TABLE 2.

Calculated biophysical parameters for WT apoA-I and the apoA-I[F225A/V227A/F229A/L230A] mutant

Mutation	Helicity	Thermal Denaturation			Chemical Denaturation	ANS Binding
ApoA-I	α-Helix	Tm (°C)	Slopea	Cooperativity Index (n)	D1/2 (M)	Fold Increaseb
WT	59.3 ± 0.5	56.0 ± 0.5	7.8 ± 0.1	6.3 ± 0.4	1.02 ± 0.06	10.2 ± 0.5
F225A/V227A/F229A/L230A	51.7 ± 0.3c	57.8 ± 0.2d	4.0 ± 0.0c	11.4 ± 0.4e	1.01 ± 0.03	6.0 ± 0.4c

Values are means ± SD of 3–4 experiments. Parameters obtained from the indicated measurements are as follows: α-helix is the % α-helical content of the protein as calculated from the molecular ellipticity of the protein sample at 222 nm; T_m, is middle point of the thermal denaturation transition (melting temperature); slope is the calculated slope of the linear component of the thermal denaturation transition, around the melting temperature; cooperativity index n is an indicator of the cooperativity of the thermal unfolding transition and is calculated using the Hill equation the Hill equation n = (log 81)/log(T_0.9/T_0.1), where T_0.9 and T_0.1 are the temperatures at which the unfolding transition has reached a fractional completion of 0.9 and 0.1; D_1/2 is the guanidine HCl concentration at which the midpoint of the chemical denaturation is achieved; fold increase is the increase in ANS fluorescence in the presence of the protein relative to free ANS in the same buffer.

^aSlope is calculated from the fit of thermal denaturation curve to a Boltzman sigmoidal model curve using the equation [Θ]₂₂₂ = Bottom + ((Top − Bottom) / (1 = exp((Tm − X) / Slope))). X describes the temperature, and slope describes the steepness of the curve, with a larger value denoting a shallow curve.

^bFold-increase in signal compared with unbound ANS.

^cP< 0.0001.

^dP< 0.05.

^eP< 0.001.