ParSe 2.0: A web tool to identify drivers of protein phase separation at the proteome level.

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.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

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.

Convergent behavior of extended stalk regions from staphylococcal surface proteins with widely divergent sequence patterns.

and are highly problematic bacteria in hospital settings. This stems, at least in part, from strong abilities to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized multicellular aggregates of bacteria, which, when formed on indwelling medical devices, lead to infections that are difficult to treat.

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins.

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein's propensity to phase separate is thought to be driven by a preference for protein-protein over protein-solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance.

Structural and Energetic Characterization of the Denatured State from the Perspectives of Peptides, the Coil Library, and Intrinsically Disordered Proteins.

The α and polyproline II (PPII) basins are the two most populated regions of the Ramachandran map when constructed from the protein coil library, a widely used denatured state model built from the segments of irregular structure found in the Protein Data Bank. This indicates the α and PPII conformations are dominant components of the ensembles of denatured structures that exist in solution for biological proteins, an observation supported in part by structural studies of short, and thus unfolded, peptides. Although intrinsic conformational propensities have been determined experimentally for the common amino acids in short peptides, and estimated from surveys of the protein coil library, the ability of these intrinsic conformational propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs), an increasingly important class of proteins in cell function, has thus far proven elusive to establish.

Impact of Heat on Coil Hydrodynamic Size Yields the Energetics of Denatured State Conformational Bias.

Conformational equilibria in the protein denatured state have key roles regulating folding, stability, and function. The extent of conformational bias in the protein denatured state under folding conditions, however, has thus far proven elusive to quantify, particularly with regard to its sequence dependence and energetic character. To better understand the structural preferences of the denatured state, we analyzed both the sequence dependence to the mean hydrodynamic size of disordered proteins in water and the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the α and polyproline II (PPII) backbone conformations can be obtained.

Evidence for the Misfolding of the A1 Domain within Multimeric von Willebrand Factor in Type 2 von Willebrand Disease.

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes.

Sequence Reversal Prevents Chain Collapse and Yields Heat-Sensitive Intrinsic Disorder.

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of "Retro-nuclease" is highly expanded in solution with characteristics similar to biological intrinsically disordered proteins.

Intrinsic α helix propensities compact hydrodynamic radii in intrinsically disordered proteins.

Proteins that lack tertiary stability under normal conditions, known as intrinsically disordered, exhibit a wide range of biological activities. Molecular descriptions for the biology of intrinsically disordered proteins (IDPs) consequently rely on disordered structural models, which in turn require experiments that assess the origins to structural features observed. For example, while hydrodynamic size is mostly insensitive to sequence composition in chemically denatured proteins, IDPs show strong sequence-specific effects in the hydrodynamic radius (R ) when measured under normal conditions.

The Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction.

Staphylococcus epidermidis is one of the primary bacterial species responsible for healthcare-associated infections. The most significant virulence factor for S. epidermidis is its ability to form a biofilm, which renders the bacteria highly resistant to host immune responses and antibiotic action.

Hydrodynamic Radii of Intrinsically Disordered Proteins Determined from Experimental Polyproline II Propensities.

The properties of disordered proteins are thought to depend on intrinsic conformational propensities for polyproline II (PPII) structure. While intrinsic PPII propensities have been measured for the common biological amino acids in short peptides, the ability of these experimentally determined propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs) has not been established. Presented here are results from molecular simulations of disordered proteins showing that the hydrodynamic radius (Rh) can be predicted from experimental PPII propensities with good agreement, even when charge-based considerations are omitted.

Fungal metabolite ophiobolin A as a promising anti-glioma agent: In vivo evaluation, structure-activity relationship and unique pyrrolylation of primary amines.

Glioblastoma, the most common form of malignant primary brain tumor, is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches based on apoptosis induction in cancer cells. Previously, a fungal secondary metabolite ophiobolin A was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, thus, offering an innovative strategy to combat this type of cancer. The current work describes the results of a preliminary evaluation of ophiobolin A in an in vivo glioblastoma model and its chemical derivatization to establish first synthetically generated structure-activity relationship.

Structural origins of misfolding propensity in the platelet adhesive von Willebrand factor A1 domain.

The von Willebrand factor (VWF) A1 and A3 domains are structurally isomorphic yet exhibit distinct mechanisms of unfolding. The A1 domain, responsible for platelet adhesion to VWF in hemostasis, unfolds through a molten globule intermediate in an apparent three-state mechanism, while A3 unfolds by a classical two-state mechanism. Inspection of the sequences or structures alone does not elucidate the source of this thermodynamic conundrum; however, the three-state character of the A1 domain suggests that it has more than one cooperative substructure yielding two separate unfolding transitions not present in A3.

Alanine and proline content modulate global sensitivity to discrete perturbations in disordered proteins.

Molecular transduction of biological signals is understood primarily in terms of the cooperative structural transitions of protein macromolecules, providing a mechanism through which discrete local structure perturbations affect global macromolecular properties. The recognition that proteins lacking tertiary stability, commonly referred to as intrinsically disordered proteins (IDPs), mediate key signaling pathways suggests that protein structures without cooperative intramolecular interactions may also have the ability to couple local and global structure changes. Presented here are results from experiments that measured and tested the ability of disordered proteins to couple local changes in structure to global changes in structure.

Temperature effects on the hydrodynamic radius of the intrinsically disordered N-terminal region of the p53 protein.

Intrinsically disordered proteins (IDPs) are often characterized in terms of the hydrodynamic radius, Rh . The Rh of IDPs are known to depend on fractional proline content and net charge, where increased numbers of proline residues and increased net charge cause larger Rh . Though sequence and charge effects on the Rh of IDPs have been studied, the temperature sensitivity has been noted only briefly.

Using the COREX/BEST server to model the native-state ensemble.

Protein structures under normal conditions exist as ensembles of interconverting, transient microstates. A computer algorithm known as COREX/BEST (Biology using Ensemble-based Structural Thermodynamics) was developed to model microstate structures and describe the native ensembles of proteins in statistical thermodynamic terms. This algorithm has been tested extensively and validated through experimental comparisons examining a range of biophysical and functional phenomena, such as structural cooperativity, pH-dependent stability, and cold denaturation.

Thermal unfolding of the N-terminal region of p53 monitored by circular dichroism spectroscopy.

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.

Mutational analysis of m-values as a strategy to identify cold-resistant substructures of the protein ensemble.

Characterizing the native ensemble of protein is an important yet difficult objective of structural biology. The structural dynamics of protein macromolecules play key roles in biological function, but the short lifetimes and low population of near-native states of the protein ensemble limit their ability to be studied directly. In part to address such issues, it was shown recently that the cooperative substructures that populate a protein ensemble could be ascertained by NMR methods performed at very cold temperatures.

Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins.

The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions.

The role of protein conformational fluctuations in allostery, function, and evolution.

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade.

Characterizing the role of ensemble modulation in mutation-induced changes in binding affinity.

Protein conformational fluctuations are key contributors to biological function, mediating important processes such as enzyme catalysis, molecular recognition, and allosteric signaling. To better understand the role of conformational fluctuations in substrate/ligand recognition, we analyzed, experimentally and computationally, the binding reaction between an SH3 domain and the recognition peptide of its partner protein. The fluctuations in this SH3 domain were enumerated by using an algorithm based on the hard sphere collision model, and the binding energetics resulting from these fluctuations were calculated using a structure-based energy function parametrized to solvent accessible surface areas.

Denatured-state energy landscapes of a protein structural database reveal the energetic determinants of a framework model for folding.

Position-specific denatured-state thermodynamics were determined for a database of human proteins by use of an ensemble-based model of protein structure. The results of modeling denatured protein in this manner reveal important sequence-dependent thermodynamic properties in the denatured ensembles as well as fundamental differences between the denatured and native ensembles in overall thermodynamic character. The generality and robustness of these results were validated by performing fold-recognition experiments, whereby sequences were matched with their respective folds based on amino acid propensities for the different energetic environments in the protein, as determined through cluster analysis.

Exploring the impact of polyproline II (PII) conformational bias on the binding of peptides to the SEM-5 SH3 domain.

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.

Ligand effects on the protein ensemble: unifying the descriptions of ligand binding, local conformational fluctuations, and protein stability.

Detailed description of the structural and physical basis of allostery, cooperativity, and other manifestations of long-range communication between binding sites in proteins remains elusive. Here we describe an ensemble-based structural-thermodynamic model capable of treating explicitly the coupling between ligand binding reactions, local fluctuations in structure, and global conformational transitions. The H(+) binding reactions of staphylococcal nuclease and the effects of pH on its stability were used to illustrate the properties of proteins that can be described quantitatively with this model.