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J Mol Biol. 2012 Nov 9;423(5):789-99. doi: 10.1016/j.jmb.2012.08.019. Epub 2012 Aug 29.

Early hydrophobic collapse of α₁-antitrypsin facilitates formation of a metastable state: insights from oxidative labeling and mass spectrometry.

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Departments of Biochemistry and Chemistry, Western University, London, Ontario, Canada N6A 5B7.


The biologically active conformation of α₁-antitrypsin (α₁AT) and other serine protease inhibitors represents a metastable state, characterized by an exposed reactive center loop (RCL) that acts as bait for the target enzyme. The protein can also adopt an inactive "latent" conformation that has the RCL inserted as a central strand in β-sheet A. This latent form is thermodynamically more stable than the active conformation. Nonetheless, folding of α₁AT consistently yields the active state. The reasons that the metastable form is kinetically preferred remain controversial. The current work demonstrates that a carefully orchestrated folding mechanism prevents RCL insertion into sheet A. Temporal changes in solvent accessibility during folding are monitored using pulsed oxidative labeling and mass spectrometry. The data obtained in this way complement recent hydrogen/deuterium exchange results. Those hydrogen/deuterium exchange measurements revealed that securing of the RCL by hydrogen bonding of the first β-strand in sheet C is one factor that favors formation of the active conformation. The oxidative labeling data presented here reveal that this anchoring is preceded by the formation of hydrophobic contacts in a confined region of the protein. This partial collapse sequesters the RCL insertion site early on and is therefore instrumental in steering α₁AT towards its active conformation. RCL anchoring by hydrogen bonding starts to contribute at a later stage. Together, these two factors ensure that formation of the active conformation is kinetically favored. This work demonstrates how the use of complementary labeling techniques can provide insights into the mechanisms of protracted folding reactions.

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