Mutation of gp42 I159 does not affect binding affinity or kinetics with gHgL or HLA-DQ2. SathiyamoorthyKarthik JiangJiansen Xiong HuYao L. RoweCynthia S. MöhlBritta ChenJia JiangWei D. MellinsElizabeth LongneckerRichard ZhouZ. Hong S. JardetzkyTheodore 2014 <p>Kinetic binding experiments were conducted similarly to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-g002" target="_blank">Figure 2</a> on the OctetRED96. For panels A and B site-specifically biotinylated HLA-DQ2 (CLIP1) was immobilized on the streptavidin (SA) biosensor tip surfaces. Binding kinetics of HLA-DQ2 (ligand) with the single mutant gp42 C114S (A) or the gp42 I159C double mutant (B) are depicted. Global curve fitting with a 2∶1 heterogeneous ligand model closely matched the experimental data. The calculated binding curves are shown overlaid on the data from the experiment. For panels C and D, biotinylated EBV gHgL was immobilized on the streptavidin (SA) biosensor surfaces. Binding kinetics of EBV gHgL with the single mutant gp42 C114S (C) or I159C double mutant (D) are depicted. Overlay curves show the global fitting results using a 1∶1 Langmuir binding model. Kinetic and thermodynamic binding constants are similar to binding gp42 wildtype (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-g002" target="_blank">Figure 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004309#ppat-1004309-t002" target="_blank">Table 2</a>). (E) Representative class averages of gHgL/gp42-PEG2K/HLA-DQ2 complexes. The gp42 I159C mutant was labeled with PEG-2K maleimide and used to form complexes with gHgL and HLA-DQ2, which were by purified gel filtration chromatography. Representative angles between the two arms of the complexes formed by gHgL and gp42/HLA are indicated as well as the number of particles included in each class.</p>