Taylor A. Poor

Post-Assembly Functionalization of Supramolecular Nanostructures with Bioactive Peptides and Fluorescent Proteins by Native Chemical Ligation

Post-assembly functionalization of supramolecular nanostructures has the potential to expand the range of their applications. We report here the use of the chemoselective native chemical ligation (NCL) reaction to functionalize self-assembled peptide amphiphile (PA) nanofibers. This strategy can be used to incorporate specific bioactivity on the nanofibers, and as a model, we demonstrate functionalization with the RGDS peptide following self-assembly. Incorporation of bioactivity is verified by the observation of characteristic changes in fibroblast morphology following NCL-mediated attachment of the signal to PA nanofibers. The NCL reaction does not alter the PA nanofiber morphology, and biotinylated RGDS peptide was found to be accessible on the nanofiber surface after ligation for binding with streptavidin-conjugated gold nanoparticles. In order to show that this strategy is not limited to short peptides, we utilized NCL to conjugate yellow fluorescent protein and/or cyan fluorescent protein to self-assembled PA nanofibers. Förster resonance energy transfer and fluorescence anisotropy measurements are consistent with the immobilization of the protein on the PA nanofibers. The change in electrophoretic mobility of the protein upon conjugation with PA molecules confirmed the formation of a covalent linkage. NCL-mediated attachment of bioactive peptides and proteins to self-assembled PA nanofibers allows the independent control of self-assembly and bioactivity while retaining the biodegradable peptide structure of the PA molecule and thus can be useful in tailoring design of biomaterials.

Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.

Significance The activities of the fusion proteins that mediate virus–cell fusion are an absolute requirement for virus entry and infectivity of enveloped viruses such as HIV, influenza virus, measles virus, and respiratory syncytia virus, among others. Viral fusion proteins are translated initially in a metastable prefusion state and, upon triggering, undergo an extensive and irreversible refolding process. Membrane fusion is coupled to the energy released by the fusion proteins adopting a stable, low-energy postfusion state. Here we use oxidative footprinting of the parainfluenza virus 5 fusion protein to reveal new details of this critical event in the viral lifecycle. A greater understanding of the dynamic nature of these metastable proteins may reveal novel opportunities for the development of targeted therapeutics. To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.

On the stability of parainfluenza virus 5 F proteins

ABSTRACT The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.

Src Family Kinase Phosphorylation of the Motor Domain of the Human Kinesin-5, Eg5

Spindle formation in mammalian cells requires precise spatial and temporal regulation of the kinesin‐5, Eg5, which generates outward force to establish spindle bipolarity. Our results demonstrate that Eg5 is phosphorylated in cultured cells by Src family kinases (SFKs) at three sites in the motor head: Y125, Y211, and Y231. Mutation of these sites diminishes motor activity in vitro, and replacement of endogenous Eg5 with phosphomimetic Y211 in LLC‐Pk1 cells results in monopolar spindles, consistent with loss of Eg5 activity. Cells treated with SFK inhibitors show defects in spindle formation, similar to those in cells expressing the nonphosphorylatable Y211 mutant, and distinct from inhibition of other mitotic kinases. We propose that this phosphoregulatory mechanism tunes Eg5 enzymatic activity for optimal spindle morphology.

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Significance Paramyxovirus fusion proteins (F), critical for viral entry and infection, initially fold into a metastable prefusion state and, upon triggering, refold irreversibly to a stable postfusion state to physically mediate membrane fusion. The large-scale conformational changes that occur in the F-refolding pathway are understood, but a detailed structural understanding of F-protein metastability remains elusive. Here, stabilizing and destabilizing mutations of the parainfluenza virus 5 fusion protein were examined to reveal that the immobilization of the N-terminal helix stabilizes paramyxovirus prefusion F proteins. The N-terminal helix, the interaction of which with domain II appears to be a critical early step in the F-protein refolding pathway, presents a novel alternative target for structure-based antiviral therapeutics. Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin–neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.