Heat-induced Aggregation Research Articles

Tracking Structural Changes in the UCS Domain of the Myosin Chaperone UNC-45 by Fluorescence Spectroscopy

Molecular chaperones are commonly identified by their ability to suppress heat-induced protein aggregation. The muscle-specific molecular chaperone UNC-45 is known to be involved in myosin folding and is trafficked to the sarcomeres A-band during thermal stress. Using a combination of fluorescence, circular dichroism, limited proteolysis and mass spectroscopy to quantitatively analyze the effect of temperature on UNC-45 we previously found that the UCS domain undergoes significant structural changes in response to temperatures within a heat-shock range (Bujalowski, et al., FEBS let 2015). In order to further analyze the regions within the UCS domain that become exposed to the solvent we used fluorescent assays based on the environment-sensitive hydrophobic fluorescent dye, Bis-ANS. We found that UCS interacts with Bis-ANS in 2:1 stoichiometry at 25oC with an apparent Kd of 46nM. We found that the number of bound ANS molecules increases ∼10-fold when exposed to temperatures above 35oC. We found that Bis-ANS stably binds to the UCS domain after heating (indicated by the asymmetry in the heating and cooling curves). We further investigated temperature-induced structural changes tryptophan fluorescence. The UCS domain has three Trp residues, W783, W823 and W863. We found that the relative Trp fluorescence signal decreases of as the temperature was raised indicating a change from an hydrophobic to an aqueous environment. In order to quantify the energetics of the ANS-UCS interaction we used isothermal titration calorimetry (ITC). Analysis of the calorimetric titration profile showed a monotonic decrease in the exothermic heat of binding. From these data we estimated an N= 4 (number of binding sites) and DS= + 70 cal/mol/K, suggesting that hydrophobic forces are dominant in the binding of ANS to UCS. In order to approximately map the regions at which ANS binds in the UCS domain we used resonance energy transfer (FRET) between Trp residues and Bis-ANS. We found that as Bis-ANS is added, Trp fluorescence is quenched as energy is transferred to the extrinsic fluorophore in a concentration dependent fashion. We are using these techniques to map the key amino acid regions in the UCS domain that mediate its myosin-binding and chaperoning functions. This work was supported by the American Heart Foundation (AHA 13GRNT17290006) and the NIH (1R01GM118534 01).

Amphiphilic copolymers reduce aggregation of unfolded lysozyme more effectively than polyethylene glycol

Certain amphiphilic block copolymers are known to prevent aggregation of unfolded proteins. To better understand the mechanism of this effect, the optical properties of heat-denatured and dithiothreitol reduced lysozyme were evaluated with respect to controls using UV–Vis spectroscopy, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. Then, the effects of adding Polyethylene Glycol (8000 Da), the triblock surfactant Poloxamer 188 (P188), and the tetrablock copolymer Tetronic 1107 (T1107) to the lysozyme solution were compared. Overall, T1107 was found to be more effective than P188 in inhibiting aggregation, while PEG exhibited no efficacy. TEM imaging of heat-denatured and reduced lysozymes revealed spherical aggregates with on average 250–450 nm diameter. Using CD, more soluble lysozyme was recovered with T1107 than P188 with β-sheet secondary structure. The greater effectiveness of the larger T1107 in preventing aggregation of unfolded lysozyme than the smaller P188 and PEG points to steric hindrance at play; signifying the importance of size match between the hydrophobic region of denatured protein and that of amphiphilic copolymers. Thus, our results corroborate that certain multi-block copolymers are effective in preventing heat-induced aggregation of reduced lysozymes and future studies warrant more detailed focus on specific applications of these copolymers.

Molecular cloning, expression and functional characterization of the 40-kDa heat shock protein, DnaJ, from Bacillus halodurans

Abstract In the present study, we identified, cloned and expressed a 40-kDa heat shock protein, DnaJ, from Bacillus halodurans. The open reading frame of the cloned gene contained 1116 bp and encoded 371 amino acid residues. The purified recombinant DnaJ contained a His-tag at the C-terminus and showed a single band at approximately 41-kDa on SDS-PAGE gel. The 3D structures of DnaJ obtained by I-TASSER showed that the overall structures of DnaJ from B. halodurans Guj1 and E. coli are very similar, with 45% sequence similarity. The present study revealed that the DnaJ protein from B. halodurans inhibits the heat-induced aggregation of insulin in a concentration-dependent manner as aggregation of the insulin B-chain was reduced by approximately 50% at 40 °C in the presence of 0.1 mg/ml of purified recombinant DnaJ. The overexpression of DnaJ improved thermotolerance properties in E. coli transformed with pET-28a + DnaJ. Salt resistance experiments indicated that the survival of E. coli transformed with DnaJ was enhanced 1.85-fold compared to that of the control cells in the presence of 0.5 M NaCl for 72 h. According to the results obtained, DnaJ from B. halodurans can potentially be used for improving the functional properties of enzymes and proteins in various applications.

The influence of ionic strength on the characteristics of heat-induced soy protein aggregate nanoparticles and the freeze-thaw stability of the resultant Pickering emulsions.

The improvement of the freeze-thaw stability of emulsions by interfacial engineering has attracted increasing attention in recent years. The present work investigated the potential of using soy protein isolate (SPI) aggregate nanoparticles as the Pickering stabilizers to improve the freeze-thaw stability of the resultant emulsions. SPI nanoparticles with different particle sizes and surface properties were fabricated through heating the SPI solutions (at a constant protein concentration of 2%, w/v) at 95 °C for 15 min, by varying the ionic strength (I) in the range of 0-500 mM. The nanoparticles fabricated at I values of 100-500 mM exhibited larger particle sizes and higher surface hydrophobicity, but poorer emulsification efficiency than those at I = 0.05 mM. The presence of NaCl during the nanoparticle fabrication resulted in the formation of a kind of gel-like emulsion with a high extent of droplet flocculation. The emulsion stabilized by SPI nanoparticles at I = 0.05 mM was highly susceptible to coalescence, flocculation and creaming upon freeze-thaw treatment, while those in the presence of NaCl exhibited excellent freeze-thaw stability. The much better freeze-thaw stability of the emulsions in the presence of NaCl (relative to that at I = 0.05 mM) was largely attributed to the gel-like network formation, rather than the salt itself. The results indicated that a kind of Pickering emulsion with excellent freeze-thaw stability, stabilized by heat-induced SPI nanoparticles, could be fabricated by heating the SPI solutions at I values of 100-500 mM. The findings would be of great relevance for providing important information about the development of food grade Pickering emulsions stabilized by protein-based particles, with potential applications in frozen food, or functional food formulations.

Physical Properties of Heat-induced Whey Protein Aggregates Formed at pH 5.5 and 7.0

Physical Properties of Heat-induced Whey Protein Aggregates Formed at pH 5.5 and 7.0

Heat-induced Aggregation of Casein in Reconstituted Skimmed Milk at pH 3.30 – 3.55

Heat-induced Aggregation of Casein in Reconstituted Skimmed Milk at pH 3.30 – 3.55

Evaluation of structural characteristics determining surface and foaming properties of β-lactoglobulin aggregates

Due to its structural versatility and remarkable surface activity, β-lactoglobulin (β-Lg) is a prominent food structuring agent in both its native state and in the form of heat-induced aggregates. In this study, we considered the properties of soluble aggregates of pure β-Lg having a median diameter of 50nm. The behavior of the aggregates at the air/water interface has been probed as a function of pH, with the aim of evaluating the controversial role of surface charge in connection with the functionality of particle-stabilized foams. Based on these new experimental results, it would appear that charge shielding around the isoelectric point (pI) leads to significantly increased foam stability as compared to samples having a significant positive or negative zeta potential. Against expectations, however, maximum foam stability was not linked to minimum foam drainage. High foamability and foam stability have been found to correlate with maxima in the rates of initial surface pressure increase and with interfacial dilatational properties. Evidence for efficient stabilization of the air/water interface was also reflected in a small average bubble size and a low rate of mean bubble area increase for aggregates with pH∼pI. Information from additional comparative experiments on systems with native β-Lg enabled an evaluation of the significance of surface hydrophobicity and aggregate particle size in relation to the foaming properties.

Effects of pH, temperature and pulsed electric fields on the turbidity and protein aggregation of ovomucin-depleted egg white

The effect of either pulsed electric fields (PEF) or thermal processing on protein aggregation of ovomucin-depleted egg white (OdEW) solutions at different pH was assessed by solution turbidity and SDS-PAGE. Heating to 60°C for 10min caused marked protein aggregation of OdEW at pH5, 7, and 9. At constant electric field strength (E=1.4–1.8kV/cm), PEF processing under high specific energy input (Wspec=260–700kJ/kg) induced some protein aggregation at pH5 and 7, but not at either pH4 or 9. Similar effects of pH on protein aggregation were observed upon PEF processing at varied E (from 0.7 to 1.7kV/cm) but with constant Wspec (713kJ/kg). Analysis by SDS-PAGE revealed that proteins in the OdEW solution at pH5 were most susceptible to both PEF- and heat-induced protein aggregation and lysozyme was only involved in the formation of insoluble aggregates under PEF. The present study shows that PEF treatment has considerable potential for minimizing protein aggregation in the processing of heat-labile egg white proteins. Retaining the OdEW proteins in solution during processing has potential industry application, for example, protein fortification of drinks with OdEW, where minimizing solution turbidity would be advantageous.

Effect of temperature, ionic strength and 11S ratio on the rheological properties of heat-induced soy protein gels in relation to network proteins content and aggregates size

The effects of heating temperature, ionic strength and 11S ratio on the rheological properties of heat-induced soy protein gels, particularly in relation to network proteins content and aggregates size were investigated. Results revealed that non-network proteins consisted of undenatured glycinin (11S) AB subunits, high levels of A and A3 polypeptides but low levels of β-conglycinin (7S) α and α′ subunits. Results further showed that a positive correlation between storage modulus (G′) and network proteins ratio of gels that were formed at different temperatures. Additionally, the G′ values of gels initially increased with increasing NaCl concentration from 0 to 0.25 M, but decreased with further increasing ionic strength from 0.25 to 0.50 M. This trend was consistent with the variation of heat-induced protein aggregates size in soy protein solution at the same ionic strength. Higher 11S/7S ratio resulted in higher storage modulus values of soy protein gels due to the formation of larger and compacter aggregates mainly by B polypeptides via hydrophobic bonds.

Limited changes in physical and rheological properties of peroxidase-cross-linked apo-α-lactalbumin after heat treatment

The current study aims at elucidating the effect of heating on the physical properties of peroxidase-cross-linked (apo) α-lactalbumin (α-LA) aggregates with hydrodynamic radii (RH) of 25 nm – 100 nm. We consider the effects of heating at the single protein level, and at the particle level for both dilute and dense dispersions of the cross-linked α-LA. From far-UV circular dichroism (CD) spectroscopy, we find that changes in the secondary structure of the cross-linked α-LA are limited upon heating but thermoreversible. Cross-linking already leads to a complete disappearance of all tertiary structure, and heating therefore leads to no further changes of tertiary structure as detected by near-UV CD. The surface hydrophobicity of the cross-linked α-LA, as probed using a fluorometric assay (ANSA), remains unchanged upon heating, whereas heating leads to a significant increase of surface hydrophobicity for uncross-linked α-LA. Prolonged heating leads to a moderate decrease of the particle size of α-LA aggregates, while heating does not affect its electrophoretic mobility. As a consequence of the insensitivity of the physical properties of the cross-linked α-LA to heating, we find that dilute dispersions of the cross-linked α-LA are stable against heat-induced aggregation over a wide range of pH values and salt concentrations. Furthermore, for dense dispersions of α-LA aggregates, that form physical hydrogels, we find that heating has a very limited impact on the rheology of the hydrogels. Our results may be of importance in designing heat-insensitive protein ingredients for novel food formulations.

Inhibition and Promotion of Heat-Induced Gelation of Whey Proteins in the Presence of Calcium by Addition of Sodium Caseinate.

Heat-induced aggregation and gelation of aqueous solutions of whey protein isolate (WPI) in the presence of sodium caseinate (SC) and CaCl2 was studied at pH 6.6. The effect of adding SC (0-100 g/L) on the structure of the aggregates and the gels was investigated by light scattering and confocal laser scanning microscopy at different CaCl2 concentration ([CaCl2] = 0-30 mM). The gelation process was studied by oscillatory shear rheology. At the whey protein concentrations studied here (34 and 60 g/L), no gels were formed in the absence of CaCl2 and SC. However, WPI solutions gelled above a critical CaCl2 concentration that increased with increasing SC concentration. In the absence of CaCl2, WPI gels were formed only above a critical SC concentration. The critical SC concentration needed to induce WPI gelation decreased weakly when CaCl2 was added. In an intermediate range of CaCl2 concentrations, gels were formed both at low and high SC concentrations, but not at intermediate SC concentrations. Finally, at high CaCl2 concentrations gels were formed at all SC concentrations. The gelation rate and the gel structure of the gels formed at low and high casein concentrations were very different. The effect of SC on the thermal gelation of WPI was interpreted by competition for Ca2+, a chaperon effect, and microphase separation.

Investigation of the molecular interactions between β-lactoglobulin and low methoxyl pectin by multi-detection High Performance Size Exclusion Chromatography

To achieve controlled physiochemical, structural, and consequently, functional properties of interacting systems containing β-lactoglobulin (βLG) and commercial standardized low methoxyl pectin (LMP, DE 36.8%), solution conditions were established and studied at the weight ratio of 3:1 (w/w), pH ∼6.50. Online multi-detection High Performance Size Exclusion Chromatography (HPSEC) was used to characterize the physicochemical properties of the interacting biopolymer fractions in detail. Results showed that upon direct mixing, ∼6.1% (w/w) of native dimeric βLG molecules formed complexes with LMP molecules of two sizes, namely [Mw] ∼ 250 and 55 kDa. The interactions produced complexes of altered shapes and hydrodynamic properties compared to those of βLG and LMP controls. Heat-induced (80 °C, 10 min) denaturation of βLG greatly increased its participation in the complexes as aggregates, to ∼10.4%, which appear to favor the LMP molecules of higher [Mw]. Pre-heating LMP, however, impeded its binding to βLG and increased the amount of unbound βLG as a result. The effect of sucrose on the interactions between βLG and LMP was also investigated using a set of similarly treated samples but with sucrose removed from LMP. These studies demonstrated that sucrose greatly facilitated the complex formation, notably between the pre-heated βLG and LMP. The structures of the complexes were found to be less flexible and less open in the presence of sucrose than its absence. It is suggested that sucrose contributed greatly to the stabilization of the heat-induced βLG aggregates as they interact with LMP through a preferential exclusion mechanism.

Dynamic Interactions of Arabidopsis TEN1: Stabilizing Telomeres in Response to Heat Stress.

Telomeres are the essential nucleoprotein structures that provide a physical cap for the ends of linear chromosomes. The highly conserved CST (CTC1/STN1/TEN1) protein complex facilitates telomeric DNA replication and promotes telomere stability. Here we report three unexpected properties of Arabidopsis thaliana TEN1 that indicate it possesses functions distinct from other previously characterized telomere proteins. First, we show that telomeres in ten1 mutants are highly sensitive to thermal stress. Heat shock causes abrupt and dramatic loss of telomeric DNA in ten1 plants, likely via deletional recombination. Second, we show that AtTEN1 has the properties of a heat-shock induced molecular chaperone. At elevated temperature, AtTEN1 rapidly assembles into high molecular weight homo-oligomeric complexes that efficiently suppress heat-induced aggregation of model protein substrates in vitro. Finally, we report that AtTEN1 specifically protects CTC1 from heat-induced aggregation in vitro, and from heat-induced protein degradation and loss of telomere association in vivo. Collectively, these observations define Arabidopsis TEN1 as a highly dynamic protein that works in concert with CTC1 to preserve telomere integrity in response to environmental stress.

Effect of chitosan on the heat stability of whey protein solution as a function of pH.

Chitosan was reported to interact with proteins through electrostatic interactions. Their interaction was influenced by pH, which was not fully characterized. Further research on the interactions between protein and chitosan at different pH and their influence on the thermal denaturation of proteins is necessary. In this research, the effect of chitosan on the heat stability of whey protein solution at pH 4.0-6.0 was studied. At pH 4.0, a small amount chitosan was able to prevent the heat-induced denaturation and aggregation of whey protein molecules. At higher pH values (5.5 and 6.0), whey proteins complexed with chitosan through electrostatic attraction. The formation of chitosan-whey protein complexes at pH 5.5 improved the heat stability of dispersions and no precipitation could be detected up to 20 days. The dispersion with a medium amount of chitosan (chitosan:whey protein 1:5) produced the most stable particles, which had an average radius of 135 ± 14 nm and a zeta potential value of 36 ± 1 mV. In contrast, at pH 6.0 only the dispersion with a high amount of chitosan (chitosan:whey protein 1:2) showed good shelf stability up to 20 days. It was possible to produce heat-stable whey protein beverages by regulating the interaction between chitosan and whey protein molecules. © 2016 Society of Chemical Industry.

Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro.

C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer’s amyloid β (Aβ) (1-40) and on that from D76N β2-microglobulin (β2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aβ(1-40) and D76N β2-m fibril formation in a Ca2+-independent manner. CRP and SAP interacted with fresh and aggregated Aβ(1-40) and D76N β2-m on the fibril-forming pathway. Interestingly, in the presence of Ca2+, SAP first inhibited, then significantly accelerated D76N β2-m fibril formation. Electron microscopically, the surface of the D76N β2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed.

Expression and localization of the Xenopus laevis small heat shock protein, HSPB6 (HSP20), in A6 kidney epithelial cells

Small heat shock proteins (sHSPs) are molecular chaperones that bind to unfolded protein, inhibit the formation of toxic aggregates and facilitate their refolding and/or degradation. Previously, the only sHSPs that have been studied in detail in the model frog system, Xenopus laevis, were members of the HSP30 family and HSPB1 (HSP27). We now report the analysis of X. laevis HSPB6, an ortholog of mammalian HSPB6. X. laevis HSPB6 cDNA encodes a 168 aa protein that contains an α-crystallin domain, a polar C-terminal extension and some possible phosphorylation sites. X. laevis HSPB6 shares 94% identity with a X. tropicalis HSPB6, 65% with turtle, 59% with humans, 49% with zebrafish and only 50% and 43% with X. laevis HSPB1 and HSP30C, respectively. Phylogenetic analysis revealed that X. laevis HSPB6 grouped more closely with mammalian and reptilian HSPB6s than with fish HSPB6. X. laevis recombinant HSPB6 displayed molecular chaperone properties since it had the ability to inhibit heat-induced aggregation of citrate synthase. Immunoblot analysis determined that HSPB6 was present constitutively in kidney epithelial cells and that heat shock treatment did not upregulate HSPB6 levels. While treatment with the proteasomal inhibitor, MG132, resulted in a 2-fold increase in HSPB6 levels, exposure to cadmium chloride produced a slight increase in HSPB6. These findings were in contrast to HSP70, which was enhanced in response to all three stressors. Finally, immunocytochemical analysis revealed that HSPB6 was present in the cytoplasm in the perinuclear region with some in the nucleus.

Complexes between ovalbumin nanoparticles and linoleic acid: Stoichiometric, kinetic and thermodynamic aspects

Stoichiometric, kinetic and thermodynamic aspects of complex formation between heat-induced aggregates of ovalbumin (ovalbumin nanoparticles, OVAn) and linoleic acid (LA) were evaluated. Extrinsic fluorescence data were fitted to modified Scatchard model yielding the following results: n: 49±2 LA molecules bound per OVA monomer unit and Ka: 9.80±2.53×105M. Kinetic and thermodynamic properties were analyzed by turbidity measurements at different LA/OVA monomer molar ratios (21.5–172) and temperatures (20–40°C). An adsorption approach was used and a pseudo-second-order kinetics was found for LA-OVAn complex formation. This adsorption process took place within 1h. Thermodynamic parameters indicated that LA adsorption on OVAn was a spontaneous, endothermic and entropically-driven process, highlighting the hydrophobic nature of the LA and OVAn interaction. Finally, Atomic Force Microscopy imaging revealed that both OVAn and LA-OVAn complexes have a roughly rounded form with size lower than 100nm.

Cross-Linking Activity of Ethanolic Coconut Husk Extract Toward Sardine (Sardinella albella) Muscle Proteins

Effects of ethanolic coconut husk extract (ECHE) at various levels (0–0.03%, based on protein content) on the heat-induced aggregation of natural actomyosin (NAM), extracted from sardine (Sardinella albella) muscle, were studied. During heating from 20 to 90C, the NAM solution showed increased turbidity, surface hydrophobicity, and disulfide bond content. Aggregation was more pronounced as ECHE concentration increased (P < 0.05), while protein solubility and total sulfhydryl groups decreased (P < 0.05). Furthermore, the Ca2+-ATPase activity of NAM decreased in the presence of ECHE, during heating up to 40C. Zeta potential analysis revealed that NAM became less negatively charged with the increasing amount of ECHE added (P < 0.05). Cross-linking of protein strands during heating was enhanced by the presence of ECHE, as evidenced by increased G′, and particle size, and denser aggregates. Thus, ECHE could serve as a cross-linking agent for fish muscle proteins. Practical Applications Coconut husk is an underutilized product from coconut processing, and generally contains several phenolic compounds including tannic acid, which acts as a protein cross-linker. Coconut husk extract could therefore increase the cross-linking of proteins, thereby enhancing the strength of surimi gel. Alternatively, coconut husk extract could be used as a new natural protein cross-linker to improve the properties of surimi as well as other muscle foods.

Effect of extraction pH on heat-induced aggregation, gelation and microstructure of protein isolate from quinoa (Chenopodium quinoa Willd)

The aim of this study was to determine the influence of extraction pH on heat-induced aggregation, gelation and microstructure of suspensions of protein isolates extracted from quinoa (Chenopodium quinoa Willd). Quinoa seed protein was extracted by alkaline treatment at various pH values (pH 8 (E8), 9 (E9), 10 (E10) and 11 (E11)), followed by acid precipitation. The obtained protein isolates were freeze dried. The protein isolates E8 and E9 resulted in a lower protein yield as well as less protein denaturation. These isolates also had a higher protein purity, more protein bands at higher molecular weights, and a higher protein solubility in the pH range of 3–4.5, compared to the isolates E10 and E11. Heating the 10%w/w protein isolate suspensions E8 and E9 led to increased aggregation, and semi-solid gels with a dense microstructure were formed. The isolate suspensions E10 and E11, on the other hand, aggregated less, did not form self-supporting gels and had loose particle arrangements. We conclude that extraction pH plays an important role in determining the functionality of quinoa protein isolates.

Effect of heat, pH and shear on digestibility and antigenic characteristics of wheat gluten

Processing-induced conformational changes may affect digestibility and antigenic potential of food proteins. In vitro gastrointestinal digestibility of gluten proteins was established after treatment at various pH (3 or 7), temperature (room temperature or 100 °C) and shear (0 or 1500 s−1). Electrophoretic patterns (SDS-PAGE) of the resulting hydrolysates and antigenicity (ELISA and immunoblotting) of gliadin fraction of the proteins were also studied. Digestibility was positively correlated with α-helix/β-sheet ratio. Lower antigenic reaction shown by digested hydrolysate of gluten samples treated at pH 3 and room temperature was ascribed to its acidic deamidation and improved digestibility. Further heating to 100 °C slightly increased antigenicity. In contrast, hydrolysate of gluten at pH 7 and room temperature exhibited highest antigenicity, attributed to partial resistance of α-/β-gliadin to digestion and appearance of some new potentially antigenic polypeptides. However, heating at 100 °C caused heat-induced protein aggregation, consequently lowered digestibility and availability of antigenic components resulting in a minimum (60 % reduction) antigenicity. Overall, shear had no effect on digestibility and antigenicity irrespective of pH and temperature. Thus, antigenic potential of gliadins can be minimized by selecting appropriate parameters during processing.