CO)9]− and Fluorinated Alcohols

The interaction of fluorinated alcohols with the anionic hydrido complex [HRe2(CO)9]- (1) has been investigated by NMR spectroscopy. According to the acidic strength of the alcohols, the interaction may result not only in the formation of dihydrogen-bonded ROH...[HRe2(CO)9]- adducts 2, but also in proton transfer to give the neutral species [H2Re2(CO)9] (3). With the weaker acid trifluoroethanol (TFE) evidence for the occurrence of the dihydrogen-bonding equilibrium was obtained by 2D 1H NOESY. The dependence of the hydride chemical shift on TFE concentration at different temperatures provided values for the constants of this equilibrium, from which the thermodynamic parameters were evaluated as deltaH(degrees) = -2.6(2) kcal mol(-1), deltaS(degrees) = -9.3(2) cal mol(-1) K(-1). This corresponds to a rather low basicity factor (E(j) = 0.64). Variable-temperature T1 measurements allowed the proton-hydride distance in adduct 2 a to be estimated (1.80 angstroms). In the presence of hexafluoroisopropyl alcohol (HFIP) simultaneous occurrence of both dihydrogen-bonding and proton-transfer equilibria was observed, and the equilibria shifted versus the protonated product 3 with increasing HFIP concentration and decreasing temperature. Reversible proton transfer between the alcohol and the hydrido complex occurs on the NMR timescale, as revealed by a 2D 1H EXSY experiment at 240 K. For the more acidic perfluoro-tert-butyl alcohol (PFTB) the protonation equilibrium was further shifted to the right. Thermal instability of 3 prevented the acquisition of accurate thermodynamic data for these equilibria. The occurrence of the proton-transfer processes (in spite of the unfavorable pK(a) values) can be explained by the formation of homoconjugated RO...HOR- pairs which stabilize the alcoholate anions.

Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.

Characterisation of Cytochrome c Unfolding by Nano-Electrospray Ionization Time-of-Flight and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Protein charge-state distributions (CSDs) in electrospray-ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%-5% is found to favor cyt c folding at pH approximately 7. Furthermore, we perform comparison of CSDs obtained by time-of-flight (ToF) and Fourier-transform-ion- cyclotron-resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kind of denaturants. In particular, experiments with 1-propanol suggest that also by FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.

In vitro study of stability and amyloid‐fibril formation of two mutants of human stefin B (cystatin B) occurring in patients with EPM1

Myoclonus epilepsy of type 1 (EPM1) is a rare monogenic progressive and degenerative epilepsy, also known under the name Unverricht-Lundborg disease. With the aim of comparing their behavior in vitro, wild-type (wt) human stefin B (cystatin B) and the G4R and the R68X mutants observed in EPM1 were expressed and isolated from the Escherichia coli lysate. The R68X mutant (Arg68Stop) is a peptide of 67 amino acids from the N terminus of stefin B. CD spectra have shown that the R68X peptide is not folded, in contrast to the G4R mutant, which folds like wild type. The wild type and the G4R mutant were unfolded by urea and by trifluoroethanol (TFE). It has been shown that both proteins have closely similar stability and that at pH 4.8, where a native-like intermediate was demonstrated, TFE induces unfolding intermediates prior to the major transition to the all-alpha-helical state. Kinetics of fibril formation were followed by Thioflavin T fluorescence while the accompanying changes of morphology were followed by the transmission electron microscopy (TEM). For the two folded proteins the optimal concentration of TFE producing extensive lag phases and high fibril yields was predenaturational, 9% (v/v). The unfolded R68X peptide, which is highly prone to aggregate, formed amyloid fibrils in aqueous solution and in predenaturing 3% TFE. The G4R mutant exhibited a much longer lag phase than the wild type, with the accumulation of prefibrillar aggregates. Implications for pathology in view of the higher toxicity of prefibrillar aggregates to cells are discussed.

Solvational Tuning of the Unfolding, Aggregation and Amyloidogenesis of Insulin

Solvational perturbations, accomplished by the addition of the three model cosolvents glycerol, ethanol and trifluoroethanol, exert pronounced and diversified effects on the unfolding, non-native assembly and fibril formation of the amyloidogenic protein insulin. Fluorescence, CD and UV-spectroscopic methods as well as atomic force microscopy imaging have been employed to reveal distinct structural and kinetic features upon the aggregation of insulin under different solvational perturbations, which ultimately manifest in morphological variations of mature aggregates and fibrils. In particular, fluorescence anisotropy studies proved to be very valuable in characterizing the corresponding aggregation nuclei. Glycerol stabilizes, through enhanced hydration, native oligomerization and retards fibrillar aggregation at all concentrations studied (up to 40% (w/w)). In contrast, both monoalcohols facilitate the formation of aggregation-prone intermediates by destabilization of the native assembly. The reversal from a kosmotropic to a merely chaotropic solvational behaviour can explain the accelerating effect on ordered fibrillation of low concentrations and the inhibitory nature of high concentrations of ethanol and trifluoroethanol, ultimately leading to amorphous aggregate structures. Mechanistically, dimer dissociation under stabilizing and nucleation under destabilizing conditions have been identified to be the rate-limiting steps that account for the non-monotonic concentration effects of the monoalcohols on the aggregation kinetics. A rationale as to how solvational constraints can tune the stability of the species on the native self-assembly and non-native aggregation pathway, and the energetic barriers that need to be overcome for the required structural interconversions has been put forward. We may propose that the concept of perturbed solvation is generally applicable to phenomena that are related to pathogenic amyloidogenesis of proteins and, in general, solvational effects, besides other aspects of the cellular environment, may play a significant role in a reshaping of the folding/aggregation funnel of proteins.

Amyloid Formation from HypF-N under Conditions in which the Protein is Initially in its Native State

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.