Vesicular stomatitis virus glycoprotein G (VSV-G) belongs to a new class of viral fusion proteins (Class III). The structure of VSV-G has been solved in two different conformations and fusion is known to be triggered by low pH. To investigate Class III fusion mechanisms, molecular dynamics simulations were performed on the VSV-G prefusion structure in two different protonation states: at physiological pH (pH 7) and low pH present in the endosome (pH 5). Domain IV containing the fusion loops, which need to interact with the target membrane, exhibits the highest mobility. Energetic analyses revealed weakened interaction between Domain IV and the protein core at pH 5, which can be attributed to two pairs of structurally neighboring conserved and differentially protonated residues in the Domain IV–core interface. Energetic calculations also demonstrated that the interaction between the subunits in the core of the trimeric VSV-G is strengthened at pH 5, mainly due to newly formed interactions between the C-terminal loop of Domain II and the N-terminus of the adjacent subunit. A pair of interacting residues in this interface that is affected by differential protonation was shown to be the main effectors of this phenomenon. The results of this study thus enhance the mechanistic understanding of the effects of protonation changes in VSV-G. Proteins 2012. © 2012 Wiley Periodicals, Inc.