Interaction of fusion peptides from SARS-CoV S glycoprotein with membranes

Author(s)

Luis Guilherme Mansor Basso

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2014-03-24

Examining board members

Antônio José da Costa Filho; Fabio Ceneviva Lacerda de Almeida; Eduardo Ribeiro de Azevêdo; Eduardo Maffud Cilli; Karin do Amaral Riske

• Advisor: Antônio José da Costa Filho

Abstract

This thesis has the general goal of consolidating in our group the use of site directed spin labeling along with pulsed electron spin resonance, in particular double electron-electron resonance (DEER), for distance measurements in biological molecules. Our specific goal is to obtain structural information on two peptides belonging to the fusion domain of the spike glycoprotein from the SARS coronavirus when in the presence of membrane model systems. We used a joint approach involving spectroscopic, calorimetric, and computational techniques to monitor conformational changes in the peptides, their most representative conformations and their effects on the structure of model membranes. Calorimetric results showed that the peptides strongly perturb the thermotropic behavior of zwitterionic and negatively-charged lipid vesicles, with the largest effects seen with the later. Not only the charge, but also the lipid headgroup structure seems to be relevant for the energetics of the interaction. Circular dichroism experiments showed that the peptides present high conformational flexibility, assuming different secondary structures in diverse environments. A mixture of conformation of SARSFP coexists in aqueous solutions and in the membrane models, suggesting large structural plasticity. This peptide also showed high auto-association tendency forming β structures and/or regular β aggregates. On the other hand, the SARSIFP peptide is predominantly an α-helix when in micelles, β structures in lipossomes, and assumes irregular conformations in water. In particular, this peptide seems to bind to membranes as an α-helix, transitioning to β structures in high concentrations. The conformational flexibility of the peptides was also studied by molecular dynamics (MD). Helices, β structures, turns, hairpins and irregular structures are visited during the trajectories of both peptides, but SARSIFP presents less structural flexibility. The free energy profile is consistent with that of a plane, broad and shallow surface, without large energetic barriers separating the different conformational states. The thermal energy at 300 K is sufficient to make the peptides visit most of the conformational space accessible for a certain choice of order parameters. Lastly, we obtained structural information from paramagnetic analogs of the peptides, which were doubly-labeled with nitroxide radicals, in different solvents and membrane mimetics. We also studied the oligomerization process of singly-labeled analogs. The distance distributions determined from the DEER traces showed that SARSIFP adopts a primarily α-helix conformation in the presence of the mimetics, with the formation of β structures in the presence of negative micelles. DEER results for SARSFP showed a broad distribution of distances in all membrane mimetics, thus reflecting the coexistence of compact conformations. Singly-labeled analogs revealed the high tendency of formation of oligomers in SDS-d25. Our results allowed us to make considerable progress in understanding the conformations of the peptides in the conditions under investigation, which contributed with relevant information on the early steps of the membrane fusion mechanism carried out by the spike glycoprotein from the SARS coronavirus. (AU)

• FAPESP's process: 09/10997-7 - Interactions between model membranes and fusion peptides derived from SARS CoV glycoprotein s
• Grantee: Luís Guilherme Mansor Basso
• Support Opportunities: Scholarships in Brazil - Doctorate