To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably called HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. In parainfluenza virus 5 (PIV5), upon binding its receptor sialic acid, HN 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 (PIV5) F was triggered to refold using temperature and 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 tandem mass spectrometry. Globally, metastable prefusion F is more extensively oxidized than postfusion F, indicating that the prefusion state is more solvent exposed and flexible. A comparison of peptide oxidation levels to solvent accessible surface area (SASA) values 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 atomic model of a double mutant of PIV5 F solved by x-ray crystallography is also presented. Mutagenesis guided by this atomic model suggests that S443P destabilizes prefusion PIV5 F by both disrupting a hydrogen bond network at the base of the globular head and by steric and electrostatic clashes. P22L stabilizes the prefusion state through interaction of the side chain with a conserved hydrophobic pocket on the side of the globular head of F, suggesting a conserved means of stabilizing paramyxovirus prefusion F proteins. The work presented here represents a dramatic step forward in understanding the dynamic function of these critical viral molecular machines.
|Advisor:||Lamb, Robert A., Mondragon, Alfonso|
|Commitee:||Kelleher, Neil, Longnecker, Richard|
|Department:||Interdepartmental Biological Sciences Program|
|School Location:||United States -- Illinois|
|Source:||DAI-B 76/02(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Virology, Biophysics|
|Keywords:||Metastable, Paramyxovirus, Protein, Refolding|
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