Dataset Information

Folding mechanism of the metastable serpin ?1-antitrypsin.

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 ?-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human ?(1)-antitrypsin (?(1)-AT). We find large differences in the folding kinetics of different regions. A key region in the metastable ? stable transition, ?-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 ?-sheets B and C is largely complete before the center of ?-sheet A begins to fold. We propose this as the mechanism for trapping ?(1)-AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which ?(1)-AT avoids polymerization during folding.

SUBMITTER: Tsutsui Y

PROVIDER: S-EPMC3311335 | biostudies-other | 2012 Mar

REPOSITORIES: biostudies-other

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Folding mechanism of the metastable serpin α1-antitrypsin.

Tsutsui Yuko Y Dela Cruz Richard R Wintrode Patrick L PL

Proceedings of the National Academy of Sciences of the United States of America 20120305 12

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-deu ...[more]

PMID: 22392975

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Project description:Protein glycosylation commonly stabilizes proteins thereby increasing protein half-lives and protecting against denaturation or proteolytic degradation. While generally beneficial, such stabilization is potentially disadvantageous in the case of inhibitory serpins. These protease inhibitors are metastable and a conformational transition to a more stable form is key to their function. Instability is therefore essential for these inhibitory serpins and mutagenesis has demonstrated that substantial stabilization results in compromised function. We have used optical spectroscopy and hydrogen/deuterium exchange and mass spectrometry to investigate the effects of glycosylation on the human serpin alpha-1 antitrypsin (α(1)-AT). Previous studies found that unglycosylated recombinant α(1)-AT populates a molten globule at low denaturant and that the ability to populate this state is correlated with efficient protease inhibition. Further, a high degree of conformational flexibility was found in several important regions. Guanidine hydrochloride denaturation monitored by circular dichroism indicates that plasma α(1)-AT, which is glycosylated at 3 sites, is substantially stabilized relative to the unglycosylated form. However, hydrogen exchange reveals complete loss of protection in plasma α(1)-AT above 1 M GuHCl, similar to what is seen for the recombinant form. Sugars therefore appear to stabilize the compact denatured state of α(1)-AT without significant stabilization of the folded state. Native state hydrogen exchange reveals minor perturbations to native flexibility, but high flexibility in key regions such as the f helix is conserved. β-strand 1c is stabilized in plasma α(1)-AT, which may confer increased resistance to forming pathogenic polymers. Overall, our results indicate that glycosylation of inhibitory serpins does not interfere with either native state flexibility or the native instability that is required for efficient function, though it may confer resistance to degradation by proteases and thus extend the half-life of circulating serpins.

Dataset Information

Folding mechanism of the metastable serpin ?1-antitrypsin.

Publications

Folding mechanism of the metastable serpin α1-antitrypsin.

Similar Datasets

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