Probing the folding pathway of a consensus serpin using single tryptophan mutants

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• Conserpin is an engineered protein based on eukaryotic serpins, known for their metastable native state and conserved structure.
• Previous studies showed conserpin adopts an inhibitory conformation, has a unique reversible folding pathway, and resists inactivation.

Purpose of the Study:

• To design a conserpin variant (cAT) with the inhibitory specificity of α1-antitrypsin.
• To investigate the folding pathway of cAT using single-tryptophan variants.

Main Methods:

• Protein engineering to create cAT.
• Generation of single-tryptophan variants of cAT.
• Analysis of thermal stability, inactivation, and folding using intrinsic fluorescence, circular dichroism, and analytical gel filtration.

Main Results:

• cAT demonstrated similar thermal stability and high kinetic stability in its native state compared to the parental conserpin.
• Inactivation of cAT was linked to oligomerization, not a transition to a latent conformation.
• Tryptophan variants elucidated an unfolding intermediate ensemble structurally similar to mesophilic serpin intermediates.
• Folding pathway analysis revealed highly cooperative structural changes that minimize aggregation-prone intermediates.

Conclusions:

• The cAT variant provides a stable, functional model for studying serpin superfamily structure-stability relationships.
• The findings offer insights into the mechanisms preventing aggregation during protein folding.
• This work lays the groundwork for future research into serpin stability and engineering.