Supporting Information
Denaturant-specific effects on the structural energetics of a protein denatured ensemble
Mahdi Muhammad Moosa†, ‡, Asha Z. Goodman†, Josephine C. Ferreon†,‡, Chul Won Lee§, Allan Chris M. Ferreon†,‡,*, Ashok A. Deniz†,*
† Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
§Department of Chemistry, Chonnam National University, Gwangju 500-757, Republic of Korea.
‡Present Address: Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
* Email:
Materials and Methods
Chemicals and reagents: Experiments were carried out in αβγ buffer (0.2 M sodium chloride (NaCl), 10 mM sodium acetate (CH3COONa), 10 mM monosodium phosphate (NaH2PO4), 10 mM glycine (NH2CH2COOH)) at pH 5.0 ± 0.05 unless otherwise stated. All chemicals used were either analytical or reagent grade, and were spectroscopically silent to ensure high signal-to-noise ratio. The concentrations of GdnHCl and urea stock solutions (in αβγ buffer) were calculated from refractive indices of respective solutions measured using Spectronic Instruments refractometer as described previously (Ferreon and Bolen 2004). Urea stock solutions were stored at -80°C and thawed prior to experiments to ensure minimal degradation (Hagel et al. 1971).
Protein expression, purification and labeling: Wild type GB1 was subcloned from GB1-p53 fusion construct (Ferreon et al. 2009) into a T7 expression vector derived from pET21a(Ferreon et al. 2009). Site-directed mutagenesis was carried out to introduce Cys at positions 22 and 56 for dye-labeling using maleimide chemistry. All proteins were expressed in Escherichia coli BL21 (DE3). Cells were grown at 37°Cuntil the absorbance reached 0.8-1.0 followed by overnight induction at 16°C. The pellet was lysed in 20 mM Tris, 8 M urea at pH 8.0, mixed with 4-fold excess 0.1% trifluroacetic acid (TFA), and centrifuged at 20,000 g for 30 minutes to remove precipitate. Protein purification was performed by reverse-phase HPLC. Protein concentrations were determined via the Edelhoch method (Edelhoch 1967) as described previously by Ferreon and Bolen (Ferreon and Bolen 2004).
Double-Cys mutant GB1 was labeled in 50 mM Tris, 6 M GdnHCl at pH 7.2 using 3-5 fold molar excess of maleimide dye with 1:3 concentration ratio of Alexa Fluor 488:Alexa Fluor 594 dyes. Labeling reactions were run for approximately 2 hours at room temperature. The dye-labeled protein was purified using analytical reverse-phase HPLC with C18 column. The purified product was checked for correct mass and incorporation of the Alexa dyes by MALDI-TOF mass spectrometry.
Protein unfolding and data analysis: Protein unfolding measurements were carried out by mixing appropriate proportions of denaturant, GB1 and αβγ buffer solutions. Protein secondary structure perturbations by denaturants were monitored using an Aviv 62DS/202SF CD spectrometer equipped with a Peltier automated temperature control unit at 25°C using 50 µM GB1. Wavelength-specific mean residue ellipticities (MRE) were calculated from the measured signal after correcting for buffer background as described previously (Ferreon and Deniz 2007). Single-wavelength readings were taken with 15 s averaging time, whereas wavelength scans were performed with an averaging time of 5 s. Details of the circular dichroism measurements are similar to the method described by Ferreon and Deniz previously (Ferreon and Deniz 2007). Data from the denaturation experiments were plotted using OriginPro 8.0 (OriginLab, Northampton, MA) and analyzed using two-state linear extrapolation method (LEM) and non-linear least-squares (NLS) fitting (Ferreon and Deniz 2012; Santoro and Bolen 1988). Some of the parameters were fixed as discussed in the main text.
FRET measurements were carried out at room temperature with dual-labeled sub-nM GB1 in αβγ buffer with respective chemical denaturants using the single-molecule instrumentation described previously (Ferreon et al. 2009; Gambin et al. 2009). Donor and accepter fluorescence from dual-labeled GB1 were recorded using simultaneous two-channel data collection system with a binning time of 500 µs. The leakage of donor emission into the acceptor channel (9%) and acceptor emission due to direct excitation (5%) were taken into account (Ferreon et al. 2012). Lower and upper thresholds of 50 and 200 were used respectively, where threshold is the sum of signals from donor and acceptor channels collected within the binning time. FRET efficiencies (EFRET) were calculated from the corrected donor (ID) and acceptor (IA) fluorescence intensities as
The value of γ was approximated to unity based on our previous measurements for the Alexa 488/594 dye-pair in a protein system (Ferreon et al. 2009). EFRET histograms were generated and the distributions were fitted to Gaussian functions in MATLAB 7.5 (The MathWorks Inc., Natick, MA) using relationships described previously (Brustad et al. 2008; Ferreon et al. 2009). EFRET histograms were plotted using OriginPro 8.0 and the denaturation data were analyzed as described above.
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