Electronic Supplementary Material (ESI) for Green Chemistry. This journal is The Royal Society of Chemistry 2017 Electronic Supplementary Information Long-term protein packaging in cholinium-based ionic liquids: Improved catalytic activity and enhanced stability of cytochrome C against multiple stresses Meena Bisht, a,b Dibyendu Mondal, b,c Matheus M. Pereira, b Mara G. Freire, b P. Venkatesu, a * and J. A. P. Coutinho b * a Department of Chemistry, University of Delhi, Delhi 110 007, India, E-mail: venkatesup@hotmail.com, pvenkatesu@chemistry.du.ac.in; Fax: +91-11-2766 6605; Tel: +91-11-27666646-142 b Chemistry Department, CICECO-Aveiro Institute of Materials, University of Aveiro, 3810-193, Aveiro, Portugal. E-mail: jcoutinho@ua.pt; quijorge@ua.pt; Fax: +351 234 370 084; Tel: +351 234 401 507 c Center for Nano and Material Science, Jain University, Bangalore, Karnataka, India-562112 *Corresponding authors
78 72 66 [Ch][Dhp]+ABTS [Ch][Dhc]+ABTS [Ch][Suc]+ABTS [Ch][Glu]+ABTS [Ch][Mal]+ABTS [Ch][Tar]+ABTS Absorbance (a.u.) 60 54 48 42 36 30 0 20 40 60 80 100 120 Time (sec) Fig. S1 Background interference of aqueous ILs (IL and water in a ratio by weight) and ABTS.
Absorbance 0.35 0.30 5 0 0.15 0.10 (a) [Ch][Dhp] Absorbance 0.35 0.30 5 0 0.15 0.10 (b) [Ch][Dhc] 1:1 Absorbance 0.35 0.30 5 0 0.15 0.10 (c) [Ch][Suc] 1:1 Absorbance 5 0 0 0.35 0.30 5 0 0.15 0.10 300 400 500 600 700 (d) [Ch][Glu] Absorbance 5 0 0.35 0.30 5 0 0.15 0.10 300 400 500 600 700 (e) [Ch][Tar] 1:1 Absorbance 5 0 0.35 0.30 5 0 0.15 0.10 300 400 500 600 700 (f) [Ch][Mal] 1:1 5 5 5 0 300 400 500 600 700 0 300 400 500 600 700 0 300 400 500 600 700 Fig. S2 UV-Vis spectra of Cyt C in aqueous solutions of bio-ils at different concentration. Absorbance 0.3 0.1 [Ch][Mal] [Ch][Dhp] [Ch][Dhc] [Ch][Suc] [Ch][Tar] [Ch][Glu] 300 400 500 600 700 Fig. S3 UV-Vis spectra of aqueous solutions of ILs at ratio by weight (IL:Water) (no enzyme added).
1.0 - -1.0 - -1.0 (a) [Ch][Dhp] 1.0 (b) [Ch][Dhc] (c) [Ch][Suc] 1:1 1:1 - -1.0 - -1.2-1.0-1.4 390 400 410 420 430 440 450 390 400 410 420 430 440 450 390 400 410 420 430 440 450 1.0 (d) [Ch][Glu] 1.0 (e) [Ch][Tar] (f) [Ch][Mal] 1:1 1:1 1:1 390 400 410 420 430 440 450 - -1.0 390 400 410 420 430 440 450 - -1.0 390 400 410 420 430 440 450 Fig. S4 CD spectra of Cyt C in aqueous solutions of cholinium-based ILs. (a) [Ch][Dhp] (b) [Ch][Dhc] (c) [Ch][Suc] 1:1 1.2 1.0 260 270 280 290 300 260 270 280 290 300 260 270 280 290 300 (d) [Ch][Glu] (e) [Ch][Tar] 1:1 (f) [Ch][Mal] 260 270 280 290 300 1.0 260 270 280 290 300 260 270 280 290 300 Fig. S5 Mid near UV CD spectra of Cyt C in presence of buffer and ILs.
Fig. S6 FTIR spectra of Cyt C in the amide I and amide II regions at 25 o C: (a) Cyt C in aqueous solutions containing weight ratio of ILs (b) Cyt C in aqueous solutions containing of [Ch][Dhp] and 1:1 of the remaining ILs. 10 8 6 4 2 0 Cyt C without GuHCl Cyt C in 4M GuHCl [Ch][Dhp] [Ch][Dhc] [Ch][Glu] [Ch][Tar] [Ch][Mal] [Ch][Suc] -2-4 -6 360 380 400 420 440 Fig. S7 CD spectra of Cyt C in presence of 4M GuHCl, with and without ILs added.
Table S1 T 50 of enzyme in presence of different ILs. Samples T 50 ( o C) 101 [Ch][Dhp] 117 [Ch][Dhc] 110 [Ch][Suc] 114 [Ch][Glu] 115 [Ch][Tar] 114 [Ch][Mal] 113 ALA15 GLU21 Figure S8 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Ch] +.
HIS18 ASN52 TRP59 Figure S9 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Dhc] -. TRY67 TRP59 ARG38 HIS18 ASN52 Figure S10 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Tar] -.
TRY67 THR78 Figure S11 CytC docking pose with the lowest absolute value of affinity (kcal/mol) with [Mal] -. TRP59 ASN52 TYR67 Figure S12 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Suc] -.
LYS27 TYR97 LYS7 Figure S13 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Adi] -. TYR67 Figure S14 Cyt C docking pose with the lowest absolute value of affinity (kcal/mol) with [Prop] -.
ALA21 Electrostatic GLU21 Figure S15 Molecular interactions representation between [Ch] + and the amino acids residues of ASN52 TRP59 HIS18 Figure S16 Molecular interactions representation of [Dhc] - and the amino acids residues of
TYR67 ARG38 ASN52 HIS18 TRP59 Electrostatic Figure S17 Molecular interactions representation of [Tar] - and the amino acids residues of THR78 TYR67 Figure S18 Molecular interactions representation of [Mal] - and the amino acids residues of
ASN52 TRP59 TYR67 Figure S19 Molecular interactions representation of [Suc] - and the amino acids residues of LYS7 LYS27 TYR97 Electrostatic Figure S20 Molecular interactions representation of [Adi] - and the amino acids residues of
TYR67 Figure S21 Molecular interactions represenation of [Prop] - and the amino acids residues of GLY45 GLY41 PHE46 LYS53 Electrostatic LYS53 Figure S22 Molecular interactions representation of [Dhp] - and the amino acids residues of
HIS18 Electrostatic Figure S23 Molecular interactions representation of [But] - and the amino acids residues of THR78 TRP59 TYR67 ASN52 Figure S24 Molecular interactions representation of [Glu] - and the amino acids residues of
Table S2. Docking affinity energy and interacting amino acids predicted by AutoDock vinna for Cyt C-ILs. IL ion Affinity / (kcal/mol) Interacting amino acids Type of interaction From To Distance / (Å) Glutamic acid21 Electostatic [Ch] + Glutamic acid - - N [Ch] + -2.6 O 4.82 Alanine15 [Ch] + - H Alanine - O 1.92 Histidine18 Histidine - C [Dhc] - - O 3.56 [Dhc] - -4.4 Asparagine52 [Dhc] - - H Asparagine- O 2.20 Tryptophan59 Tryptophan - N [Dhc] - - O 3.15 Glycine41 [Dhp] - - H Glycine - H 2.75 Glycine45 Glycine - N [Dhp] - - O 3.08 [Dhp] - -2.7 Phenylalanine46 [Dhp] - Phenylalanine - - H O 2.21 Lysine53 Lysine -C [Dhp] - - O 3.55 Electostatic Lysine -N [Dhp] - - O 3.96 Arginine38 Electostatic Arginine - H [Tar] - - O 5.05 Histidine18 Histidine - C [Tar] - - O 3.56 [Tar] - -4.1 Asparagine52 [Tar] - - H Asparagine - O 2.05 Tryptophan59 Tryptophan - N [Tar] - - O 3.18 Tyrosine67 Tyrosine -OH [Tar] - - O 2.98 [Tar] - - H Tyrosine -OH 2.74 Asparagine52 Asparagine- N [Suc] - - O 3.08 [Suc] - -3.9 Tryptophan59 Tryptophan - N [Suc] - - O 2.98 Tyrosine67 Tyrosine -OH [Suc] - - O 2.94 [Mal] - 3.3 Tyrosine67 Tyrosine -OH [Suc] - - O 2.95
[Suc] - - O 2.96 Threonine78 Threonine -O [Suc] - - O 3.22 IL ion Affinity / (kcal/mol) Interacting amino acids Type of interaction From To Distance / (Å) [Glu] - -4.2 [Adi] - -3.4 Asparagine52 Asparagine - N [Glu] - - O 3.03 Tryptophan59 Tryptophan - N [Glu] - - O 3.03 Tyrosine67 Tyrosine -OH [Glu] - - O 2.91 Threonine78 Threonine -O [Glu] - - O 3.14 [Glu] - - O 3.11 Electostatic [Adi] - - O 3.13 Lysine27 Lysine -N [Adi] - - O 5.21 Tyrosine97 Tyrosine - OH [Adi] - - O 2.90 [But] - -3.2 Histidine18 Electostatic [But] - - O Histidine - C 3.36 [Prop] - -2.8 Tyrosine67 Tyrosine - OH [Prop] - - O 3.13