SUPPORTING INFORMATION Water Dynamics in Cytoplasm-like Crowded Environment Correlates with the Conformational Transition of the Macromolecular Crowder Pramod Kumar Verma,, Achintya Kundu,, Jeong-Hyon Ha, and Minhaeng Cho *,, Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul 84, Republic of Korea Department of Chemistry, Korea University, Seoul 84, Republic of Korea. Space-Time Resolved Molecular Imaging Research Team, Korea Basic Science Institute, Seoul 36-75, Korea Corresponding Author *Email: mcho@korea.ac.kr In this supporting information (SI), we present () Tabulated details of wt% of PEGDME and corresponding number of water molecules per PEGDME (Table S) () Experimental methods of the fitting procedure of mid-ir PP (pump-probe) data and heating contribution removal procedure (3) The fitting of the FTIR data of the OD stretch of HDO at different wt% of crowder (Figure S) (4) Representative figures depicting uncorrected femtosecond IR PP spectra of OD of HDO in neat water and 8 wt% crowder and the decomposed water (HDO) IR PP spectrum and thermal component (Figure S) (5) Representative figures depicting femtosecond IR PP spectra of HN 3 in neat water and 8 wt% crowder solution (Figure S3) (6) Representative figures depicting uncorrected and corrected femtosecond mid-ir PP spectra of PEGME-N 3 in dichloromethane, 3 and 8 wt% crowder solutions (Figure S4) (7) Anisotropy decays of the OD band of HDO in neat water and various wt% of crowder solutions (Figure S5) (8) Anisotropy decays of the azido stretch band of HN 3 in neat water and various wt% of crowder solutions (Figure S6) (9) Anisotropy decays of the azido stretch band of PEGME-N 3 in dichloromethane and various wt% of crowder solutions (Figure S7) S
Table S. Number of water molecules per PEGDME depending upon the wt% of PEGDME. [PEGDME] wt% [water] wt% water per PEG water per ether [PEGDME] (molal effective) - - - 9 349. 5.9.6 3 7 9.5 4..6 5 5 38.8.8.43 7 3 6.6.8 3.34 8 9.7 5.7 85 5 6.8.3 8. A number average molecular weight of corresponds to approx. (n) ethylene oxide (labelled as e ) monomer units. The molal effective term is used because the effective molecular weight is generally smaller than number average weight of PEGDME which is. The effective molecular weight of 699 is taken from reference. S
Experimental Methods The first fs of the IR PP signal lies within the cross-correlation of the pump and probe pulses. In addition, fast librational motions also contribute to the signal at delay times smaller than.5 ps., hence the fitting was performed from ps. The vibrational relaxation dynamics of HDO in water often includes an in-growing heating contribution. The heating contribution originates from the temperature increase in the sample arising from the absorption and subsequent dissipation of the excited vibrational energy. As the energy of the excited vibration is transformed to heat, this signal grows in with delay and appears as residual signals at long delay times. Since an increase in temperature shifts the OD band to the blue, the long-time absorption consists of an induced absorption on the blue side of the spectrum and a bleach on the red side. This heating contribution is eliminated by fitting the isotropic data to a model proposed by Bakker et al. Mid-IR PP measurement using hydrazoic acid (HN 3 ) as vibrational probe shows negligible in-growing heating contribution and hence does not require any correction for the heating signal. The large anharmonicity of the NNN asymmetric stretch band compared to its linewidth gives rise to two well separated positive and negative peaks. During the fitting procedure, all the kinetic traces (at all the wavenumbers) were fitted globally to an exponential function with a shared time component but different amplitude. Thus obtained vibrational lifetime is plotted in the inset of Figure H and I (main manuscript). Mid-IR PP measurement using PEGME-N 3 as vibrational probe shows a finite residual signal originating from pump-induced heating effect (Figure S3) only in, 3 and 5 wt% crowder solutions. In the case of dichloromethane and water-poor solutions of crowder, we do not observe any appreciable residual signal originating from pump-induced heating effect. This absence of heating effect confirms that the heating contribution to the IR PP signal at long delay times originates from the dissipation of the vibrationally excited water combination band not from that of azido stretch mode. For low crowder solutions, the heating contribution to the isotropic IR PP signal was eliminated by assuming that it is a rising function, exp. Since the mid-ir PP signal at long delay times has mostly the heating contribution, the corrected mid-ir PP signal is obtained by subtracting the scaled signal at long times from the raw signal as Δ, =,,. Since the (local heating time) is mainly due to the relaxation of the OH stretching mode of water solvent molecules, we can apply the literature value of.84 ps. 3 The mid-ir PP signals before and after subtraction with this time constant are presented in Figure S4, which shows that the heating contribution can be successfully removed. Then, all the kinetic traces (at all the wavenumbers) were fitted globally (with OriginPro software) to an exponential function with two shared time components but different amplitudes. The fit results are shown in Figure F and G in the main text. S3
Fitting analyses of the FTIR data of the OD band of HDO at different wt% of crowder using a Gaussian function as available in OriginPro software.....8.6 HDO (5%) Gaussian fit.8.6 % PEGDME Gaussian fit 35 4 45 5 55 6 65 35 4 45 5 55 6 65 35 4 45 5 55 6 65 Wavenumber / cm - Wavenumber / cm - Wavenumber / cm -. 5% PEGDME OD-ether OD-water.8 Cumulative fit.6. 7% PEGDME OD-ether OD-water.8 Cumulative fit.6.8.6 3% PEGDME OD-ether OD-water Cumulative fit 35 4 45 5 55 6 65 35 4 45 5 55 6 65 35 4 45 5 55 6 65 Wavenumber / cm - Wavenumber / cm - Wavenumber / cm -. 85% PEGDME OD-ether.8 OD-water Cumulative fit.6 35 4 45 5 55 6 65 Wavenumber / cm -. 8% PEGDME OD-ether.8 OD-water Cumulative fit.6 Figure S. FTIR absorption spectra of the OD stretch of 5 % HDO (v/v) in different wt% of crowder. The symbols are the raw data and the solid lines are the fitted curves using Gaussian functions. From 3 wt% onwards, the fitting requires two different Gaussian peaks, corresponding to OD-water (fixed at 59 cm - ) and OD-ether, respectively. S4
Femtosecond mid-ir PP spectra of HDO in neat water and 85 wt% crowder solution along with the representative kinetic traces after removing heat contribution. Isotropic signal ( S) / mod 6 4 HDO (neat water) - 4 45 5 55 6 65 - (A) ps ps ps 4 ps 6 ps 3 ps HDO (85% PEGDME) ps ps ps 4 ps 6 ps 3 ps (C) 4 45 5 55 6 65 Wavenumber / cm - 5 4 3 5 5 5 3 (B) HDO (neat water) kinetic trace (raw data) kinetic trace (thermal subtracted) ingrowing thermal trace HDO (85% PEGDME) (D) kinetic trace (raw data) kinetic trace (OD-w; thermal subtracted) kinetic trace (OD-e; thermal subtracted) ingrowing thermal trace 3 4 5 6 7 8 9 Time delay / ps Figure S. Experimental isotropic mid-ir PP signal of HDO in neat water (A) and 85 wt% crowder solution (C). Kinetic traces at 59 (neat water) and 564 (85 wt%) cm - from the experimentally measured IR PP signals are shown in symbol in (B) and (D), respectively. The kinetic trace after removing the heating contributions and the ingrowing heating signals are shown in solid lines ((B) and (D)). S5
Representative femtosecond mid-ir PP spectra of HN 3 in neat water and 8 wt% crowder solution Isotropic signal ( S) of HN 3 / mod 8 6 4 - -4-6 8 4 6 8 Wavenumber / cm - 4 -.3 ps ps.6 ps.8 ps ps ps 3 ps 4 ps ps 5 ps HN 3 (neat water) HN 3 (8% PEGDME) -4 8 4 6 8 Wavenumber / cm - Figure S3. Experimental isotropic mid-ir PP signal of HN 3 in neat water (upper) and 8 wt% crowder solution (lower)..3 ps ps.6 ps.8 ps ps ps 3 ps 4 ps ps 5 ps S6
Representative femtosecond mid-ir PP spectra of PEGME-N 3 in dichloromethane, 3 wt% (with and without heat subtraction) and 8 wt% crowder solution Isotropic signal ( S) / mod - - - -.3 ps.5 ps.7 ps ps ps 4 ps 8 ps 6 ps 5 ps 3% PEGME-N 3 (raw data) 3% PEGME-N 3 (heat corrected data) 8% PEGME-N 3 (heat correction not needed) (dichloromethane) PEGME-N 3 A (heat correction not needed) 4 6 8 4 Wavenumber / cm - B C D Figure S4. Isotropic mid-ir PP signal of PEGME-N 3 in dichloromethane (A), 3 wt% with (C) or without heat subtraction (B) and 8 wt% crowder solution (D). Heat corrections were not required in case of dichloromethane and 8 wt% of crowder because of negligible residual thermal contribution at long delay times. S7
Anisotropy decays of the HDO (at center wavenumber of the OD band) for aqueous solutions of crowder at different wt% and in neat water. Oreintataion relaxation of water, r(t) HDO (5%) % PEGDME 3% PEGDME 5% PEGDME 7% PEGDME 8% PEGDME 85% PEGDME 3 4 5 6 7 8 9 Time delay / ps Figure S5. Orientational relaxation decays of HDO in neat water and different wt% of crowder. The solid lines represent fit to a single exponential function (τ r ) with an offset (R slow ), = / +. The offset represents much slower decay component (> ps). S8
Anisotropy decays of the HN 3 (at center wavenumber of the azido band) for aqueous solutions of crowder at different wt% and in neat water. Oreintataion relaxataion of HN 3.3..3..3..3..3..3. HN 3 (neat water) % PEGDME 3% PEGDME 5% PEGDME 7% PEGDME 8% PEGDME 3 4 5 6 7 8 9 Time delay / ps Figure S6. Orientational relaxation decays of HN 3 in neat water and different wt% of crowder. The solid lines represent fit to a single exponential function (τ r ) with an offset,, = / +. The offset represent much slower decay component (> ps). S9
Anisotropy decays of the PEGME-N 3 (at center wavenumber of the azido band) for aqueous solutions of crowder at different wt% and in neat water. PEGME-N 3 (dichloromethane) % PEGME-N 3 r(t), Orientational relaxation of PEGME-N 3 3% PEGME-N 3 5% PEGME-N 3 7% PEGME-N 3 8% PEGME-N 3 3 4 5 6 7 8 9 Time dealy / ps Figure S7. Orientational relaxation decays of PEGME-N 3 in dichloromethane and different wt% of crowder. The solid lines represent fit to a single exponential function (τ r ) with an offset (R slow ), = / +. The offset represent much slower decay component (> ps). S
Supporting References () Reid, C.; Rand, R. P. Biophys. J. 997, 7,. () Post, S. T. v. d.; Bakker, H. J. Phys. Chem. Chem. Phys., 4, 68. (3) Chieffo, L.; Shattuck, J.; Amsden, J. J.; Erramilli, S.; Ziegler, L. D. Chem. Phys. 7, 34, 7. S