Supporting Information Section:
pH Induced Structural Change of a Multi-Tryptophan Protein MPT63 with
Immunoglobulin Like Fold: Identification of Perturbed Tryptophan Residue/Residues
Manini Mukherjeea, Ranendu Ghosh b,1, Krishnananda Chattopadhyayb*,Sanjib Ghosh a*
a
Department of Chemistry, Presidency University, Kolkata 700 073, India.
b
Structural Biology & Bio-Informatics Division,CSIR-Indian Institute of Chemical Biology,
Kolkata-700 032, India.
1
Present Address: Department of Chemical and Biomolecular Engineering, University of
Delaware, Newark, DE 19716, USA.
*Author
for correspondence:
Tel.:+91-9836940620 (S.G.), +9133-2241 3893 (S.G.); Fax:+9133-2241 3893 (S.G.);
E-mail: sanjibg@cal2.vsnl.net.in (S.G.); pchemsg@gmail.com (S.G.);
E-mail: krish@iicb.res.in(K.C.).
Figure S1: ANS fluorescence emission spectra of folded (0 M guanidine hydrochloride) and
unfolded (4.0 M guanidine hydrochloride) MPT63 in 20 mM sodium phosphate, pH 7.5.
Protein concentration was 1µM.
Figure S2: Phosphorescence spectra of 5 µM WT MPT63 in aqueous buffer (pH 1.5)+ 40%
EG matrix at 77 K with different excitation wavelength. Excitation bandpass = 10 nm and
emission bandpass =1 nm in each case except with λexc=305nm, where the emission band
pass=2.5 nm.
Figure S3: Phosphorescence spectra of 5 µM WT MPT63 with acrylamide in aqueous
buffer (pH 1.5)+ 40% EG matrix at 77 K with different excitation wavelength. Excitation
bandpass = 10 nm and emission bandpass =1 nm in each case except with λ exc=305nm,
where the emission band pass=2.5 nm. (A) for pH 7.5, (B) for pH 1.5.
Heat capacity (kcal.K-1.mol-1)
10
5
0
300
320
340
360
Temperature (K)
Figure S4: DSC thermogram of MPT 63 in 20 mM sodium phosphate buffer pH 7.5. The
filled circle symbols are experimental heat capacity values and the solid line is the best fit of
the two – state model resulting Tm value of 338 K (650C). Protein concentration was 1
mg/mL.
Figure S5: Continuous C(s) distribution of heat treated (at 800C for 20 minutes) and native
(at 250C) MPT63 at different pH conditions. Positions of monomer and possible aggregate
are also indicated. The residual distribution of the fit obtained by SEDFIT analysis is shown
in the inset.
Figure S6: Effect of pH on MPT63 amyloid fibril formation and the associated
change in thioflavin T (ThT) fluorescence. MPT63 in different solvent system (pH
1.5 and 7.5) incubated at 800C for different time periods (zero to 90 minutes) and
then mixed with ThT solution (pH 7.5, 250C). ThT solution with the protein was
subjected to spectroscopic analysis. Heat treated MPT63 at pH 1.5 shows an
increase in ThT fluorescence. Inset: Thioflavin T (ThT) fluorescence spectra when
mixed with buffer and heat treated MPT63 (heated at 800C for 20 minutes) at pH 1.5
at room temperature (250C) showing increase in fluorescence intensity as well as the
shift in the emission maxima. λexc=440nm.
2.6. Thioflavin T Assay:
Thioflavin T (Th T) stock solution was prepared in 20 mM sodium phosphate buffer, pH 7.5.
The solution was filtered and the concentration was measured using an excitation coefficient of
26,620 M-1 at 416 nm. (Poduslo et al., 2010) Protein solution containing 40 µM of MPT63 was
incubated at 800C in respective buffer solutions. Aliquots were taken at different time of heating
and diluted 40 times (final protein concentration 1 µM) with Thioflavin T solution (final Th T
concentration 5 µM) and incubated for 5 minutes before fluorescence measurements.
Fluorescence measurements were performed at 250C by exciting the sample at 440 nm and
monitoring the fluorescence emission at 482 nm.
2.7. ANS Fluorescence Measurements:
1–aniline–8–naphthalene sulfonic acid (ANS) stock solution was prepared in methanol. The
solution was filtered and the concentration was measured using excitation coefficient of 5,000 M1
at 350 nm. (Qadeer et al., 2012) Final ANS and protein concentration used in the experiments
were 20 µM and 1 µM respectively. Prior to the experiments ANS was added to the samples and
the solutions were incubated for another 5 minutes before the measurements of ANS
fluorescence. Fluorescence spectra of ANS containing samples were recorded using an excitation
wavelength of 370 nm and emission spectra were recorded between 450 nm and 550 nm. For pH
dependence experiments, ANS fluorescence intensities at 470 nm were plotted at different pH
conditions.
2.9. Analytical Ultracentrifugation:
Sedimentation velocity experiments were performed using a Beckman Optima XL-I analytical
ultracentrifuge (Beckman Coulter, Inc. U.S.A.) at 20 °C using an An-50 Ti rotor. Protein
solutions (concentration of 40 µM) were incubated at 800C for 20 minutes at pH 7.5 and pH
1.5.To avoid any suspended particle, protein samples as well as the reference buffer solutions
were centrifuged at 5000 r.p.m for 4 minutes at room temperature in a table – top centrifuge
(Tarson, India) immediately before the experiments. Aggregated protein samples of 400 μL were
loaded against 420 μL of equivalent buffer as a reference into 12 mm charcoal-filled Epon
centerpieces and centrifuged at 42000 r.p.m for 10 hours. Sedimentation velocity data were
analyzed using SEDFIT using the continuous c(s) distribution model. Collections of the first 60
scans were used for the analysis with s – values ranging between 2 and 40. The positions of the
cell bottom and meniscus were determined manually and then refined in the final fit.