Figure 3: (A) Secondary structure assessment for Tf C and different mutants. (B) Size exclusion chromatography of binding altered variants of Tf C. (C) SDS-PAGE analysis for partitioning of Tf C after 40 h incubation with PET film. Lane 1: G62A bound to PET, Lane 2: G62A in solution, Lane 3: G62A/L90F bound to PET, Lane 4: G62A/L90F in solution, Lane 5: Protein marker, Lane 6: G62A/F209I bound to PET, Lane 7: G62A/F209I in solution, Lane 8: G62A/F249R bound to PET, Lane 9: G62A/F249R in solution. (D) Densitometric quantification of proteins in lanes 1, 3, 6, 8.
3.4 Thermal and chemical (kinetic and thermodynamic) stability of Tf C
Thermal unfolding of Tf C was examined using CD and DSC. Changes in Tf C’s secondary structure were measured during heating (20 °C to 90 °C), at 1 °C/min, and 0.2 °C/min, respectively (Figure 4A). At the faster unfolding rate of 1 °C/min, ~70% denaturation was observed at 90°C, whereas at the slower unfolding rate of 0.2 °C/min, ~100 % unfolding was observed, with a Tm of ~85 °C. This indicates thatTf C is kinetically (thermally) stabilized, displaying a resistance to unfolding that leads to unfolding equilibrium being achieved only through slower heating. To further Tf C’s unfolding kinetics, changes in CD were monitored over 120 min incubations, at temperatures flanking the Tm (Figure 4B). The Arrhenius plot obtained from rates of unfolding in the range of 68-87.5 °C (Supporting Information Figure S6A) yielded an activation energy, Ea, of 217.557 kJ/mol (i.e. 52 kcal/mol). Thermodynamic parameters determined through construction of an Eyring plot (Supporting Information Figure S6B) were: (1) ΔH = 214.643 kJ/mol; (2) ΔS = 314.4346 J/mol*K; and (3) ΔG = 120.96 kJ/mol (i.e. 28.9 kcal/mol). DSC measurements (Figure 4C) corroborated the Tm determined by CD, while establishing two proximal unfolding transitions at ~78.5 °C and ~86.7 °C, supporting the high thermal, kinetic, and thermodynamic stability of Tf C, and causing Tf C to be suited to PET degradation at temperatures close to PET’s glass transition temperature over hours, or tens of hours.Tf C’s chemical stability was explored through overnight incubation in urea (0 to 8 M), or guanidium hydrochloride, Gdm.HCl (0 to 6M), based on measurements of changes in the CD signal at 222 nm.Tf C is resistant to urea, but not to Gdm.HCl (Figure 4D), which is a more potent denaturant (disrupting electrostatic interactions and hydrogen bonding, unlike urea which primarily disrupts only hydrogen bonding). Gdm.HCl causes unfolding with an apparent Cmof 3.9 M. Kinetics of denaturation by Gdm.HCl were determined through 2 h incubations of Tf C at 5M, 5.5M, 6M, 6.5M and 7M Gdm.HCl (Figure 4E). The rate of unfolding in absence of denaturant, Ku,w, was determined from a half-chevron plot (Supporting Information Figure S7), to be 3.77*10-11s-1, indicative of slow unfolding. This resistance to thermal and chemical denaturation bodes well for Tf C’s utility under industrial conditions.