Figure 2. Characteristics of the mutants and heat-induced polymerization monitored by FRET.

(A) Assessment of the integrity of wild-type and reactive loop mutants by CD. CD spectra of the reactive loop mutants, at 0.5 mg/ml in 10 mM Na₂HPO₄/NaH₂PO₄ pH7.4, recorded between 260 and 190 nm, show a similar profile to that of the wild-type protein. The spectra are the average of at least four independent experiments. (B) Monomer and samples containing predominantly dimer or trimer were separated by gel filtration of polymerized material using a Superdex 200 column (inset gel). Different ratios of the oligomer fractions were mixed with monomer such that the total concentration of each sample was 0.1 mg/ml, and combined with 4 μM NTA-ATTO550 and 4 μM NTA-ATTO647N. The ratio of fluorescence at 665 nm to that at 575 nm upon excitation at 488 nm was recorded. The measurements shown are from two separate experiments, each using the two different oligomer preparations. The FRET signal varied inversely with the amount of monomer on non-denaturing PAGE. A linear relationship between oligomer concentration and FRET signal is in keeping with previous biophysical studies of α₁-antitrypsin polymerization [14]. Inset fluorescence spectra, normalized for the emission maximum of ATTO550, show a distinct FRET peak (indicated by an asterisk ‘*’) for recombinant α₁-antitrypsin heated at 60°C for 10 min in the presence of 4 μM NTA-ATTO550 and 4 μM NTA-ATTO647N dyes, but not for the unheated control or in the absence of either dye. (C) A 600 μl sample of recombinant α₁-antitrypsin (thick lines) or plasma antitrypsin (thin lines) at 0.1 mg/ml in 10 mM Na₂HPO₄/NaH₂PO₄, 100 mM NaCl in the presence (unbroken lines) or absence (broken lines) of 4 μM NTA-ATTO550/4 μM NTA-ATTO647N was heated from 20 to 95°C at a rate of 1°C min⁻¹ in a Jasco-J810 spectropolarimeter and the ellipticity at 222 nm measured across a 2 mm pathlength. Values were scaled to occur between 0 and 1.0; curves shown are the average of two experiments and error bars indicate the difference between the duplicate measurements. The midpoint of denaturation of recombinant α₁-antitrypsin in these buffer conditions is 59.6±0.1°C in the absence of the reporter dyes and 58.0±0.4°C in the presence of the dyes, and the respective midpoints for the plasma protein are 64.0±0.1 and 63.8±0.1°C. (D) 2 μg α₁-antitrypsin (lane A) was combined with different molar ratios of bovine α-chymotrypsin (indicated by numbers at the top of the gel) in chymotrypsin assay buffer for 15 min at room temperature. Samples were mixed with SDS loading buffer without boiling and separated on an SDS/PAGE(4–12% gel) bis-Tris PAGE, and visualized by Coomassie Brilliant Blue. The position of the α₁-antitrypsin–bovine α-chymotrypsin complex and α₁-antitrypsin in the native and cleaved forms are indicated by the E–I, I_N and I_C labels, respectively.