Insights into the mechanism of membrane fusion induced by cytoplasmatic dehydrogenases: role of the positively charged crevices

CHEHÍN RN

Málaga- España

Conferencia; Internacional Workshop oin Collaborative Bioinformatics- EMBnet-Rib; 2007

EMBnet

The fusion of biological membranes is one of the most fundamental reactions of life. Although the precise mechanism of protein mediated membrane fusion is not yet fully understood, it is becoming clear that fusion proteins are nanomachines that exert mechanical force upon the membranes destined to fuse. It involves the initial recognition of the membranes, the close apposition of the membranes followed by the generation of nonbilayer intermediates, and finally the opening of an aqueous fusion pore. While the intermediates in various fusion reactions share common features, diverse classes of proteins evolved independently to carry out the task. Structural and functional analyses have also revealed similarities between viral and intracellular fusion where folding of a four helical bundle leads to the apposition of two membranes and provides the energy for the fusion reaction. During the viral fusion process, the interaction with the target membrane involves an hydrophobic stretch of about 15 residues called “the fusion peptide”. Several NAD(P)H dehydrogenases can promote the aggregation and fusion of phospholipid vesicles despite of they not have the characteristic four helix bundle topology neither the fusion peptide and then, they constitute a suitable and simple model to study the molecular process. Sequence analyses indicate the absence of any conserved region attributable to the fusogenic capability. In order to shed light on the energetic and structural requirements of these redox proteins to induce membrane fusion, the first steps of the fusion process i.e. protein membrane binding and membranes docking were dissected and subjected to in silico free energy calculations and structural analysis of the protein-membrane system.  In this way, finite difference Poisson Boltzmann calculations are used to describe the electrostatic interaction between proteins and phospholipid membranes. The most probable orientation of fusion proteins over the membrane was deduced from the electrostatic free energy of protein-membrane association. The aggregation capability of proteins was predicted by using a bidimentional representation of the free energy of association as a function of the protein rotation angles. Fluorescence spectroscopy assays were used as counterpart to validate the theoretical predictions. The hemifusion intermediate was not detected but membrane perturbation, crucial to trigger this event was studied and a relationship between previously undescribed deep positive crevices and membrane destabilization was suggested.