High Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine Rich Repeat Protein

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High Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine Rich Repeat Protein

Publication date: Available online 13 March 2018
Source:Journal of Molecular Biology

Author(s): Yi Zhang, Melanie Berghaus, Sean Klein, Kelly Jenkins, Siwen Zhang, Scott A. McCallum, Joel Morgan, Roland Winter, Doug Barrick, Catherine A. Royer

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well-documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine rich repeat protein, pp32, by monitoring the pressure and urea induced unfolding of an N-cap deletion mutant, pp32-?N-cap, and a C-cap destabilization mutant pp32-Y131F/D146L, using residue specific NMR and SAXS. Destabilization of the C-cap motif resulted in higher cooperativity for the unfolding transition compared to wild type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-terminal capping motif led to strong deviation from two-state unfolding. In both urea and pressure-induced unfolding, residues in repeats 1–3 of pp32-?N-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-?N-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-?N-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-?N-cap variant arise from their distinct mechanisms of action.
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