Evolutionary conservation of the polyproline II conformation surrounding intrinsically disordered phosphorylation sites.

Intrinsically disordered (ID) proteins function in the absence of a unique stable structure and appear to challenge the classic structure-function paradigm. The extent to which ID proteins take advantage of subtle conformational biases to perform functions, and whether signals for such mechanism can be identified in proteome-wide studies is not well understood. Of particular interest is the polyproline II (PII) conformation, suggested to be highly populated in unfolded proteins. We experimentally determine a complete calorimetric propensity scale for the PII conformation. Projection of the scale into representative eukaryotic proteomes reveals significant PII bias in regions coding for ID proteins. Importantly, enrichment of PII in ID proteins, or protein segments, is also captured by other PII scales, indicating that this enrichment is robustly encoded and universally detectable regardless of the method of PII propensity determination. Gene ontology (GO) terms obtained using our PII scale and other scales demonstrate a consensus for molecular functions performed by high PII proteins across the proteome. Perhaps the most striking result of the GO analysis is conserved enrichment (P < 10(-8) ) of phosphorylation sites in high PII regions found by all PII scales. Subsequent conformational analysis reveals a phosphorylation-dependent modulation of PII, suggestive of a conserved "tunability" within these regions. In summary, the application of an experimentally determined polyproline II (PII) propensity scale to proteome-wide sequence analysis and gene ontology reveals an enrichment of PII bias near disordered phosphorylation sites that is conserved throughout eukaryotes.

Temperature and urea have opposing impacts on polyproline II conformational bias.

The native states of globular proteins have been accessed in atomic detail by X-ray crystallography and nuclear magnetic resonance spectroscopy, yet characterization of denatured proteins beyond global metrics has proven to be elusive. Denatured proteins have been observed to exhibit global geometric properties of a random coil polymer. However, this does not preclude the existence of nonrandom, local conformational bias that may be significant for protein folding and function. Indeed, circular dichroism (CD) spectroscopy and other methods have suggested that the denatured state contains considerable local bias to the polyproline II (PII) conformation. Here, we develop predictive models to determine the extent that temperature and the chemical denaturant urea modulate PII propensity. In agreement with our predictive model, PII propensity is observed experimentally to decrease with an increase in temperature. Conversely, urea appears to promote the PII conformation as determined by CD and isothermal titration calorimetry. Importantly, the calorimetric data are in quantitative agreement with a model that predicts the stability of the PII helix relative to other denatured state conformations based upon solvent accessible surface area and experimentally measured Gibbs transfer free energies. The ability of urea to promote the PII conformation can be attributed to the favorable interaction of urea with the peptide backbone. Thus, perturbing denatured states by temperature or cosolutes has subtle, yet opposing, impacts on local PII conformational biases. These results have implications for protein folding as well as for the function of signaling proteins that bind proline-rich targets in globular or intrinsically disordered proteins.