Complexation Behavior of Î²-Lactoglobulin with Surface Active Ionic

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B: Fluid Interfaces, Colloids, Polymers, Soft Matter, Surfactants, and Glassy Materials

Complexation Behavior of #-Lactoglobulin with Surface Active Ionic Liquids in Aqueous Solutions: An Experimental and Computational Approach Gagandeep Singh, Gurbir Singh, Srinivasarao Kancharla, and Tejwant Singh Kang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11610 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 17, 2019

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The Journal of Physical Chemistry

Complexation Behavior of β-Lactoglobulin with Surface Active Ionic Liquids in Aqueous Solutions: An Experimental and Computational Approach Gagandeep Singh,a Gurbir Singh,a Kancharla Srinivasa Rao,b Tejwant Singh Kang* ,a Department of Chemistry, UGC-Centre for Advance Studies – II, Guru Nanak Dev University, Amritsar, 143005, India. b Department of Earth Resources Engineering, Faculty of Engineering, Kyushu University, Fukuoka 8190395, Japan a

Abstract: The nature of functionalization of alkyl chain of imidazolium based surface active ionic liquids (SAILs) with amide- of ester-moiety led to contrasting complexation behavior towards globular protein, bovine serum albumin (BSA). This prompted us to further investigate the SAIL dependent colloidal behavior of another globular protein, -lactoglobulin (LG) to probe the origin of varying structural transformations in globular proteins induced by SAILs. Herein, we investigated the colloidal systems of βLG, rich in β-sheet structure, in the presence of four structurally different SAILs using a multi-technique approach. The complexation behavior, both at air-solution interface as well as in bulk, is supplemented by different techniques. Docking studies has complemented the obtained experimental results. The specificity of structure, Hbonding ability of SAILs and inherent structure of protein is found to govern their complexation behavior in terms of size, shape and polarity of protein-SAIL complexes along with varying degree of structural alterations in globular proteins. The present work is expected to be very useful in establishing a deep understanding of structure–property relationship between the nature of proteins and SAILs for their complexation and colloidal behavior for various biomedical applications.

*To whom correspondence should be addressed: Email: [email protected]; [email protected] Tel: +91-183-2258802-Ext-3207

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1. Introduction Among various globular proteins,1-4 β-lactoglobulin (βLG) is relatively abundant and is easy to purify and hence it has been investigated as a model globular protein for different investigations.5-10 βLG contains 162 amino acid residues with a molecular mass of 18.4 kDa. It is commonly found in whey protein (~65%) and belongs to class of lipocalins. 11 Native βLG is rich in β-sheet structure (51%) along with some α-helical (7%) content.12,13 At pH corresponding to its isoelectric point 4.8,14,15 βLG remain in dimeric form, whereas at pH below 3.5 and above 6.5, it exists in monomeric form.16,17 βLG possess two binding sites, one of which is present in internal cavity of globule formed by eight anti-parallel β-sheets converging into calyx geometry known as β-barrel whereas another binding site exists at outer surface between α-helix and βbarrel.8-10,18 This makes βLG susceptible to bind with a broad spectrum of biologically important hydrophobic ligands.8,19-21 A better control over binding affinity and release kinetics of various agents complexed with proteins18-22 can be achieved by using protein in conjugation with surfactants. In this regard, physico-chemical investigations on interactional behavior of βLG with variety of surfactants have been reported. 23-30 During past few years, surface active ionic liquids (SAILs) have attracted a great interest from the scientific community due to their better surface active properties over conventional ionic surfactants31-37 along with possibility of tailoring their hydrophilicity/hydrophobicity balance via functionalization of alkyl chain. 38,39 Owing to such properties, SAILs were conceived to offer more potentiality for exploiting surfactant-protein applications as compared to conventional ionic surfactants and therefore studies on protein-SAIL interactions have been derived.40-52 Most of these studies pertained to BSA, where stabilization of BSA by biamphiphilic SAIL, 40 significant alterations in secondary (2o) structure of BSA depending on chain length of imidazolium based SAILs ([Cnmim][Br] n = 4, 6, 8, and 10),44 and nature of SAIL has been observed.43,45 On the other hand, 1-tetradecyl-3-methylimidazolium bromide, [C14mim][Br], has been found to alter tertiary structure of BSA without affecting its secondary structure when used in very low concentraitons.42 The effect of functionalization of alkyl chain via ester or amide moiety near cationic head group on their interactional behavior with BSA has been investigated by our group for the first time.46 It was observed that the varying H-bonding ability of ester or amide group along with the flexible nature of ester moiety affects the interactional process. This resulted in concentration dependent formation of right handedly twisted long helical amyloid structures only


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in case of ester-functionalized SAIL. On the other hand, the stabilization of 2o structure of βLG with [C12 mim][Br] and [C6mim][C12OSO3] in lower concentration of SAIL was reported. 48 From literature, it is apparent that there is scarcity of investigations on colloidal behavior of βLG in aqueous solutions of SAILs, where a few reports highlighting the influence of short chain ILs on conformation of βLG has been reported in a wide concentration range of ILs.49,50 This along with the contrasting behavior of investigated SAILs towards complexation with a globular protein, BSA,46 and a linear protein Gelatin,51,52 prompted us to extend study to another globular protein i.e. βLG. The results obtained from this study would provide detailed insights about the origin of varying interactional processes between proteins and SAILs that led to morphologically different SAIL-Protein complexes, in past.46,51 Herein, the complexation phenomenon of LG in the presence of four different SAILs, 1dodecyl-3-methylimidazolium

chloride,

[C12mim][Cl],

3-(2-(dodecylamino)-2-oxoethyl)-1-

methyl-1Himidazol-3-ium chloride, [C12Amim][Cl], 3-methyl-1-dodecyloxycarbonylmethyl imidazolium

chloride,

[C12Emim][Cl],

and

1-dodecyl-3-methylimidazoliumoctylsulfate,

[C12mim][C8SO4] (a biamphiphilic SAIL) using multi-technique approach was investigated both at air-solution interface as well as in bulk. Besides this, the location of binding sites of SAILs with βLG and corresponding interactional energy was probed by computer simulation using AutoDockVina software. The results obtained from different techniques corroborate well with each other and are also supported by computational studies. The obtained results are compared with that reported in literature to get insights into the phenomenon, wherever possible. It is expected that this study along with previous investigations on interactional behavior of SAILs with structurally different proteins46,48,51 would establish structure-property relations between the nature of SAILs and the nature of proteins, required to develop highly controlled, stable and functional colloidal systems. 2. Materials and Methods: Scheme 1 shows the molecular structure of βLG and SAILs under investigation. SAIL-1, SAIL-2 and SAIL-3 were used from the same lot as reported in our earlier work,46 whereas SAIL-4 was synthesized using the reported protocol.53 A detailed account of materials used, synthetic procedure of SAIL along with characterization details are provided in Annexure S1 (Supporting Information, SI). The interactional behavior of SAILs and βLG, both at the air-solution interface



as well as in bulk, was investigated by titration method. In brief, concentrated solutions of SAILs were added to aqueous solution of βLG (10 μM) in phosphate buffer (pH = 7.2, I = 5 mmol L-1) and the observations were made using various state of art techniques.

Scheme 1: Molecular representation of β-lactoglobulin and SAILs used in the present study.

3. Result and Discussion: 3.1 Interfacial Behavior of Colloidal Systems: Figure 1(A-D) represents the comparative adsorption isotherms of investigated SAILs in the presence and absence of βLG in aqueous phosphate buffer solution. The surface tension (γ) of aqueous solution of βLG is less than that of βLG free solution owing to surface active nature of βLG.46,51 In concentration profiles of γ, three transitions marked as C1, C2 and C3(cmc), corresponding to different interactional processes in different concentration regimes (Table 1) of SAILs, were observed. In dilute concentration regimes of SAILs, γ decreases till C1 owing to the formation of surface active βLG-SAIL monomer complex (MC) at the air-solution interface. To confirm the occurrence of complexation, γ was measured at different concentrations of βLG in the presence of SAILs (Figure S1, SI). It is observed that up to C1 different SAILs reduce the γ to different extent and a change in concentration of βLG does not have a visible influence on the value of γ at C1. Further, the variation of γ with concentration of βLG in the absence of SAILs has been measured (Figure S2A, SI). The aqueous solutions of βLG exhibits γ ≈ 55.9 mN m-1, which is lower than that of water but higher than that observed for different SAILs in buffer at cmc. This along with the varying difference in γ of respective SAILs and βLG-SAIL systems (Figure 1) at C1 suggests the


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formation of differently surface active MCs, which lowers the γ. However, the possible role of additive effect of γ of βLG and SAILs cannot be ruled out at this stage. A lower value of C1 signifies the ease of saturation of binding sites of βLG by SAILs and follows the order: SAIL-4 < SAIL-2 < SAIL-3 < SAIL-1. The hydrophobic interactions offered by two alkyl chains, one each for cation and anion, of SAIL-4 along with electrostatic interactions seems to govern the monomeric saturation of βLG, whereas co-operative H-bonding interactions along with other set of interactions in case of SAIL-2 and SAIL-3 drives the interactional process in this concentration regime. Such interactions are further supported by FTIR (Figure S3, SI) measurements. For this, Amide I region of βLG (1700-1600 cm-1) has been explored as Amide III region of protein (1350-1200 cm-1) gives very weak signal. The presence of other bands such as C=O stretching vibrations54 from SAIL-2 and SAIL-3 along with skeleton vibrations of imidazole ring55 in the same region poses a difficulty in analysis and would be problematic for further discussion. However, a relative shift in the position of Amide I band of βLG and C=O stretching vibrations from SAIL-2 and SAIL-3 in βLG-SAIL systems as compared to pure βLG or SAILs suggests the presence of H-bonding interactions. This is further justified by computer simulation studies (discussed later).

Figure 1(A-D): Variation of surface tension (γ) in aqueous buffer solutions of different SAILs as function of concentration of SAILs in the absence and presence of βLG at 298.15 K.



Between C1 to C2, γ remain nearly constant followed by a decrease between C2 to C3, in case of SAIL-1 and SAIL-2. Almost constant γ is suggestive of either no change in surface activity of formed MC complex at air-water interface or the dominance of complexation of SAILs in bulk over air-solution interface.51 A decrease in γ between C2 to C3 is assigned to increased surface activity of formed SAIL-βLG (Aggregate) complex (AC). The partial dissolution of AC into bulk where SAILs consecutively occupy the air-solution interface51 could also lead to such decrease in γ. On the other hand, no transition corresponding to C2 was observed in case of SAIL-3 and SAIL-4, for which, γ goes on decreasing till C3(cmc). After C3(cmc), γ attains almost constant value for all investigated SAILs except in SAIL-1 system, where γ increases marginally.51 A constant γ after C3(cmc) is assigned to the saturation of air-solution interface followed by formation of micelles in the bulk solution. Almost similar interactional behaviour has been observed in case of [C12mim][Br] with the exception that the γ increases between C1 to C2 in previous report and indicates the role of varying size and polarity of counter-ions of respective SAILs.48 The relative affinity of different SAILs for binding with βLG is quantified by employing tensiometric measurements at different concentrations of βLG (Figure S1, SI). The number of SAIL molecules bound to βLG at cmc has been obtained from the slope of linear fit between the concentration of βLG and obtained values of cmc of SAILs (Figure S2B, SI) using equation 1:56 [S]cmc =[S]free + N*[P]

(1)

where [S]cmc is the concentration of SAIL at cmc, [S]free is concentration of free SAIL, [P] refers to protein concentration under investigation, and N is the number of surfactant units bound to βLG. The cmc data and the parameters calculated using equation 1 are provided in Table S1 (SI). SAIL-2 and SAIL-3 has been found to have higher affinity for βLG as manifested by relatively higher number of SAIL units bound (SAIL-2; 113 and SAIL-3; 257) to βLG as compared to SAIL-4 (SAIL-4; 38). This is in line with previous report on binding of SAIL-2 and SAIL-3 with BSA46 and is ascribed to co-operative H-bonding ability of these SAILs accompanied by unfolding of βLG, which results in more number of active sites for SAILs to interact with βLG (discussed later). The quantification of SAIL-1 bound to βLG using this method could not be made as the obtained cmc at different concentrations of βLG decreases marginally instead of expected increase. It is natural to assume that with increasing concentration of βLG, proportionally large amount of water hydrates βLG. This along with a weaker set of interactions


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of SAIL-1 with βLG favors micellization of SAIL-1. Various parameters i.e. surface tension at cmc (γcmc), Gibbs surface excess(Γmax), minimum area per molecule (Amin) and standard free energy of adsorption (∆𝐺𝑜𝑎𝑑𝑠 ) at air-solution interface were calculated using standard equations (Annexure S2, SI), the values of which are provided in Table S2 (SI). Table 1. The values of concentration corresponding to various transitions namely C1, C2, and C3(cmc) obtained during the complexation of βLG-SAIL from different techniques i.e. Surface tension, γ, Conductivity, , Pyrene fluorescence, Pyr. Flr., Intrinsic fluorescence, Int. Flr., Isothermal titration calorimetry, ITC., Turbidity, Turb., Zeta potential, ζ-pot., Dynamic light scattering, DLS in aqueous solutions. Ca, Cb and Cc are the concentration values corresponding to different transitions observed from zeta potential, count rate and hydrodynamic diameter measurements. Conc.

γ



Pyr. Flr.

Int. Flr.

ITC

Turb.

DLS Ca

(mmol L-1)

Cb

Cc

LG-SAIL-1 C1

1.2

C2

2.8

C3(cmc)

12.2

̶ 10.8

1.5

1.0

1.6

1.3

1.2

0.9

0.9

3.3

2.5

3.1

3.81

3.5

2.5

2.5

5.7

11.2

12.2

13.7

12.7

14.8

11.3

LG-SAIL-2 C1

0.4

0.5

0.5

0.6

0.9

0.22

0.5

0.3

0.4

C2

1.8

̶

1.6

2.0

3.1

1.22

1.8

1.0

0.8

C3(cmc)

5.4

5.9

4.6

4.9

6.6

4.41

6.1

3.7

5.5

LG-SAIL-3 C1

0.5

0.5

0.6

0.5

0.7

0.4

0.4

0.3

C2

̶

̶

̶

̶

3.6

1.3

1.9

1.6

0.8

C3(cmc)

3.90

7.0

4.0

6.2

5.9

4.9

6.4

6.5

4.2

LG-SAIL-4 C1

0.2

̶

0.2

̶

0.3

0.7

0.2

0.5

0.4

C2

̶

̶

0.4

0.5

0.4

1.0

̶

0.7

1.0

C3(cmc)

1.1

1.0

0.8

1.1

0.6

1.6

0.9

1.1

1.7

The γcmc is found to be more in presence of βLG as compared to βLG free systems for SAIL-1 and SAIL-2, whereas no significant difference is observed in cases of SAIL-3 and SAIL-4. This suggests that beyond C2, incoming surfactant ions (SAIL-1 or SAIL-2) prefers to interact with ACs in bulk and do not fully replace the surface active ACs from air-solution interface. However, in SAIL-3 and SAIL-4 systems, ACs dissolves into bulk from air-solution interface, which is



consecutively populated by the surfactant ions leading to γcmc close to that observed in the absence of βLG (Table S2, SI). This is assigned to relatively more surface active nature of SAIL3 and SAIL-4 as compared to SAIL-1 and SAIL-2 as manifested by observed lower values of γcmc for the former ones. Further, C3(cmc) for all investigated SAILs (Table S2, SI) is found to increase in presence of βLG except for SAIL-1, where marginal decrease is observed. It is natural to observe an increase in C3(cmc) of ionic surfactants as well as SAILs in presence of polymers as more number of surfactant ions are required to first saturate the polymer backbone followed by micellization in bulk.46,51 In the presence of βLG, Amin increases in case of SAIL-1 and SAIL-2, decreases in case of SAIL-4 and remains almost constant in case of SAIL-3 as compared to βLG free systems. This supports the fact that ACs tends to populate the air-solution interface even beyond C2 in case of SAIL-1 and SAIL-2, whereas there is almost complete removal of ACs from air-solution interface by SAIL-3 and SAIL-4 as also reported in case of biamphiphilic SAIL, [C6mim][C12OSO3].48 Almost similar or greater negative values of ∆𝐺𝑜𝑎𝑑𝑠 in case of SAIL-1 and SAIL-2 respectively, support the dominance of surface adsorption of SAIL ions over micellization in bulk. On the other hand, marginally lower values of ∆𝐺𝑜𝑎𝑑𝑠 in the presence of βLG in case of SAIL-3 and SAIL-4 are suggestive of no change in adsorption efficacy of these SAILs at air-solution interface even in the presence of βLG, which supports the dissolution of ACs from air-solution interface into bulk. 3.2. Bulk Behavior of Colloidal Systems: 3.2.1. Ionic Conductivity and Zeta-Potential measurements: Alternations in ionic environment of colloidal systems comprising βLG and SAILs were explored using ionic conductivity (Figure 2A and Figure S4, SI) and zeta-potential (ζ-potential) measurements (Figure 2B and C).46,51 The concentrations corresponding to different transitions obtained from specific conductivity (κ) and ζ-potential measurements (Table 1) are in good agreement with that observed from tensiometry. Only one transition corresponding to C3(cmc) in conductivity profile of SAIL-1 and SAIL-4 was observed in the presence of βLG (Figure S4, SI). The absence of C1 suggests relatively weaker interactions between βLG and SAIL-1 in bulk contrary to that observed the air-solution interface. Two transitions corresponding to C1 and C3(cmc) in case of SAIL-2 (Figure S4B, SI) and SAIL-3 (Figure 2A) were observed. The transition at C1 is assigned to the formation of MCs and indicates the presence of relatively stronger interactions of these


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SAILs with βLG, owing to presence of H-bonding prone amide and ester-moieties, respectively. This behaviour is in line with that observed for Gelatin-SAIL-2 and Gelatin-SAIL-3 systems.51 Below C3(cmc), SAIL-1 and SAIL-2 interacts with βLG in a different manner, however exhibit similar ionic environment as suggested by similarity of slope change of  in the absence and presence of βLG. It is important to discuss that the specific conductivity data of SAIL-1 and SAIL-2 (Figure S4, SI), with and without βLG, seems similar. This suggests that SAIL-1 and SAIL-2 almost retain their mobility in bulk, however interacts strongly with βLG as observed from various other techniques. This could be possible via negligible counter-ion condensation onto backbone of βLG, which otherwise adsorb at micelle-water interface. Further, a high exchange rate between monomers of SAIL in bulk and in adsorbed state as compared to the measurement time scale of conductivity could lead to such behavior. With further increase in concentration, κ rises following a relatively lower slope as compared to βLG free solutions in case of SAIL-3, which indicates the prevalence of interactions between SAIL-3 and formed MCs. A similar slope change in , in the absence and presence of βLG, above C3(cmc), in case of SAIL-1, SAIL-2 and SAIL-4 supports the formation of βLG freemicelles, whereas micelles of SAIL-3 in presence of βLG remains complexed with βLG in ACs.

Figure 2(A-C): (A) The variation of specific conductance () of SAIL-3 in aqueous solution with and without βLG; (B) Zeta potential profile of the investigated SAILs in aqueous solution of βLG; and (C) zoomed out view of zeta-potential profiles in lower concentration regime of SAILs.

These results are further supported by ζ-potential measurements (Figure 2B and C), where the magnitude of ζ-potential decreases from -16.4 mV and approaches zero at C1 in all the studied colloidal systems except for SAIL-4. This decrease in magnitude of ζ-potential indicates the predominant adsorption of imidazolium cation onto βLG, without condensation of Cl− which is



contrary to that observed in case of gelatin. 51 Considering the observations made from conductivity measurements, it is natural to assume that SAIL-1 and SAIL-2 adsorbs onto βLG mainly via weak hydrophobic interactions and retains the charged cationic head group towards bulk solvent without appreciate counter-ion condensation. In SAIL-4 system, an increase in overall negative potential from -16.4 mV to -18.4 mV till C1 could be due to dominance of [C8OSO3]- over [C12mim]+ in interacting with βLG. Greater hydrophilic-lipophilic balance57 (HLB) in SAIL-4 reflects highly polar nature of [C8OSO3]- that interacts strongly with positively charged residue of βLG in addition to hydrophobic interactions. Further, till C2, ζ-potential increases towards positive values following the order: SAIL-1 < SAIL-2 < SAIL-3 < SAIL-4, indicating the adsorption of SAILs onto forming ACs, the order which is also followed thereafter between C2 and C3. After C3(cmc), negligible change in ζ-potential is observed except for SAIL-3 system, where ζ-potential decreases. This ascertains that complexation of micelles formed by SAIL-3 with βLG-ACs occurs even beyond C3(cmc), which is in line with observations made from conductivity measurements. 3.2.2. Size and Morphological Investigations: The complexation phenomenon between SAILs and βLG was further investigated by turbidity (Figure 3A and Figure S5A, SI) and dynamic light scattering (DLS) measurements (Figure 3B and Figure S5B, SI). In monomeric regime of SAILs, till C1, the rise in turbidity and hydrodynamic diameter (Dh) for different SAILs follows the order: SAIL-1 < SAIL-2 < SAIL-4 < SAIL-3 and suggests the extent of interactions between SAILs and βLG. Negligible change in turbidity and Dh in case of SAIL-1, till C1, supports the presence of relatively weaker interactions of SAIL-1 with βLG as also indicated by κ and ζpotential measurements. An increase in Dh from 3.78 nm to ≈ 220, ≈ 710, and ≈ 210 nm, is observed for SAIL-2, SAIL-3 and SAIL-4, respectively, till C1. SAIL-2 and SAIL-3 bears relatively rigid H-bond donor/acceptor amide58 and flexible H-bond acceptor ester-moiety58 respectively that synergistically assists their interactions with βLG along with hydrophobic and electrostatic forces of interactions. It seems that H-bond donor and acceptor capability of SAIL-2 stabilizes the H-bonding network of βLG resulting in relatively lesser expansion of forming ACs. On the other hand, the possibility to adopt varying orientations 58 by SAIL-3 enhances its interactions with βLG leading to unfolding of βLG. This unfolding opens up more number of interacting sites leading to formation SAIL-3 mediated self-assembled MCs in this concentration regime as only unfolding is not expected to produce such large sized MCs. Similar phenomenon


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has been observed in case of BSA and G in the presence of SAIL-3.46,51 SAIL-4 due to its catanionic nature and greater hydrophobicity interacts strongly with βLG and stabilizes the forming self-assembled structures (MCs).

Figure 3 (A-F): (A) Variation of turbidity and (B) hydrodynamic diameter (Dh) of colloidal complexes of SAILs with βLG in their aqueous solution as a function of concentration of SAILs. (C-F) SEM images SAIL-3-βLG complexes at different concentration of SAIL-3 i.e. (C) below C1, 0.4 mmol L-1; (D) around C1, 1 mmol L-1; (E) Above C2, (1.61 mmol L-1) and (F) below C3(cmc), (4.08 mmol L-1) at 298.15K.

Between C1 to C2, turbidity and Dh for different SAILs follows almost same trend as below C1 with the exception of SAIL-4 that exhibit highest turbidity. After C2, for all the investigated systems, Dh remains constant in a certain concentration range before decreasing and a minima in Dh is observed at C3(cmc), with the exception of SAIL-4, where Dh begin to decrease immediately



after C2 (Figure S5, SI). A decrease in Dh before C3(cmc) is assigned to dissolution of formed ACs by the forming micelles that also results in decrease in turbidity. It seems that [C12mim][C8OSO3] interacts with βLG more strongly as compared to [C6mim][C12OSO3]48 owing to greater hydrophobicity as suggested by a comparison of turbidity measurements, whereas the nature of counter-ions in [C12mim][Cl] and [C12 mim][Br]48 also affects the interactional process. Morphology of SAIL-βLG complexes at different concentration of SAILs was explored using SEM measurements (Figure 3C-F and Figure S6-S9, SI). Native βLG in aqueous solution shows a bimodal size distribution with Dh ≈ 10 and 250 nm from DLS measurements (Figure S6A, SI), which is supported by presence of large sized agglomerates from SEM imaging (Figure S6B, SI). In case of SAIL-1, below C1, nearly spherical MCs of size 100-150 nm (Figure S7A, SI) are formed, which further grows up to 200-400 nm between C1-C2 via SAIL mediated selfassembly of MCs (Figure S7C, SI). After C3(cmc), smaller ACs become more spherical and compact (Figure S7D, SI). On the other hand, morphology of SAIL-LG complexes formed in case of SAIL-2 and SAIL-3 differs significantly. In SAIL-2, below C1, MCs are relatively larger (≈200-250 nm, Figure S8A, SI) as compared to that observed in SAIL-1, and near C2, relatively small ACs transforms into elongated to near spherical ACs of size ≈ 480 nm (Figure S8B, SI). In SAIL-3 system, even at very low concentration i.e. at C1 (0.4 mmol L-1) micron sized ≈ 932 nm (Figure 3C) aggregates (MCs) begin to form, which is also confirmed by DLS measurements (Figure 2B).The formed MCs further grow to larger ACs of size ≈ 1200 nm, having rod like architectures near C2 (Figure 3D). At higher concentration, just below C3(cmc) (4.08 mmol L-1) rod-like architectures disappears and near spherical aggregates of variable size were observed (Figure 3E).In case of SAIL-4, the formed SAIL-βLG complexes remains near spherical in shape in different concentration regimes (Figure S9B, SI) and the size of formed complexes is well supported by DLS measurements. For all the investigated systems, near C3(cmc), aggregates of variable sizes were observed having more number of smaller ACs (≈10-100 nm) as compared to larger aggregates (Figure 3 and Figure S7-S9, SI), which is supported by DLS measurements (Figure S10-S12, SI). The presence of free micelles and micelles complexed with LG give rise to such distribution.


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On comparison, it is found that both LG and BSA46 forms morphologically similar complex with SAIL-1 till respective C2 values, however ACs in case of BSA developed into compact elongated ACs whereas LG-SAIL ACs disintegrate beyond micellization. The behavior of SAIL-2 towards LG and BSA is found to be almost similar with the exception of presence of both rod-shaped as well as spherical aggregates in case of LG. Interestingly, no formation of elongated helical rods rich in amyloid structure has been observed in case of LG as observed in case of BSA.46 LG forms relatively small rod-like aggregates that seem to be originated form layered growth of small sheet-like structures (Inset, Figure 3E). This suggests that globular proteins may interact with SAILs in a similar fashion macroscopically, however the specific interactions between two owing to dissimilarities in nature of amino acid residues and their packing in 2o structure in protein governs the formation of discrete self-assembled structures. Therefore the role of inherent structure of protein in terms of relative content of α-helix and βsheet in governing their complexation with SAILs cannot be ruled out. 3.2.3. Spectroscopic Investigations into Complexation Behavior: Dynamic interplay of hydrophilic-hydrophobic interactions involved in protein-SAIL complexation is explored using steady-state fluorescence by using pyrene as an external probe. Figure 4(A-D) shows the variation in ratio of intensity of first (I1) to third (I3) vibronic band of pyrene as a function of concentration of SAILs in absence and presence of βLG. The value of I1/I3 of pyrene was found to be 1.42 in presence of βLG, much lower than that observed in water (1.72).59 This shows the adsorption of pyrene in relatively hydrophobic environment of globular proteins.45 In dilute concentration regime below C1, I1/I3 increases from 1.42 to 1.63 at concentration marked as Cs (Figure 4E), and is assigned to replacement of pyrene molecules by SAIL ions from hydrophobic sites of βLG.48 Similar finding has been reported in the earlier report where such abrupt rise in I1/I3 was attributed to conformational changes in βLG in presence of SAILs.48 We have confirmed the replacement of pyrene by incoming SAILs as the reason for increase in hydrophilicity as experienced by pyrene by performing docking studies for pyrene in the presence of βLG (Figure S13A-D, SI).



Figure 4 (A-E): (A-D) Variation in ratio of I1/I3 of pyrene in the absence and presence of βLG in aqueous solution as function of different SAILs at 298.15K; and (E) expanded view of obtained data (AD) in low concentration range of SAILs (0 to 2 mmol L-1).

Higher docking scores for βLG and SAILs as compared to βLG and pyrene suggest relatively stronger binding of SAILs as compared to pyrene (Table S3 and S4, SI). On comparison, it is observed that increase in I1/I3 takes place at relatively lower pace in presence of BSA45 as compared to βLG for SAILs i.e. [C8mim][Cl] and [C4 mim][C8OSO3] which are homologous with the SAIL-1 and SAIL-4. The variation in preferential adsorption site of pyrene in two proteins dictates such behaviour (Figure S13, SI). After Cs, the decay pattern of I1/I3 (Figure 4A-D) clearly indicates the variation in pace, nature and extent of interactions between SAILs and βLG due to difference in structure and polarity of head groups of SAILs. I1/I3 decreases till C1 and changes marginally thereafter until C2 is reached with the exception of SAIL-4, where a relatively smaller decrease in I1/I3 is observed till C2. For different SAILs, the value of I1/I3 at C1 follows the order: SAIL-4 (1.52) > SAIL-1 (1.35) ≈ SAIL-2 (1.33) > SAIL-3 (1.20). In case of SAIL-2 and SAIL-3, functional group assisted hydrophobic interactions between SAILs and βLG results in the formation of highly hydrophobic MCs and ACs. This reflects that the pyrene is localized in highly hydrophobic environment of formed MCs and ACs till C1 and between C1 and C2 respectively for SAILs with the exception of SAIL-4, which is assigned to strong hydrophobic interactions between SAIL-4 and βLG that


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accompanies other set of interactions. The hydrophobic interactions between these SAIL-4 decorated MCs results in growth of MCs to ACs via mediation of incoming [C 12mim]+ and leads to charge reversal. Further the decay of I1/I3 for [C6mim][C12OSO3]48 and [C12mim][C8OSO3] (SAIL-4) in the presence of βLG is found to be very much similar, whereas contrasting behavior of [C12mim][Br] in comparison to [C12mim][Cl] is in line with observations made form turbidity measurements in both studies. Further, between C2 to C3(cmc), I1/I3 remains constant for SAIL-1 and SAIL-3 but decreases marginally in case of SAIL-2 and to a large extent in case of SAIL-4 (Figure 5). This is assigned to a breakdown of ACs in case of SAIL-4 (Figure 3B) which provides more hydrophobic sites for interaction with incoming SAIL-4 cations and lowers I1/I3 to a greater extent. Above C3(cmc), although I1/I3 represents the average value of SAIL-βLG complexes as well as of formed micelles, however it is observed that the formed ACs beyond C3(cmc) are even more hydrophobic than free micelles in case of SAIL-4 and the hydrophobicity index follows the order: SAIL-4 (0.92) < SAIL-3 (1.14) ≈ SAIL-2 (1.13) < SAIL-1 (1.31). The obtained values of cmc differs from surface tension data as different techniques sense different stages of micellization or aggregation, which results in such observations in many biopolymer-SAIL systems.45,52 3.3.4. Conformational Changes in  LG: 3.3.4.1. Intrinsic fluorescence: The variations in Trp (Figure S14, SI) fluorescence have been exploited in past to extract information about conformational changes occurring in proteins. 46,48 Figure 5A shows the variation in intrinsic fluorescence of βLG in aqueous solution as a function of SAILs concentration, mainly owing to presence of Trp-19 present in internal cavity of βLG30 as the fluorescence of Trp-61 present in close proximity of Cys66–Cys160 disulfide sulfurbridge30 is quenched by disulfide sulfur bridges.60 Initially, the fluorescence intensity increases and follows the order: SAIL-2 > SAIL-1> SAIL-3 ≈ SAIL-4. Different reasons such as (i) replacement of solvating water molecules by relatively less polar [C12 mim]+ head groups; (ii) binding of SAIL cations to Glu-62 and in its vicinity closer to Trp-61, which could weakens the quenching action of disulphide sulphur bridge; and (iii) enhancement in hydrophobicity around fluorophore by localization of alkyl chains, can be accounted for such an increase. The maximum fluorescence enhancement in case of SAIL-2 could be due to clustering of Trp-61 present in internal cavity of βLG by SAIL-2 via relatively stronger H-bonding interactions with amino acid



residues, which shields it from disulphide sulphur bridge quenching 30,60 along with enhanced hydrophobic interactions around fluorophores.

Figure 5 (A-E): (A) Emission spectra of βLG at excitation wavelength of 280nm in presence of different SAILs; (B-D) far-UV and (E) near-UV spectra of βLG in aqueous buffer solutions of different SAILs at 298.15 K.

On similar lines, the interaction of SAIL-1 with βLG via electrostatic interactions supported by hydrophobic interactions (Figure 2B and C) creates relatively hydrophobic environment near both Trp-19 and Trp-61 resulting in larger increase in fluorescence intensity. On the other hand, SAIL-3 and SAIL-4 causes less changes in secondary structure of βLG where exposure of Trp residues towards relatively less polar environment results in lesser increase in fluorescence intensity.30,45,46 Such an increase in fluorescence intensity below C1 has not been observed in case of BSA46 and is assignable to difference in 2o structure and relative location of fluorescent Trp residues in BSA and βLG. After C1, fluorescence intensity decreases with a relatively steeper slope till C3(cmc) after which it decreases marginally. An enhanced, unfolding of βLG expose the Trp residues towards relatively polar imidazolium cation, which also brings greater hydration leading to fluorescence quenching.45,46,48 Beyond C3(cmc), relatively smaller change in fluorescence intensity suggests marginal change in environment of Trp residues. No shift in emission wavelength has been observed in investigated SAIL systems which are contrary to


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interaction of BSA with SAILs.40,45 This observation negates the existence of any significant changes in conformation of tertiary structure of βLG. 3.3.4.2. CD spectroscopy: The far-UV CD spectra of proteins exhibit peculiar spectral features for proteins varying in content of -helices and -sheets and based on this, the proteins may be divided into 4 types.61,62 The investigated protein, βLG, falls in the category of all-β proteins and is rich in β-sheet with a considerable amount of α-helices.63,64 The CD spectra of βLG showed a dependence on molecular structure and concentration of SAIL in aqueous solutions (Figure 5B, C, D and S12, SI). SAIL-1 is found to alter the 2o structure of βLG after a concentration of 0.4 mmol L-1 as indicated by blue shifting of CD band and increased half-line width (Figure S15, SI). This suggests the retention of 2o structure of βLG in low concentration regime of SAIL-1 as also observed in case of [C12mim][Br],48 which is followed by disordered anti-parallel β-sheets with marginal enhancement in content of α-helices at higher concentration of SAIL-1 between C1 and C2. Relatively weaker hydrophobic interactions between βLG and SAIL-1, as mentioned earlier, don’t influence the inherent structure of protein to a greater extent even during formation and growth of MCs and ACs. Beyond C2, CD spectra of βLG became shapeless which indicates the complete unfolding of protein. Similarly, βLG retains its 2o structure in the presence of SAIL-4 even at higher concentrations up to C3(cmc) (Figure 5D). Such stabilization is assignable to biamphiphilic nature of SAIL-4. It is evident from ζ-potential and conductivity measurements that both [C12mim]+and [C8OSO3]-interacts with βLG to oppositely charged amino acid residues below C1, and stabilizes the 2o structure via hydrophobic interactions between their alkyl chains that could act like a bridge in stabilizing the 2o of βLG.40 On comparing two H-bonded prone SAILs, SAIL-2 and SAIL-3, it is observed that monomeric adsorption of SAIL-2 onto βLG induces un-ordering of -sheet structure below C1, and a complete breakdown of -sheet structure to unordered random coils is observed near C1 (Figure 5B). Interestingly, with further increase in concentration, the formation of ACs between C1 and C2, are accompanied by refolding of protein towards original structure. Such complete breakdown and reversal of 2 o structure of proteins in the presence of SAILs have never been observed. Contrary to this, SAIL3, don’t exert much influence on secondary structure of βLG till C1,whereas marginal unordering of β-sheets as indicated by increased half-line width accompanied by a blue shift in CD band between C1 and C2 (Figure 5B), is observed. Further, a changeover of 2o structure from allβ protein toα + β structure is observed between C2 and C3.



The change in tertiary structure (3o structure) of βLG was evaluated with the help of CD spectra in the near-UV region (Figure 5E). Native βLG showed four prominent bands at 266 nm, 277 nm, 286 nm and 295 nm. The bands at 266 nm and 277 nm could be attributed to tyrosine and the bands at 286nm and 295 nm to tryptophan vibrational fine structure. 65 In case of SAIL-1, SAIL-2 and SAIL-3, 3o structure was evaluated up to a concentration below C2, where the SAIL interacts with the protein majorly in its monomeric form, whereas for bi-amphiphilic SAIL-4, the structural evaluation was done up to C3(cmc). As can be seen from Figure 5E, in case of SAIL-1, in all the concentration regimes, the bands in the tryptophan and tyrosine region are found to be retained, however a blue and red shift is observed, respectively. This suggests the retention of 3 o structure of βLG. Ester functionalized SAIL-3 induced dramatic alterations in 3 o structure of βLG, where up to 0.1 mmolL-1 of SAIL-2, 3o structure was partially retained in the tryptophan region. However, further increase in concentration to 0.5 mmolL -1 (C1), there occur the complete loss of 3o structure as evidenced by sign reversal of molar ellipticity with large positive values (Figure 5E). A further increase in the concentration up to 1.0 mmol L -1 of SAIL-2, partial recovery of the protein’s 3 o structure is observed. This is in line with the complete loss of 2 o structure from a β-sheet structure to random coil followed by reversal of β-sheet structure (Figure 5B). The addition of SAIL-3 induced a gradual change in 3 o structure. At a relatively higher concentration (2.0 mmol L-1), the bands at 266 and 286 nm were retained, however the other two bands could not be observed and indicates partial sustenance of 3o structure. In the presence of biamphiphilic SAIL-4, the tertiary structure of the βLG was almost retained up to C2, however further increase in the concentration up to C3(cmc) resulted in the total collapse of the 3o structure and attained a structure characteristic of molten globular state.66 Such a huge structural change could be due to the increased hydrophobic interactions of protein with the SAIL-4 caused by the two longer alkyl chains present in both the cation and anion unlike in the other three SAILs. It is inferred that there is a contrasting variation between SAIL-2 and SAIL-3 towards induction of changes in 2o structure of βLG, whereas both of these SAILs behaved almost similarly towards another globular protein BSA.46 Although, SAIL-2 and SAIL-3 exhibit different H-bonding characteristics along with varying flexibility near cationic head group, however, it is clear that it is the nature of protein (linear or globular) 46,52 and inherent 2o and 3o structure of globular (BSA and βLG) proteins that governs their complexation and formation of different self-assembled architectures in the presence of SAILs. The CD spectra studies-suggests that polar functional


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groups that interacts with the protein through H-bonding interactions plays a key role in inducing the conformational changes in the protein structure. 3.3.5.

Thermodynamics

of

Complexation:

Differential

power

and

corresponding

enthalpograms of investigated SAILs in the absence and presence of βLG are provided in Figure S16 and S17, SI. ITC profiles of investigated SAILs in the presence of LG seems similar to that observed in buffer (Figure S17, SI), however significant differences are observed till C3(cmc). There are different factors which govern the enthalpy changes. These factors include the nature of ionic head group, hydration/dehydration phenomenon, degree of counter-ion condensation, partial molar enthalpy of monomers, aggregation number, dilution of micelles and unfolding/refolding of protein.67-70 A reversal of sign in enthalpy from exothermic to endothermic, with increase in concentration of SAIL-1, both in the absence (Figure S16A) and the presence (Figure S16E), near cmc, is observed. This can be ascribed to greater extent of dehydration (endothermic) and enhanced electrostatic interactions (endothermic) upon micellization as compared to other SAILs. After cmc, the dilution of micelles mainly dominates the enthalpy changes and hence not much variation in enthalpy change is observed beyond cmc.

Figure 6 (A-D): The difference plots of enthalpogram of βLG-SAIL systems highlighting the interactions between βLG and SAILs in aqueous solution of βLG at 298.15 K.



To have better insight into the phenomenon, difference plots of enthalpy changes signifying the complexation of SAILs with LG are provided in Figure 6 (A-D).Thermodynamics of complexation depends on many factors such as electrostatic, hydrophobic and H-bonding interactions which includes hydration/dehydration of surfactant head groups and alkyl chain. For the sake of simplicity, the obtained thermodynamic data (Table S5 and S6, SI) is discussed according to different concentration regimes as between C0-C1 (∆𝐻𝑜1 ), C1-C2 (∆𝐻𝑜2 ), C2-C3(cmc) ( ∆𝐻𝑜3 ). In monomeric regime C0-C1, exothermic enthalpy changes are observed in all investigated SAILs, except SAIL-4, and is accounted to dominance of electrostatic forces of attraction between oppositely charged groups of SAILs and βLG. In this concentration regime, the role of disaggregation of micelles in governing exothermic enthalpy changes cannot be ruled out. On the other hand, an endothermic enthalpy change in case of SAIL-4 suggests the dominance of electrostatic forces of interactions predominantly between negatively charged amino acid residues of LG and [C12mim]+ of SAIL-4 as indicated by ζ-potential measurements. A greater extent of dehydration of long alkyl chains as compared to other SAILs is also thought to contribute to exothermic enthalpy changes. The extent of unfolding of βLG induced by SAILs also plays an important role towards enthalpy changes. In line with greater degree of destabilization of 2o structure of βLG caused by SAIL-2 along with possibility of H-bonding interactions (exothermic), largest exothermic enthalpy changes in case of SAIL-2 are observed, whereas the retention of 2o structure of βLG in case of SAIL-4 give rise to endothermic enthalpy change. The enthalpy change between C1-C2 follows the order SAIL-4 (∆𝐻𝑜2 = -3.22 kJ mol-1) > SAIL-3 (∆𝐻𝑜2 = -1.15 kJ mol-1) >SAIL-2 (∆𝐻𝑜2 = -0.35 kJ mol-1) >SAIL-1 (∆𝐻𝑜2 = -0.04 kJ mol-1). This is in line with the growth of the formed ACs (Figure 3B) mediated by SAIL ions. Thereafter, between C2 and C3(cmc), the endothermic enthalpy change follows the order: SAIL-4 (∆𝐻𝑜3 = +1.42 kJ mol-1) > SAIL-3 (∆𝐻𝑜3 = +0.62 kJ mol-1) > SAIL-2 (∆𝐻𝑜3 = +0.16 kJ mol-1) > SAIL-1 (∆𝐻𝑜3 = -0.13 kJ mol-1). This, in line with almost constant ζ-potential values in this concentration regime, suggests that the absorption of SAILs on formed complexes predominantly occurs via hydrophobic interactions and results in endothermic enthalpy changes.


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3.3.6. Molecular Docking Studies: To generalize the extent of interactions between investigated SAILs and LG along with probing the most preferable binding site of SAILs, molecular docking studies has been performed.71-75 Among investigated SAILs, SAIL-2 possesses highest free energy of binding with βLG due to presence of H-bonding acceptor-donor interactions along with other set of interactions and such stronger interactions led to unfolding of protein (Figure 5B). βLG exists as a mixture of two forms i.e. βLG-A and βLG-B, and the free energy of binding of SAILs with βLG-A is found to be higher than βLG-B, however the order representing the extent of interactions between SAILs and protein remains same for both the states of βLG (Table S4).

Figure 7(A-D): SAIL-2 in blue, (A-C) and (D-F) cartoon representation of βLG-A and βLG-B respectively; (A,B) showing SAIL-2 cation occupying the internal binding site (B, E) SAIL-2 cation in stick representation making H-bonds with amino acid residue present at binding site and (C, F) An enlarged view of H-bonding interaction of SAIL-2 with Proline-38 (Red) and Lysine-60 (Blue) of βLGA and with Thr-6 (Green), Lys-8 (Red) and Leu-95 (Blue) of βLG-B.

In βLG-A, SAILs occupies internal hydrophobic binding sites of protein, which results in greater number of favorable interaction of SAIL with βLG-A (Figure 7A, S18A, and S19A, SI),whereas SAILs resides mainly in contact with external binding site in case of βLG-B. The structured nature of binding site of βLG-A also highlights the impact of varying polarity and flexibility of



head group of SAIL-2 and SAIL-3, whereas such distinction seems to be absent in βLG-B due to greater occupancy of external binding site (Figure 7D, S18C and S19D, SI) by respective SAILs. In βLG-A, head group of SAIL-2 (Figure 7B) is pointing towards opening of hydrophobic cavity where it donate one H-bond to Pro-38 (Red) via NH group and accept one H-bond from Lys-60 (Blue) via C=O carbonyl part of amide group and remains in close vicinity of Trp-61.This also supports the maximum fluorescence enhancement (Figure 5A) observed till C1 in case of SAIL-2. Similarly SAIL-3 also occupies same inner hydrophobic site (Figure S19B, SI) but interact with (see magnified view Figure S19C, SI) Lys-60 (blue) and Lys-69 (red) by accepting two H-bond via C=O group of ester group. However, in βLG-B (Figure 7E), SAIL-2 remains present on external hydrophobic binding site where it is interacting with three amino acid residues namely Thr-6 (Green), Lys-8 (Red) and Leu-95 (Blue) through H-bonding interactions. Similarly SAIL3, interact with Glu-59 (Figure S19F, SI, green) present at external binding site. In both cases SAIL-1 being a non-functional analogue, interact mainly via hydrophobic interactions without polar interaction with side chain residues.SAIL-4 begin cationic system comprises of two ligand and therefore docking SAIL-4 in βLG implies docking of two ligands simultaneously which is not possible in Autodock Vina Program. In nut-shell, the complexation behavior of SAILs with βLG is investigated using different techniques which complemented each other. The observed behavior at air-solution interface is significantly different from that observed in bulk. The molecular structure of SAILs in terms of absence (SAIL-1 and SAIL-4) or presence of H-bonding prone moieties (SAIL-2 and SAIL-3) along with biamphiphilic nature of SAIL-4 has been found to exert a great influence on their interactional behavior with βLG and led to structure specific changes in 2o structure of protein. Conclusions: A contrasting behavior of SAILs differing in molecular structure towards complexation with a globular protein, βLG, is observed. SAIL-2 appended with H-bond donor and acceptor amide moiety is found to interact strongly with βLG both at air-solution interface as well as in bulk and forms relatively stable ACs at air-water interface. On the other hand, owing to higher surface activity, SAIL-3 and SAIL-4 occupies the air-solution interface at the cost of dissolution of ACs into bulk. The stronger interactions of SAIL-2 with βLG are also manifested by complete destruction of 2 o structure of βLG in lower concentration regime, which regains to some extent in higher concentration regime. On the other hand, the favorable interactions


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between βLG and SAIL-4 ions ([C12OSO3]- and [C12 mim]+) as also observed earlier in case of BSA40 and cellulose41 don’t alter the 2o structure of βLG, which is also supported by fluorescence measurements. The behavior of SAIL-2 and SAIL-3 towards complexing with βLG is found to be different as compared to that observed in case of BSA. 46 SAIL-2 and SAIL-3 displayed almost similar behavior in modifying the 2 o structure of BSA, where the formation of long helically twisted amyloid fibers in the presence of SAIL-2 was observed. The increased disorder among β-sheets induced via SAIL-2 results in non-formation of such structures in case of βLG, however relatively long rod-like structures self-assembled from thin sheets have been observed. The study along with previous reports on gelatin, 51 a linear polyampholyte, and a globular protein, BSA,45,46 suggests that both the nature of SAILs and 1o structure of proteins along with content of -helix and -sheets (BSA contain 56% α-helical and 11% β-sheet while

LG 7% α-helical and 51% β-sheet structure)12,13,46 in 2o structure and relative positions of amino-acid residues govern the complexation behavior of SAILs with proteins. However, it is clear that the ester functionalized SAILs possess peculiar tendency to form rod-like architectures especially with globular proteins. Acknowledgement: This work was supported by the DST, Govt. of India wide project number EMR/2017/002656. Supporting Information: Annexure S1-S2, Figure S1-S19, and Table S1-S6 are available at pubs.acs.org.



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Varying self-assembled structures of beta-lactoglobulin and SAILs are observed depending upon the molecular structure of SAILs. 94x44mm (300 x 300 DPI)


Complexation Behavior of Î²-Lactoglobulin with Surface Active Ionic

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