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We have developed an algorithm, ParSe, which accurately identifies from the primary sequence those protein regions likely to exhibit physiological phase separation behavior. Originally, ParSe was designed to test the hypothesis that, for flexible proteins, phase separation potential is correlated to hydrodynamic size. While our results were consistent with that idea, we also found that many different descriptors could successfully differentiate between three classes of protein regions: folded, intrinsically disordered, and phase-separating intrinsically disordered. Consequently, numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. Built from that finding, ParSe 2.0 uses an optimal set of property scales to predict domain-level organization and compute a sequence-based prediction of phase separation potential. The algorithm is fast enough to scan the whole of the human proteome in minutes on a single computer and is equally or more accurate than other published predictors in identifying proteins and regions within proteins that drive phase separation. Here, we describe a web application for ParSe 2.0 that may be accessed through a browser by visiting https://stevewhitten.github.io/Parse_v2_FASTA to quickly identify phase-separating proteins within large sequence sets, or by visiting https://stevewhitten.github.io/Parse_v2_web to evaluate individual protein sequences., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

It has been estimated that 30% of eukaryotic protein and 70% of transcription factors are intrinsically disordered (ID). The biochemical significance of proteins that lack stable tertiary structure, however, is not clearly understood, largely owing to an inability to assign well-defined structures to specific biological tasks. In an attempt to investigate the structural character of ID protein, we have measured the circular dichroism spectrum of the N-terminal region of p53 over a range of temperatures and solution conditions. p53 is a well-studied transcription factor that has a proline-rich N-terminal ID region containing two activation domains. High proline content is a property commonly associated with ID, and thus p53 may be a good model system for investigating the biochemical importance of ID. The spectra presented here suggest that the N-terminal region of p53 may adopt an ordered structure under physiological conditions and that this structure can be thermally unfolded in an apparent two-state manner. The midpoint temperature for this thermal unfolding of the N-terminal region of p53 was at the near-physiological temperature of 39°C, suggesting the possibility of a physiological role for the observed structural equilibrium., (Copyright © 2012 The Protein Society.)

The left-handed polyproline II helical structure (P(II)) is observed to be a dominant conformation in the disordered states of protein and small polypeptide chains, even when no prolines are present in the sequence. Recently, in work by Ferreon and Hilser, the energetics associated with Ala and Gly substitutions at a surface exposed proline site were determined calorimetrically by measuring the binding energetics of Sos peptide variants to the C-terminal Src Homology 3 domain of SEM-5. The results were interpreted as a significant conformational bias toward the bound conformation (i.e., P(II)), even when the ligand is unbound. That study was not able to determine, however, whether the conformational bias of the peptides could be explained in terms other than that of a P(II) preference. Here, we test, using a computer algorithm based on the hard sphere collision (HSC) model, the notion of whether a bias in the unbound states of the peptide ligands is specific for the P(II) conformation, or if a bias to any other region of (phi, psi) space can also result in the same observed binding energetics. The results of these computer simulations indicate that, of the regions of (phi, psi) modeled for bias in the small peptides, only the bias to the P(II) conformation, and at rates of bias similar to the experimentally observed rates, quantitatively reproduced the experimental binding energetics.