FIGURE 4. Structure and stability of MF1.

(A) Gel Filtration elution profile of MF1. The elution was monitored by measuring the absorbance at 280 nm as a function of elution volume. MF1 was run on a calibrated ToyoPearl HW-55F (Tosoh Biosciences LLC) packed column with a diameter of 2.2 cm and height of 78 cm in 25 mM HEPES (pH 7.4), 50 mM NaCl, and 0.1 mM EGTA. A total of 1 mL of 0.5 – 1 mg/mL MF1 was loaded on the column with a flow rate of 1.5 mL/minute and 4 mL fractions were collected. The Stokes radius of MF1 was determined to be 2.74±0.34 nm (n=3). (B) MF1 Far-UV CD spectrum. CD spectra were recorded in the far- (190–250 nm) UV region using a JASCO J-815 spectrophotometer with an automated temperature controller and a temperature-jacketed spectral cell. A path length of 1 mm was used with spectra recorded at 1 nm intervals for 10 uM MF1 in phosphate buffer pH 7.0 at 25°C. Baseline scans were obtained using the same acquisition parameters with buffer alone; which were subtracted from the respective CD data scans of MF1. The raw CD signal θ_λ (millidegrees of ellipticity) was converted to mean residue molar ellipticity.with spectra analyzed using CDPro Analysis Software ³⁰. Experimental CD spectrum (solid line) and best fit spectrum from CDPro Analysis (dotted line). (C) Thermal denaturation curve of MF1 measured by changes in mean residue ellipticity. The temperature was increased from 10 to 70°C with a step size of 1°C with the CD in millidegrees of ellipticity measured at each temperature after incubation for 2 min.