Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization

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Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization of Collagen Aafiya Tarannum, Charuvaka Muvva, Ami Mehta, Jonnalagadda Raghava Rao, and Nishter Nishad Fathima J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b02723 • Publication Date (Web): 21 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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The Journal of Physical Chemistry

Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization of Collagen Aafiya Tarannum, Charuvaka Muvva, Ami Mehta, J. Raghava Rao, N. Nishad Fathima* Chemical Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600020, India

*Corresponding Author Chemical Laboratory, CSIR-Central Leather Research Institute Adyar, Chennai 600020, India Tel.: +91 44 24437188; fax: +91 44 24911589 E-mail addresses: [email protected], [email protected] (N. N. Fathima)

ACS Paragon Plus Environment

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ABSTRACT. Ions play a key role in destabilization of collagen. This study explores the effect of diethyl methyl ammonium methane sulfonate, an ionic liquid on the different hierarchical ordering of collagen viz., at molecular and fibrillar level. The rheological behavior and secondary structural changes reveal changes in the hydrogen bonding environment of collagen, leading to alterations in the triple helical structure of collagen. Increase in concentration of AMS resulted in swelling of RTT fibers and also decreased thermal stability signifies that ions are obliged for destabilizing collagen at fibrillar level. Molecular modeling studies confirm that anion is judiciously held responsible for structural deformity in collagen, whereas the cation shows tenuous effect. Thus, the preferential role of ions present in ammonium ionic liquid has been elucidated in this study.


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1. INTRODUCTION Ionic liquids (ILs) are molten salts at room temperature, composed of cations and anions1. In the last few decades, they have been captured quite justifiably, as medium for green synthesis2, electrolyte phases for biosensors3, mobile or stationary phases for separation studies4 and precipitating agents or additives for protein crystallization5. In the last few years, studies related to protein-DNA6, 7 and protein-IL8 interaction are nesting interest in the scientific community. The effect of ILs on several proteins and nucleic acids has been investigated extensively by varying anions, cations and alkyl chains. The stability of cytochrome C is strongly maintained in alkylammonium formate ionic liquids in contrast with its denaturation in similar solutions of methanol or acetonitrile solvents9. The role of phe-IL in reversible folding-unfolding patterns of myoglobin suggested that beyond certain concentration, there is a complete transition from helical to beta sheet formation10 whilst, the structural and chemical stability of nucleic acids is maintained in ammonium ILs for extended storage periods without any loss of its activity11. Therefore, it can be summarized that ILs plays an important role in the stabilization and destabilization phenomena of biomolecules12. Ethylammonium nitrate (EAN), the first protic ionic liquid dates back to 191413 with its potential in improving the solubility of protein and mostly construed as precipitating agents14, 15. Various studies on ammonium ILs have shown that they act as stabilizers or destabilizers for biomolecules. The stability of amino acids in different ammonium-based ILs demonstrated that these ILs stabilizes the three dimensional protein structures due to the interplay of ions with amino acids of the protein16. Furthermore, triethyl ammonium acetate and phosphate have also been reported as biocompatible for α-chymotrypsin stability17. In contrast, the heme proteins such as Hb and Mb were strongly destabilized by tetramethyl ammonium hydroxide, which


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could be owing to low viscosity and smaller alkyl chain length compared to a series of tetra-alkyl ammonium cation and hydroxide anion having bulkier alkyl chains18. The ammonium-based protic ILs has been proved as novel solvent for preventing insulin from self-aggregation and stabilizing it against thermal influence19. Also, triethyl ammonium sulfate and phosphate has been shown to be employed in developing biocompatible and biodegradable materials such as cytotoxic agents against T98G brain cancer cells20 and pH sensitive drug delivery systems21. Collagen, a significant structural protein has extensive applications in the field of tissue engineering such as injectable matrices, scaffolds intended for bone regeneration. Collagen is the most widely used biomaterial because of its low immunogenicity. It is also being used in food and beverage industry to improve the elasticity, consistency and stability of food products. It has been used in protein dietary supplements, food additives, edible film and coatings apart from it being the primary leather making protein. This emphasizes the need to understand the interaction of collagen with various additives, as these studies will have significant implications in food, pharma and leather industry22-24. The interaction of collagen has been studied with various ionic liquids such as imidazolium, phosphonium and choline dihydrogen phosphate. Imidazolium IL was demonstrated to influence collagen at different hierarchical ordering25. On the other hand choline dihydrogen phosphate, a biocompatible ionic liquid was ascertained to stabilize collagen by exerting an electrostatic force on collagen, thus making it a potential biocompatible crosslinker26. Phosphonium ionic liquid with variable anions also substantiates destabilizing effect on collagen due to the chaotropicity of anions27. The interaction of collagen with ILs will open more avenues in field of tissue and biomedical engineering. The purpose of the current study is to investigate the influence of ammonium ionic


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liquid, diethyl methyl ammonium methane sulfonate (AMS) on the stability of collagen. The choice of anion was based on chaotropic and kosmotropic behavior of ions as described in Hofmeister series. This study elucidates the role of ions on interaction with collagen by various characterization techniques viz., Fourier transform infrared spectroscopy (FT-IR), circular dichroism studies (CD), viscosity analysis, UV and fluorescence, thermal and dimensional stability. Further, molecular dynamics simulation studies were carried out to complement experimental studies, in order to understand the preferential role of ions in destabilization of collagen. 2. EXPERIMENTAL 2.1. Materials Diethyl methyl ammonium methane sulfonate [(AMS), figure 1] was procured from Ionic Liquid Technologies GmbH (IoLiTec, Germany) and used. Type I collagen was extracted from rat tail tendons (RTT) teased from six month old albino rats (Wistar strain). Millipore water was used for this study.

Figure 1. Structure of diethyl methyl ammonium methane sulfonate (AMS) 2.2. Isolation of type I soluble collagen Tails of six month old albino rats (Wistar strain) were teased for the extraction of high purity type I collagen and ethical guidelines were followed accordingly. The teased collagen fibers were washed thoroughly with 0.9% NaCl at 4°C, purified using 5% Sodium chloride followed by centrifugation for garnering the precipitates. The precipitate obtained was dialyzed against 50 mM phosphate buffer with pH of 6.5±0.3. After dialysis, the collagen was re-dissolved in 0.5 M


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acetic acid followed by final dialysis against 0.05 M acetic acid. For further experiments the collagen was diluted with 50 mM acetate buffer of pH 4. Woessner28 method was followed for ascertaining the collagen concentration by estimating the hydroxyproline content. Purity of collagen solution was confirmed using SDS-PAGE electrophoresis. The collagen solution was stored at 4˚C. 2.3. Preparation of collagen-AMS The collagen solution in acetate buffer was treated with four different concentrations of diethyl methyl ammonium methane sulfonate (AMS), 1:0.05% (v/v) – C-AMS-1; 1:0.5% (v/v) – CAMS-2; 1:5% (v/v) – C-AMS-3; 1:10% (v/v) – C-AMS-4 and the solution was stirred at 4°C for 3 h throughout to prevent denaturation of collagen due to heat generated during mixing. The working concentration of collagen was taken as 2.7 µM at pH 4.0. The concentration of collagen was kept constant and AMS concentration was varied. 2.4. Viscosity measurements Ostwald type Viscometer of 3 ml capacity was used to determine the effect of AMS on the rheological property of collagen. The flow time of collagen with and without AMS was measured after a thermal equilibration of 30 minutes using a digital stopwatch at least three times and the average was taken. The relative viscosity (ηrel) was calculated from the equation, = / where, η is the time flow of buffer and η0 is the time flow of sample. The collagen treated AMS were plotted against ηrel on x-axis and on y-axis. [(ηrel=1/R), (R=Additive/Collagen)].


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2.5. UV absorbance studies To determine the changes for native and AMS treated collagen, all the samples were incubated at 4°C for overnight and was recorded using UV-1800 Shimadzu UV-Visible spectrophotometer. 2.6. Circular dichroic studies The circular dichroic spectra of collagen can be detected under nitrogen atmosphere in the far UV region ranging from 190 to 260 nm for native and AMS treated collagen using Jasco 815 circular dichroism spectropolarimeter. Approximately, 400 µl of native collagen and collagen treated AMS was scanned with 0.2 nm intervals with a path length 1 mm at 25°C with computer averaged three scans for each sample. The data were obtained in milli degrees and further converted to molar ellipticity (deg.cm2.dmol-1). Molar ellipticity was plotted against wavelength in nanometers (nm). The concentration of collagen used was 2.7 µM. 2.7. FT-IR Studies The lyophilized samples of native and AMS treated collagen were taken to study the interactions between collagen and AMS using Jasco FT/IR-4200 (Fourier Transform Infrared Spectrometer). 60 scans in the range of 4000-400 cm-1with resolution of 4 cm-1 was taken by KBr pellet method at 25°C. 2.8. Dimensional and thermal stability of RTT collagen fibers treated with AMS Collagen fibers (Rat tail tendons, RTT) were treated with ammonium methane sulfonate for 24 h at 25˚C. The changes in the dimensional stability was monitored under Aven Inc., Digital Mighty Scope, 1.3 M (Product code: 48708-25, Made in Taiwan) of 10x resolution. The denaturation temperature (Td) of native and AMS treated RTT treated were studied using differential scanning calorimeter (Netzsch-DSC 204 F1 phoenix). Samples were blot-dried and


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were sealed in the hermetic aluminum Tzero pans and heated from 30 to 80˚C at the rate of 5˚C min−1. 2.9. Molecular modeling studies Molecular dynamics simulations of all systems were carried out to comprehend the effect of collagen at varying concentrations of diethyl methyl ammonium methane sulfonate (AMS) using GROMACS (version 4.6.2)29, 30. The structure of collagen-like peptides was constructed using GENCOLLAGEN package31. All the systems were solvated with TIP3P water molecule32. Simulations were performed employing AMBER03 force field33 and wang et al, developed force field using antechamber module of amber package34,

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and RESP charges were generated for

each model. The potentials was calculated using GAUSSIAN 09 package36 and geometries of each AMS compound were optimized using B3LYP/6-31G (d, p) level. Employing the steepest descent algorithm, the systems were incurred to energy minimization. 100 nanoseconds (ns) run was implemented using 2 femto seconds time step for integration of equation of motion at 300 K and at 1 atmospheric pressure in NPT ensemble exerting a V-rescale thermostat and Parrinello−Rahman Barostat respectively37-39. A cutoff of 10 Å accounts for the electrostatic interaction using particle-Mesh Ewald (PME) method. VMD package helps to visualize the trajectories obtained from MD simulations40. The tools in the GROMACS package was used for further analyzing the trajectories. The collagen-like peptide sequence used in this study, GPOGKOGPOGPOGPOGROGPOGAOGHOGSO GPOGKOGPOGPOGPOGROGPOGAOGHOGSO GPOGKOGPOGPOGPOGROGPOGAOGHOGSO


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GENCOLLAGEN package was brought forth by Object Technology Framework (OTF), and the sequence was constructed41. These amino acid residues in the collagen-like peptides were preferred owing to the significant role on interaction with the AMS compound, which in turn effectuates the microenvironment of collagen. 3. RESULTS AND DISCUSSION 3.1. Viscosity studies Relative viscosity was measured for collagen in the presence of varying concentration of AMS. The plot of relative viscosity 1/R, [R= (collagen)/ (AMS)] indicates the changes in the intermolecular as well as intra-molecular forces of collagen42. Increase in concentration of AMS leads to increase in viscosity of collagen as seen from (S1). At higher concentrations viz., C-AMS-3 and C-AMS-4 are in close proximity resulting in aggregation or entanglement, thereby being resistant to flow. AMS influences the interaction by changing the microenvironment of the collagen, thereby restricting the mobility. 3.2. UV–Vis spectroscopic studies UV absorption studies for native and AMS treated collagen were performed to study the change in the tyrosine environment, thus exploiting the spectroscopic behavior of protein, which provides an in-depth knowledge of how the conformation of a protein changes in the microenvironment. In the UV region, the peak at 200 nm indicates peptide absorption peak in protein, whereas a second weak absorption peak at about 278 nm is due to aromatic amino acids43, 44. Aggregation in the protein structure may lead to the decrease in absorbance values indicating the strong interaction. At 278 nm, the absorbance may be primarily due to the presence of feeble tyrosine residues, as tryptophan is absent in type I collagen and the absorbance of phenylalanine is negligible (S2). At lower concentrations, (C-AMS-1 and C-AMS-


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2) absorbance was observed to be slightly higher when compared to native, indicating the bonding within collagen, whereas the absorbance for C-AMS-3 and C-AMS-4 show a decrease with increasing concentration of AMS, suggesting likely of aggregation and resulting in masking of tyrosine residues. The decrease in absorbance will primarily lead to alteration in hydrogen bonding environment of collagen, indicative of changes in secondary structure of protein. 3.3. CD spectral studies The effect of AMS on secondary structure of collagen was determined using CD spectroscopy. Collagen evidences signals at 197 nm (minima) and 222 nm (maxima), exhibiting a typical Polyproline type II (PP II) conformation45. Figure 2 shows a decrease in molar ellipticity value at 222 nm for C-AMS-3 and C-AMS-4 indicating the interaction of ammonium IL with functional groups of collagen resulting in structural deformity. There was a negligible increase in molar ellipticity values for C-AMS-1 and C-AMS-2, when compared to native collagen. This could be owing to the least interaction of collagen with AMS, which results in increased molar ellipticity. This outcome clearly suggests that the triple helical structure of collagen is affected on interaction of AMS at higher concentration. The ratio of positive to negative peak (Rpn), a characteristic of the triple helical conformation of collagen, possess slight variation in values for C-AMS(1-4), as shown in figure 3a. The effect of ionic liquid on the thermal stability of collagen was studied at 222 nm by increasing the temperature from 25°C to 50°C and denaturation for native collagen was observed at 39°C. Albeit, there exhibited a slight decrease in thermal stability at 38°C for C-AMS-1 to C-AMS-4. It could be owing to the changes in the microenvironment resulting in alteration in hydration network of collagen. CD spectra for native and ammonium methane sulfonate treated collagen with variable temperature are shown in supplementary figure (S3).


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Figure 2. CD spectra of native and ammonium methane sulfonate (AMS) treated collagen illustrating the conformational changes [concentration of collagen-0.8mg/ml], (Molar Ellipticity values at 222 nm for native and ammonium methane sulfonate (AMS) treated collagen at different molar ratios in the inset), Collagen: Ammonium Methane Sulfonate, C-AMS-1 to CAMS-4 (1:0.05to 1:10%)

Figure 3.a) Rpn (ratio of positive to negative) values from the CD spectrum for native and ammonium methane sulfonate (AMS) treated collagen at 250C, (concentration of collagen-0.8 mg/ml), Collagen: Ammonium Methane Sulfonate, C-AMS-1 to C-AMS-4 (1:0.05 to 1:10%), b) Temperature dependent molar ellipticity (at 222 nm) for native and ammonium methane sulfonate (AMS) treated collagen from 250C to 500C, (concentration of collagen-0.8mg/ml), Collagen: Ammonium Methane Sulfonate, C-AMS-1 to C-AMS-4 (1:0.05 to 1:10%)


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3.4. FT-IR spectral studies The triple helical structure of collagen has its characteristic absorption bands at 3300 cm-1 for Amide A (N-H and O-H stretching), at 2948 cm-1 for amide B (C-H stretching), at 1640 cm-1 for amide I (C=O stretching), at 1560 cm-1 for amide II (N-H bending and C-H stretching), at 1240 cm-1 for amide III (carboxyl OH). Amide I and II bands are attributed to polyproline type II structure of collagen46. As seen from figure 4, amide A show shift in peaks for C-AMS-(1-4) comparable to native, whose signal is centered at 3279 cm-1. This might be due to the interaction of ions with collagen. Amide I band, which is due to the heterogeneity of its peptide carbonyl groups in a triple helix, discloses the C=O stretching for collagen-AMS composite. A polyproline peak for native collagen was found around 1638 cm-1 considering that the region is characteristic of imine carbonyls, which could be due to the hydroxyproline concentration in collagen. There was a shift in peak for C-AMS-3 and C-AMS-4 to 1635 cm-1, although there were minimal changes observed for C-AMS-1 and C-AMS-2. The change is probably due to the alteration in microenvironment of collagen when molecules come in contact with each other. For amide II, N-H bending and C-H stretching was witnessed at 1561 cm-1 for native collagen and there was no marked shift in peak seen for C-AMS-1, but there was a significant variation was observed for C-AMS-2, C-AMS-3 and C-AMS-4 around 1556-1550 cm-1 and this conformational change suggests the modifications in structure due to the alteration in hydrogen bonding environment of collagen. The amide III band for collagen was centered at 1241cm-1 and no changes were observed for C-AMS-1 and C-AMS-2. For higher concentrations C-AMS-3 and C-AMS-4, there was a red shift in peak around 1239-1229 cm-1, which could be due to the ions which possibly interact with the carboxyl group of proteins causing a shift in bond making between collagen and ionic liquid. The shift in amide I and amide II bonds suggest that the AMS


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may be detrimental to the stability of collagen, as it may result in alteration of hydrogen bonds, which is confirmed further using molecular simulation studies.

Figure 4. FT-IR spectra depicting the changes in the functional groups of native and ammonium methane sulfonate (AMS) treated collagen (concentration of collagen-0.8mg/ml), Collagen: Ammonium methane sulfonate, C-AMS-1 to C-AMS-4 (1:0.05% to 1:10%). 3.5. Dimensional and thermal stability of RTT collagen fibers Rat tail tendons (RTT) were teased from albino rats, due to its abundancy of type I collagen. It exhibits banding patterns disclosing the helicity of fibrils in aqueous medium47. Figure 5a connotes treatment of RTT with ammonium methane sulfonate (AMS), which shows the effect on RTT collagen fibers at increasing concentrations. Concentration dependent disruption and distortion were noticed in banding patterns after incubating it for an hour. It was steadily monitored for the succeeding 24 hours and no changes were sighted further. This could be due to the chaotropicity of anions and high ionic strength that alter the structure of water molecule, which leads to distortion in wave pattern. Thus, it results in agglomeration and destabilization of


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RTT collagen fibers. On a comparative note, it was observed that the phosphonium based ILs was found to be more chaotropic than AMS27. The thermal stability of collagen treated with ionic liquids was assessed using differential scanning calorimeter (Figure 5b). The denaturation temperature (Td) of RTT collagen fiber is 63˚C as reported in literature48. The denaturation temperature and enthalpy values for native RTT and RTT treated AMS have been reported in Table 1. The enthalpy values decrease with the increase in concentration of AMS. Also, a decreasing trend in denaturation temperature was observed with an increase in the concentration of ILs. The decrease in thermal stability indicates that it is likely that the ions alter the hydration network of collagen and there occurs a reorientation in the H-bonding network of collagen within the helices and its surrounding water molecules49. The decrease in thermal stability of ILs treated RTT fibers shows the effect of ILs on collagen, which was confirmed with the swelling of RTT fibers from optical micrographs.

Figure 5a. Optical micrographs for native RTT collagen fibers and RTT treated with ammonium methane sulfonate (AMS), [AMS-1 (0.05%), AMS-2 (0.5%), AMS-3 (5%) and AMS (10%)]


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Figure 5b. DSC thermograms for native RTT collagen fibers and RTT treated with ammonium methane sulfonate (AMS), [AMS-1 (0.05%), AMS-2 (0.5%), AMS-3 (5%) and AMS (10%)]

Table 1. Denaturation temperature (Td) and ∆H values for native RTT and RTT treated with ammonium methane sulfonate (AMS), [AMS-1 (0.05%), AMS-2 (0.5%), AMS-3 (5%) and AMS (10%)]

a

RTT in

Td (˚C)a

∆H (J/g)b

Water

63˚C

-16.27 J/g

AMS-1

61˚C

-17.53 J/g

AMS-2

59˚C

-17.04 J/g

AMS-3

55˚C

-14.32 J/g

AMS-4

48˚C

-11.76 J/g

Td Denaturation temperature

b

∆H Enthalpy


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3.6. Molecular modeling studies To substantiate the changes in structural behavior of collagen like peptides and to study the alterations on interaction of collagen with ammonium methane sulfonate (AMS) at varying concentrations, MD simulations were carried out for 100 ns using the AMBER99 force field. Visual inspection of the trajectories obtained from MD simulation show that the interaction of AMS and collagen is stable throughout the 100 ns. MD studies such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonding studies, radial and spatial distribution analysis were brought in to confirm the structural changes, which was observed at experimental levels. 3.6.1. Root mean square deviation (RMSD) and Root mean square fluctuation (RMSF) analysis The backbone RMSD was calculated with respect to initial conformation as a function of time. The variation of RMSD with time is plotted in Figure 6a. Slight deviations were observed in the models with reference to its initial structure of collagen-like peptides in the presence of varying concentration of AMS, albeit RMSD fluctuations were not observed for collagen. The increasing concentration of AMS did not resulted in significant changes in RMSD. However, RMSD for all the systems were observed up to 6-7 Å, it would be owing to the winding and unwinding of triple helical structure of collagen-like peptides throughout the simulation. Further, these RMSD values of all the models show that the systems are well-equilibrated50. Figure 6b explicates the RMSF of Cα residues in collagen-like peptides from the time averaged position. It illustrates the residual fluctuations which increase with an increase in concentration of AMS, whereas there were least fluctuations in RMSF values of collagen. These


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results suggest that there was a marked change in structural deviation with increasing concentration of AMS.

Figure 6a. RMSD for collagen-like peptides and ammonium methane sulfonate (AMS), CAMS-1 to C-AMS-4 (1:0.05% to 1:10%) derived by molecular dynamics simulations

Figure 6b. The calculated RMSF values per residue for collagen-like peptides and ammonium methane sulfonate (AMS), C-AMS-1 to C-AMS-4 (1:0.05% to 1:10%) 3.6.2. Hydrogen bonding analysis It is postulated that stabilization of the collagen triple helical helix is majorly gained through H-bonding. In order to characterize the hydrogen bonding interaction between collagen with AMS (cation and anion) models MD simulation was carried out51, 52. Figure 7a and 7b exhibits


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least hydrogen bonding at low concentration in the case of AMS (cation), and slight increase in hydrogen bonding was observed on increasing the concentration of AMS (cation). For AMS (anion), more number of hydrogen bonds was observed comparable to AMS (cation), and it has increased with increase in concentration. For C-AMS-3 and C-AMS-4 averaged 20 hydrogen bonds were observed, which interacted with the collagen-like peptides. From hydrogen bonding analysis, it was evident that there was increased interaction between SO3- anion and the functional groups of collagen. This suggests that increased reorientation of H-bonds resulted in loss of hydrophilic environment as the anion strongly bonds to the collagen, thus ripping apart its stability with the surrounding water milieu leading to deformation and destabilization of collagen. These results were in accord to experimental techniques, where significant alteration was observed on increasing the concentration of AMS.

Figure 7. Hydrogen bonds formed between collagen-like peptides and (a) cation and (b) anion of C-AMS-1 to C-AMS-4 (1:0.05 to 1:10%) 3.6.3. Radial distribution function (RDF) To appraise the distribution of ammonium methane sulfonate (AMS) cation and anion of varying concentrations and water molecules around collagen-like peptides, the radial distribution


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analysis was calculated. It was noticed from the figure 8a and 8b, at lower concentration of AMS (cation) the peak was found to be below 1.5 nm. A slight increase in the peak was witnessed with the increase in concentrations of AMS (cation). In the case of AMS (anion) the value of g (r) is high for C-AMS-1 and C-AMS-2, whereas for C-AMS-3 and C-AMS-4 the g (r) values accentuated and it slackened up to 0.7 nm. These outcomes entails that there was increased interaction between collagen-like peptides with that of anion, albeit there was negligible changes in the case of cation interaction with collagen. This is likely due to readily available lone pair of electrons present in SO3- anions that can bond with free hydrogen atoms present in glycine backbone as well as the other functional groups present in collagen. On the other hand, ammonium cation has only hydrogen atoms to interact with collagen. Certainly, O-H bonds of anion-collagen interact strongly compared to H-H bonds of cation-collagen. Hence, the increased interaction of anion with collagen causes the structural deformity.

Figure 8. Radial distribution function between collagen-like peptides and (a) cation (b) anion of C-AMS-1 to C-AMS-4 (1:0.05% to 1:10%) 3.6.4. Spatial distribution function (SDF) Spatial distribution analysis was performed to elucidate the interplay and distribution of cations, anions and water molecules around collagen-like peptides52. Figure 9a and 9b depict the distributions of ions around collagen with a production run of 100 ns simulations. Increased


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interaction of AMS anion with that of positively charged residues present in collagen was noted around collagen-like peptides. This was represented by different isovalues, which shows the distribution of cation and anion around collagen-like peptides. Higher concentration of AMS (anion) showed stronger interaction indicative of structural deformation, whereas the prevalence of AMS (cation) did not change accordingly. Higher concentrations of AMS (anion) showed favorable and strong interaction between SO3- anions and functional groups of collagen. In case of phosphonium based ILs cations as well as anions are responsible for the structural disruption27 albeit for AMS, anions are held responsible for alteration in the structure of collagen at higher concentration and it was in accord with other experimental techniques.

C-AMS-1

C-AMS-2

C-AMS-3

C-AMS-4

Figure 9a. Spatial distribution function between collagen-like peptides (light blue) and cation (blue) of C-AMS-1 to C-AMS-4 (1:0.05% to 1:10%)


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C-AMS-1

C-AMS-2

C-AMS-3

C-AMS-4

Figure 9b. Spatial distribution function between collagen-like peptides (light blue) and anion (gold) of C-AMS-1 to C-AMS-4 (1:0.05% to 1:10%) 4. CONCLUSION The current study investigates the role of ions in collagen-AMS interaction. It is shown that the anion is responsible for the structural disorientation of collagen at higher concentrations. CD, FT-IR, UV-Vis, fluorescence and viscosity studies (in solution) show aggregation at higher levels, whereas at lower concentration there were tenuous changes in the secondary structure of collagen. At higher concentration of AMS, decreased thermal stability and the increased dimensions of RTT collagen fibers indicates structural disorientation of collagen. Overall, the stability of collagen has decreased in the presence of ammonium methane sulfonate. The main reason behind the observed structural deformity of collagen is that the SO3anion interacts preferentially with that of hydrogen atoms of glycine and other functional groups. They reorient the collagen-water bonds to form new bonds with that of anion, eventually leading to structural deformation and weaker interactions were observed between NH4+cation and collagen functional groups.


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The experimental results are in accordance with molecular dynamics simulation studies suggesting that due to strong interaction of anions with collagen resulted in structural deformity, whereas at lower concentration there is least interaction between collagen and SO3-anion of IL. Thus, the preferential role of SO3- anion influences the secondary structure of collagen bringing about its deformation. ACKNOWLEDGEMENT We, the authors thank CSIR – Research Initiatives for Waterless Tanning (RIWTCSC0202), a 12th five year plan project for the financial assistance. We extend our gratitude to Dr. V. Subramanian for his immense support and aid towards our research. We would like to acknowledge C-MMACS for providing super-computing facility to accomplish this work. CSIRCLRI communication code: 1198. Supporting Information Available: This study proves the role of ions of diethyl methyl ammonium methane sulfonate for destabilization of collagen through experimental and molecular dynamics simulation studies. This material is available free of charge via the Internet at http://pubs.acs.org.


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TABLE OF CONTENTS


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Role of anions in destabilization of collagen 263x121mm (96 x 96 DPI)


Role of Preferential Ions of Ammonium Ionic Liquid in Destabilization

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