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.