The consequences of cavity creation on the folding landscape of a repeat protein depend upon context

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 35; pp. E8153 - E8161
• Main Authors: Jenkins, Kelly A., Fossat, Martin J., Zhang, Siwen, Rai, Durgesh K., Klein, Sean, Gillilan, Richard, White, Zackary, Gerlich, Grayson, McCallum, Scott A., Winter, Roland, Gruner, Sol M., Barrick, Doug, Royer, Catherine A.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 28.08.2018
• Series: PNAS Plus
• Subjects: Biological Sciences; Capping; Cavities; Folding; Free energy; Holes; Humans; Intracellular Signaling Peptides and Proteins - chemistry; Intracellular Signaling Peptides and Proteins - genetics; Leucine; Mutation; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Perturbation; PNAS Plus; Pressure; Pressure dependence; Pressure distribution; Protein Domains; Protein Folding; Protein Stability; Protein structure; Proteins; Scattering, Small Angle; Small angle X ray scattering; Structural stability; Structure-Activity Relationship; Urea; X-Ray Diffraction; X-ray scattering; repeat protein folding; NMR; cooperativity; high pressure; SAXS
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1807379115

The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1–4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.