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. 2012 Mar 20;109(12):4467-72.
doi: 10.1073/pnas.1109125109. Epub 2012 Mar 5.

Folding Mechanism of the Metastable Serpin α1-antitrypsin

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Free PMC article

Folding Mechanism of the Metastable Serpin α1-antitrypsin

Yuko Tsutsui et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded β-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α(1)-antitrypsin (α(1)-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, β-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into β-sheets B and C is largely complete before the center of β-sheet A begins to fold. We propose this as the mechanism for trapping α(1)-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α(1)-AT avoids polymerization during folding.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Metastable (A) and stable (B) forms of α1-antitrypsin. Beta sheets A, B, and C are shown in green, magenta, and yellow, respectively. Helix F is shown in orange and the reactive center loop/strand 4A is shown in red.
Fig. 2.
Fig. 2.
Mass spectra of refolded and polymerized α1-antitrypsin. Data for peptides 352–372 and 173–182 are shown. Black line, α1-antitrypsin after 3,000 s of refolding; gray line, native α1-antitrypsin; dashed line, α1-antitrypsin polymers formed by heating at 45 °C.
Fig. 3.
Fig. 3.
Mass spectra of cooperative and noncooperative folding regions. Mass spectra of representative peptides at different refolding times. (A) A mass spectrum of a peptide derived from 120–142 in helix E. Two well-separated mass envelopes corresponding to the folded and unfolded populations are evident. (B) Deuterium uptake of a peptide covering 38–51 in the C-terminal half of helix A, a loop, and a portion of β-strand 6B. (C) Peptide covering 352–372 including the C-terminal portion of the RCL, and β-strands 1C and (partial) 4B.
Fig. 4.
Fig. 4.
Kinetics of cooperative folding in α1-AT. Fraction folded was calculated as described in Methods for cooperatively folding peptides at different refolding times. Data for two repeat experiments is shown. Solid lines are trend lines only.
Fig. 5.
Fig. 5.
Beta-strand 5A folds more slowly than β-strand 1C. Data for 325–338 (green triangles and red circles) and 352–372 (black squares). The solid line represents the fit of the 352–372 data to two exponentials (see text).
Fig. 6.
Fig. 6.
Visualizing α1-AT folding. (A) The order of the folding is mapped onto the crystal structure of α1-AT in the metastable state (6). (A) Cooperatively folding regions are shown in blue: dark blue, no lag phase; medium blue, lag phase < 300 s; light blue, lag phase > 300 s. Noncooperatively folding regions are shown in green: dark green, t1/2 < 250 s (t1/2 estimated from centroid mass shift); light green, t1/2 > 250 s. Regions for which no peptic fragments were identified are gray. Trp 194 is shown as red sticks. Sites of major pathogenic mutations are shown as magenta sticks. (B) Regions that display a lag phase in their folding kinetics are shown as colored spheres.

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