Conditionally disordered proteins: bringing the environment back into the fold.

For many proteins, biological function requires the folding of the polypeptide chain into a unique and persistent tertiary structure. This review concerns proteins that adopt a specific tertiary structure to function, but are otherwise partially or completely disordered. The biological cue for protein folding is environmental perturbation or minor post-translational modification. Hence, we term these proteins conditionally disordered. Many of these proteins recognize and bind other molecules, and conditional disorder has been hypothesized to allow for more nuanced control and regulation of binding processes. However, this remains largely unproven. The sequences of conditionally disordered proteins suggest their propensity to fold; yet, under the standard laboratory conditions, they do not do so, which may appear surprising. We argue that the surprise results from the failure to consider the role of the environment in protein structure formation and that conditional disorder arises as a natural consequence of the marginal stability of the folded state.

Energy drink exposures reported to Texas poison centers: Analysis of adverse incidents in relation to total sales, 2010-2014.

The ill-defined term "energy drink" includes a disparate group of products (beverages, shots, concentrates, and workout powders) having large differences in caffeine content and concentration and intended use. Hence, inaccurate conclusions may be drawn when describing adverse events associated with "energy drinks". The FDA is considering new regulation of these products but product specificity is needed to evaluate safety. To help address this, we queried Texas Poison Center Network data for single substance exposures to "energy drinks" from 2010 to 2014, then analyzed adverse events by product type. We specifically compared energy beverage exposures with sales data for the same time period to evaluate the safety profile of this category of energy drinks. Among 855 documented "energy drink" exposures, poison center-determined outcome severity revealed 291 with no/minimal effects, 417 judged nontoxic or minor/not followed, 64 moderate and 4 major effects, and no deaths. Serious complications included 2 seizures and 1 episode of ventricular tachycardia. Outcome severity by category for beverages: 11 moderate/1 major effects (none in children <17 years); shots: 19 moderate/2 major; non-liquids: 16 moderate/1 major; concentrates: 7 moderate; unknown: 10 moderate. Call incidence to poison centers for beverage type exposures was 0.58 (for moderate effects) and 0.053 (for major) per hundred million units sold. Small volume and concentrated products were associated with a greater number of adverse effects than beverage versions of "energy drinks".

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.

Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein.

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 310 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses.

Iterative projection algorithms for ab initio phasing in virus crystallography.

Iterative projection algorithms are proposed as a tool for ab initio phasing in virus crystallography. The good global convergence properties of these algorithms, coupled with the spherical shape and high structural redundancy of icosahedral viruses, allows high resolution phases to be determined with no initial phase information. This approach is demonstrated by determining the electron density of a virus crystal with 5-fold non-crystallographic symmetry, starting with only a spherical shell envelope. The electron density obtained is sufficiently accurate for model building. The results indicate that iterative projection algorithms should be routinely applicable in virus crystallography, without the need for ancillary phase information.

Identifying protein domains by global analysis of soluble fragment data.

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation "hotspots" indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α.

A note on the Hendrickson-Lattman phase probability distribution and its equivalence to the generalized von Mises distribution.

Hendrickson & Lattman [Acta Cryst. (1970), B26, 136-143] introduced a method for representing crystallographic phase probabilities defined on the unit circle. Their approach could model the bimodal phase probability distributions that can result from experimental phase determination procedures. It also provided simple and highly effective means to combine independent sources of phase information. The present work discusses the equivalence of the Hendrickson-Lattman distribution and the generalized von Mises distribution of order two, which has been studied in the statistical literature. Recognizing this connection allows the Hendrickson-Lattman distribution to be expressed in an alternative form which is easier to interpret, as it involves the location and concentration parameters of the component von Mises distributions. It also allows clarification of the conditions for bimodality and access to a simplified analytical method for evaluating the trigonometric moments of the distribution, the first of which is required for computing the best Fourier synthesis in the presence of phase, but not amplitude, uncertainty.

Copolymers of Gelatin-graft-poly(3-hexylthiophene) for Transient Electronics.

As transient electronics continue to advance, the demand for new materials has given rise to the exploration of conducting polymer (CP)-based electronic materials. The big challenge lies in balancing conductivity while introducing controlled degradable properties into CP-based transient materials. In response to this, we present in this work a concept of using conducting polymers attached to an enzymatically biodegradable biopolymer to create transient polymer electronics materials. Specifically, poly(3-hexyl thiophene) (P3HT) is covalently grafted onto biopolymer gelatin, affording graft copolymer gelatin-graft-poly(3-hexyl thiophene) (termed Gel-g-P3HT). The thin films of Gel-g-P3HT that were produced by optimized processing solvent (THF/H2O cosolvent) showed enhanced π-π stacking domains of P3HT, resulting in semiconducting thin films with good electroactivity. Due to the presence of amide bonds in the gelatin backbone, Gel-g-P3HT underwent degradation over a period of 5 days, resulting in the formation of amphiphilic micellar nanoparticles that are biocompatible and nontoxic. The potential of these conductive and degradable graft copolymers was demonstrated in a pressure sensor. This research paves the way for developing biocompatible and enzymatically degradable polymer materials based on P3HT, enabling the next generation of transient polymer electronics for diverse applications, such as skin, implantable, and environmental electronics.

Isocyanate exposure assessment combining industrial hygiene methods with biomonitoring for end users of orthopedic casting products.

Previous studies have suggested a potential risk to healthcare workers applying isocyanate-containing casts, but the authors reached their conclusions based on immunological or clinical pulmonology test results alone. We designed a study to assess potential exposure to methylene diphenyl diisocyanate (MDI) among medical personnel applying orthopedic casts using two different application methods. Air, dermal, surface, and glove permeation sampling methods were combined with urinary biomonitoring to assess the overall risk of occupational asthma to workers handling these materials. No MDI was detected in any of the personal and area air samples obtained. No glove permeation of MDI was detected. A small proportion of surface (3/45) and dermal wipe (1/60) samples were positive for MDI, but were all from inexperienced technicians. Urinary metabolites of MDI [methylenedianiline (MDA)] were detected in three of six study participants prior to both a 'dry' and 'wet' application method, five of six after the dry method, and three of six after the wet method. All MDA results were below levels noted in worker or general populations. Our conclusion is that the risk of MDI exposure is small, but unquantifiable. Because there is some potential risk of dermal exposure, medical personnel are instructed to wear a minimum of 5-mil-thick (5 mil = 0.005 inches) nitrile gloves and avoid contact to unprotected skin. This could include gauntlets, long sleeves, and/or a laboratory coat.

Galidesivir Triphosphate Promotes Stalling of Dengue-2 Virus Polymerase Immediately Prior to Incorporation.

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 µM and 47 ± 5 µM, respectively, at an ATP concentration of 20 µM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.

Recombinant expression of the coat protein of Botrytis virus X and development of an immunofluorescence detection method to study its intracellular distribution in Botrytis cinerea.

Botrytis cinerea is infected by many mycoviruses with varying phenotypical effects on the fungal host, including Botrytis virus X (BVX), a mycovirus that has been found in several B. cinerea isolates worldwide with no obvious effects on growth. Here we present results from serological and immunofluorescence microscopy (IFM) studies using antiserum raised against the coat protein of BVX expressed in Escherichia coli fused to maltose-binding protein. Due to the high yield of recombinant protein it was possible to raise antibodies that recognized BVX particles. An indirect ELISA, using BVX antibodies, detected BVX in partially purified virus preparations from fungal isolates containing BVX alone and in mixed infection with Botrytis virus F. The BVX antiserum also proved suitable for IFM studies. Intensely fluorescing spots (presumed to be virus aggregates) were found to be localized in hyphal cell compartments and spores of natural and experimentally infected B. cinerea isolates using IFM. Immunofluorescently labelled sections through fungal tissue, as well as fixed mycelia grown on glass slides, showed aggregations of virions closely associated with fungal cell membranes and walls, next to septal pores, and in hyphal tips. Also, calcofluor white staining of mature cell walls of virus-transfected Botrytis clones revealed numerous cell wall areas with increased amounts of chitin/glycoproteins. Our results indicate that some BVX aggregates are closely associated with the fungal cell wall and raise the question of whether mycoviruses may be able to move through the wall and therefore not be totally dependent on intracellular routes of transmission.

Proton-driven assembly of the Rous Sarcoma virus capsid protein results in the formation of icosahedral particles.

In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic resolution structural analysis. CA hexamers and pentamers are the fundamental building blocks of the capsid, however the pentameric state, in particular, remains poorly characterized. We have developed an efficient in vitro protocol for studying the assembly of Rous sarcoma virus (RSV) CA that involves mild acidification and produces structures modeling the authentic viral capsid. These structures include regular spherical particles with T = 1 icosahedral symmetry, built from CA pentamers alone. These particles were subject to cryoelectron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM-derived three-dimensional density map. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned underneath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra are the only detectable species and that these exist in reversible equilibrium at pH 5. These same acidic conditions are known to promote formation of a RSV CA CTD dimer, present within the icosahedral particle, which facilitates capsid assembly. The results are consistent with a model in which RSV CA assembly is a nucleation-limited process driven by very weak protein-protein interactions.

Proton-linked dimerization of a retroviral capsid protein initiates capsid assembly.

In mature retroviral particles, the capsid protein (CA) forms a shell encasing the viral replication complex. Human immunodeficiency virus (HIV) CA dimerizes in solution, through its C-terminal domain (CTD), and this interaction is important for capsid assembly. In contrast, other retroviral capsid proteins, including that of Rous sarcoma virus (RSV), do not dimerize with measurable affinity. Here we show, using X-ray crystallography and other biophysical methods, that acidification causes RSV CA to dimerize in a fashion analogous to HIV CA, and that this drives capsid assembly in vitro. A pair of aspartic acid residues, located within the CTD dimer interface, explains why dimerization is linked to proton binding. Our results show that despite overarching structural similarities, the intermolecular forces responsible for forming and stabilizing the retroviral capsid differ markedly across retroviral genera. Our data further suggest that proton binding may regulate RSV capsid assembly, or modulate stability of the assembled capsid.

Evolutionary history of cotranscriptional editing in the paramyxoviral phosphoprotein gene.

The phosphoprotein gene of the paramyxoviruses encodes multiple protein products. The P, V, and W proteins are generated by transcriptional slippage. This process results in the insertion of non-templated guanosine nucleosides into the mRNA at a conserved edit site. The P protein is an essential component of the viral RNA polymerase and is encoded by a faithful copy of the gene in the majority of paramyxoviruses. However, in some cases, the non-essential V protein is encoded by default and guanosines must be inserted into the mRNA in order to encode P. The number of guanosines inserted into the P gene can be described by a probability distribution, which varies between viruses. In this article, we review the nature of these distributions, which can be inferred from mRNA sequencing data, and reconstruct the evolutionary history of cotranscriptional editing in the paramyxovirus family. Our model suggests that, throughout known history of the family, the system has switched from a P default to a V default mode four times; complete loss of the editing system has occurred twice, the canonical zinc finger domain of the V protein has been deleted or heavily mutated a further two times, and the W protein has independently evolved a novel function three times. Finally, we review the physical mechanisms of cotranscriptional editing via slippage of the viral RNA polymerase.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes at physiological temperature.

During a proteolytically-driven maturation process, the orthoretroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at near-physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.

The metalation of hen egg white lysozyme impacts protein stability as shown by ion mobility mass spectrometry, differential scanning calorimetry, and X-ray crystallography.

Metalation of hen egg white lysozyme (HEWL) with organometallics was studied with physicochemical methods in solid state, solution and the gas phase. While metalation did not affect the crystal structure of HEWL significantly, protein destabilisation was detected in gas phase and solution.

Iterative projection algorithms in protein crystallography. II. Application.

Iterative projection algorithms (IPAs) are a promising tool for protein crystallographic phase determination. Although related to traditional density-modification algorithms, IPAs have better convergence properties, and, as a result, can effectively overcome the phase problem given modest levels of structural redundancy. This is illustrated by applying IPAs to determine the electron densities of two protein crystals with fourfold non-crystallographic symmetry, starting with only the experimental diffraction amplitudes, a low-resolution molecular envelope and the position of the non-crystallographic axes. The algorithm returns electron densities that are sufficiently accurate for model building, allowing automated recovery of the known structures. This study indicates that IPAs should find routine application in protein crystallography, being capable of reconstructing electron densities starting with very little initial phase information.

Transient dimerization of human MxA promotes GTP hydrolysis, resulting in a mechanical power stroke.

Myxovirus resistance (Mx) proteins restrict replication of numerous viruses. They are closely related to membrane-remodeling fission GTPases, such as dynamin. Mx proteins can tubulate lipids and form rings or filaments that may interact directly with viral structures. GTPase domain dimerization is thought to allow crosstalk between the rungs of a tubular or helical assembly, facilitating constriction. We demonstrate that the GTPase domain of MxA dimerizes to facilitate catalysis, in a fashion analogous to dynamin. GTP binding is associated with the lever-like movement of structures adjacent to the GTPase domain, while GTP hydrolysis returns MxA to its resting state. Dimerization is not significantly promoted by substrate binding and occurs only transiently, yet is central to catalytic efficiency. Therefore, we suggest dimerization functions to coordinate the activity of spatially adjacent Mx molecules within an assembly, allowing their mechanical power strokes to be synchronized at key points in the contractile cycle.