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Supplementary material
The following formulae were used for the Cp,pr(T) function simulation:
a)
A simple monomolecular “two-state” scheme of the HK022 Xis denaturation was chosen:
ND
b) The native state heat capacity, CpN, was calculated as a linearly growing function:
CpN(T)=CpN(0)+(T-T0)*b0
1
where the CpN(0) is the value of the native state heat capacity at the reference temperature
(0 °C) and b0 represents the temperature dependency of CpN.
c)
The denatured state heat capacity, CpD, was approximated for simplicity by a linear
function as in the work of Viguera and co-authors [1], even though a binomial behavior
of CpD has been defined for proteins [2,3]. It was found that linear approximation of the
CpD does not significantly affect the thermodynamic parameters of Xis denaturation (H
andG) and can be used for the small span of Tm observed for Xis:
CpD(T)=CpN(T)+Cp(Tm)+(T-Tm)*b1
2
where the denaturation heat capacity increment, Cp=CpD-CpN, was determined at the
denaturation midpoint temperature, Tm, and b1 is the temperature dependency coefficient
of CpD. The Cp value was determined from the temperature dependence of the Xis_wt
denaturation enthalpy.
d) The denaturation enthalpy, H, was calculated based on the assumption that Cp
depends on the temperature (formulae 1 and 2):
H(T)=H(Tm)+Cp(Tm)*(T-Tm)+½(b1*(T-Tm)2)
3
H(T)specific=H(T)/MW
3a
where H(Tm) is the denaturation enthalpy at Tm, H(T)specific is a specific denaturation
enthalpy expressed in J/g, and MW the molecular weight in kDa.
e)
The denaturation entropy, S, was calculated in an analogous manner:
1
S(T)=H(Tm)/Tm+(Cp(Tm)-b1*Tm))*ln(T/Tm)+ b1*(T-Tm)
f)
4
The free energy of protein denaturation, G, was calculated using the standard equation:
G(T)=H(T)-T*S(T)
5
g) The population of the proteins native state, Fn, was calculated using the following
equation:
K=Fn/1-Fn
6
References
1.
Viguera, A.R., Martinez, J.C., Filimonov, V.V., Mateo, P.L. & Serrano, L. (1994)
Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding
transition. Biochemistry 33, 2142–2150.
2.
Makhatadze, G.I. & Privalov, P.L. (1990) Heat capacity of proteins. I. Partial molar
heat capacity of individual amino acid residues in aqueous solution: hydration effect. J. Mol.
Biol. 213, 375–384.
3.
Privalov, P.L. & Makhatadze, G.I. (1990) Heat capacity of proteins. II. Partial molar
heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects. J. Mol.
Biol. 213, 385–391.s
2