Citations

ABSTRACTMolecular 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. Using the intrinsically disordered N‐terminal region of the p53 protein as an experimental model, a set of proline (PRO) and alanine (ALA) to glycine (GLY) substitution variants were designed to modulate backbone conformational propensities without introducing non‐native intramolecular interactions. The hydrodynamic radius (Rh) was used to monitor changes in global structure. Circular dichroism spectroscopy showed that the GLY substitutions decreased polyproline II (PPII) propensities relative to the wild type, as expected, and fluorescence methods indicated that substitution‐induced changes in Rh were not associated with folding. The experiments showed that changes in local PPII structure cause changes in Rh that are variable and that depend on the intrinsic chain propensities of PRO and ALA residues, demonstrating a mechanism for coupling local and global structure changes. Molecular simulations that model our results were used to extend the analysis to other proteins and illustrate the generality of the observed PRO and alanine effects on the structures of IDPs. Proteins 2014; 82:3373–3384. © 2014 Wiley Periodicals, Inc.

ABSTRACTThe ionic liquid 1‐ethyl‐3‐methyl imidazolium chloride (EMIM Cl) and the amino acid l‐arginine hydrochloride (l‐ArgHCl) have been successfully used to improve the yield of oxidative refolding for various proteins. However, the molecular mechanisms behind the actions of such solvent additives—especially of ionic liquids—are still not well understood. To analyze these mechanisms, we have determined the transfer free energies from water into ionic liquid solutions of proteinogenic amino acids and of diketopiperazine as peptide bond analogue. For EMIM Cl and 1‐ethyl‐3‐methyl imidazolium diethyl phosphate, which had a suppressive effect on protein refolding, as well as for l‐ArgHCl favorable interactions with amino acid side chains, but no favorable interactions with the peptide backbone could be observed. A quantitative analysis of other ionic liquids together with their already published effects on protein refolding showed that only solvent additives within a certain range of hydrophobicity, chaotropicity and kosmotropicity were effective for the refolding of recombinant plasminogen activator. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1129–1140, 2014.

The ionic liquid N‐ethyl‐N′‐methyl imidazolium chloride (EMIMCl) has been described as being very efficient in promoting refolding of the recombinant plasminogen activator rPA. Our study reveals that molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains rather than favorably interacting with the peptide backbone. This delicate balance of favorable interactions with side chains and unfavorable interactions with the peptide backbone provides a molecular explanation of how EMIMCl suppresses protein aggregation and simultaneously promotes refolding. By contrast, high concentrations of EMIMCl denature proteins because of a reduced water content and strong favorable interactions with amino acid side chains. This denatured species is not soluble and aggregates because, in contrast to the classical denaturants, guanidine hydrochloride and urea, EMIMCl does not solubilize the peptide backbone.Structured digital abstract PNP and PNP bind by molecular sieving (1, 2, 3, 4)

ABSTRACTClinical mutations in patients diagnosed with Type 2A von Willebrand disease (VWD) have been identified that break the single disulfide bond linking N‐ and C‐termini in the vWF A1 domain. We have modeled the effect of these mutations on the disulfide‐bonded structure of A1 by reducing and carboxy‐amidating these cysteines. Solution biophysical studies show that loss of this disulfide bond induces a molten globule conformational state lacking global tertiary structure but retaining residual secondary structure. The conformational dependence of platelet adhesion to these native and molten globule states of A1 is quantitatively compared using real‐time high‐speed video microscopy analysis of platelet translocation dynamics under shear flow in a parallel plate microfluidic flow chamber. While normal platelets translocating on surface‐captured native A1 domain retain the catch‐bond character of pause times that increase as a function of shear rate at low shear and decrease as a function of shear rate at high shear, platelets that interact with A1 lacking the disulfide bond remain stably attached and do not translocate. Based on these findings, we propose that the shear stress‐sensitive regulation of the A1‐GPIb interaction is due to folding the tertiary structure of this domain. Removal of the tertiary structure by disrupting the disulfide bond destroys this regulatory mechanism resulting in high‐strength interactions between platelets and vWF A1 that are dependent only on residual secondary structure elements present in the molten globule conformation. Proteins 2014; 82:867–878. © 2013 Wiley Periodicals, Inc.

AbstractWe have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea‐induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two‐state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m‐value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea‐denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions.

AbstractPlatelet attachment to von Willebrand factor (vWF) requires the interaction between the platelet GP1bα and exposed vWF‐A1 domains. Structural insights into the mechanism of the A1‐GP1bα interaction have been limited to an N‐terminally truncated A1 domain that lacks residues Q1238 − E1260 that make up the linker between the D3 and A1 domains of vWF. We have demonstrated that removal of these residues destabilizes quaternary interactions in the A1A2A3 tridomain and contributes to platelet activation under high shear (Auton et al., J Biol Chem 2012;287:14579–14585). In this study, we demonstrate that removal of these residues from the single A1 domain enhances platelet pause times on immobilized A1 under rheological shear. A rigorous comparison between the truncated A1‐1261 and full length A1‐1238 domains demonstrates a kinetic stabilization of the A1 domain induced by these N‐terminal residues as evident in the enthalpy of the unfolding transition. This stabilization occurs through site and sequence‐specific binding of the N‐terminal peptide to A1. Binding of free N‐terminal peptide to A1‐1261 has an affinity and this binding although free to dissociate is sufficient to suppress the platelet pause times to levels comparable to A1‐1238 under shear stress. Our results support a dual‐structure/function role for this linker region involving a conformational equilibria that maintains quaternary A domain associations in the inactive state of vWF at low shear and an intra‐A1‐domain conformation that regulates the strength of platelet GP1bα‐vWF A1 domain associations in the active state of vWF at high shear.