Perplexing cooperative folding and stability of a low-sequence complexity, polyproline 2 protein lacking a hydrophobic core

Abstract

The burial of hydrophobic side chains in a protein core generally is thought to be the major ingredient for stable, cooperative folding. Here, we show that, for the snow flea antifreeze protein (sfAFP), stability and cooperativity can occur without a hydrophobic core, and without α-helices or β-sheets. sfAFP has low sequence complexity with 46% glycine and an interior filled only with backbone H-bonds between six polyproline 2 (PP2) helices. However, the protein folds in a kinetically two-state manner and is moderately stable at room temperature. We believe that a major part of the stability arises from the unusual match between residue-level PP2 dihedral angle bias in the unfolded state and PP2 helical structure in the native state. Additional stabilizing factors that compensate for the dearth of hydrophobic burial include shorter and stronger H-bonds, and increased entropy in the folded state. These results extend our understanding of the origins of cooperativity and stability in protein folding, including the balance between solvent and polypeptide chain entropies.protein folding | cooperativity | kinetics | PP2 | hydrogen bonding T he basis of protein-folding stability and cooperativity remains a topic of great interest (1, 2). Most studies have focused on proteins with hydrophobic cores containing α-helices and β-sheets. Here, we study the folding of snow flea antifreeze protein (sfAFP), a globular protein that lacks these features. The 81-residue protein has a novel fold that is distinct from other proteins (3-6), containing only polyproline 2 (PP2) helices and turns with a core filled with H-bonds and no hydrophobic groups (Fig. 1).The H-bond network between the PP2 helices (6), PP2 1-6 , requires close packing, which would be precluded by the presence of a C β atom. Thus, the PP2 helices are defined by glycine (Gly) repeats (GXX or GGX, where X is any other amino acid), which enable the hydrogen-bond network. As a consequence, the sfAFP molecule has a low-complexity glycine-rich sequence [37/81 Gly residues, or 46%; typical is 6, or 7% (7)]. The sfAFP core is well packed with essentially no interior void volumes, even when probed using a 0.5-Å radius sphere (8). sfAFP also contains two disulfide bonds, C1-C28 and C13-C43.Notably, no side chains are buried in sfAFP's core (2). All 12 hydrophobic side chains (V 4 , K 2 , and P 6 ) are surface exposed. Upon folding, sfAFP buries about 20 Å 2 ·res −1 of hydrophobic surface area compared with an average of 50 Å 2 ·res −1 for a set of 34 proteins of similar size (Fig. 2A). The difference equates to a decrease in stability of ∼1 or 1.4 kcal·mol −1 ·res −1 , assuming a surface tension coefficient of γ ∼ 34 or 47 cal·mol −1 ·Å −2 based on classical (9) or Flory-Huggins theory (10), respectively. Even the lower bound of 1 kcal·mol −1 ·res −1 represents a significant stability loss, and it is not obvious which energetic terms could compensate for the reduction in hydrophobic burial.Because hydrophobic burial is generally regarded as the basis of protein stability a...

Glycine in Water Favors the Polyproline II State

“…Instead, this protein with 46 glycine residues forms stable bundles of pPII helices connected by HBs. The authors attributed this peculiar fold to the high level of pPII dihedral angle bias [92], which is consistent with findings reported here.…”

Calcium-Mediated Allostery of the EGF Fold

“…Indeed, packing interactions within the hydrophobic core can have a marked effect on the stability of a protein domain, 13 although there are noteworthy examples of protein stability without a hydrophobic core. 14 Here, we demonstrated that the calcium-binding fold was sensitive to the 'hydrophobicity' of the solvent and that an EGF-like peptide with a richer core was more structurally stable.…”

Sequence effects on size, shape, and structural heterogeneity in Intrinsically Disordered Proteins

“…[C] increases, so that at high [C], the statistical properties of the denatured state ensemble (DSE) of globular proteins and IDPs, such as R g , would exhibit the scaling R g ≈ 0.20N ν T , with ν ≈ 0.588. 81 Because the R g scaling of DSEs of globular proteins at high denaturant concentrations is statistically equivalent to those for IDPs (hence the plausible relevance of IDPs to the SAXS-FRET controversy), it is not surprising that the optimal values for the SOP-IDP model (Eq. 3) differ from the [C] = 0 values.…”