An undergraduate biochemistry laboratory experiment has been developed using biolayer interferometry (BLI), an optical biosensing technique similar to surface plasmon resonance (SPR), in which students obtain and analyze kinetic data for a protein‐protein interaction. Optical biosensing is a technique of choice to determine kinetic and affinity constants for biomolecular interactions. Measurements can be made in real‐time without labels, making biosensing particularly appropriate for the teaching laboratory. In the described exercise, students investigate the kinetics of Protein A‐human Immunoglobin G binding under conditions that mimic simple 1:1 binding. Students prepare appropriate serial dilutions of IgG and set up a microplate for the experiment by aliquotting biotinylated Protein A, buffer, and IgG solutions. A commercial BLI sensor, the FortéBio Octet QK, is used to measure binding. While data are collected students prepare a spreadsheet with which they will simulate the data to determine kon, koff, and KD. Raw data from the sensor are then exported to the spreadsheets for analysis. Optimized experiment timing, regeneration methods and other parameters are described to increase throughput and reduce cost. The experiment is readily adaptable to other biosensing platforms such as SPR instruments. Biochemistry and Molecular Biology Education Vol. 38, No. 6, pp. 400‐407, 2010

While interactions among proteins of the flagellar Type III secretion (T3S) export apparatus have been reported, few have been examined with respect to the dynamic relationships that must govern the mechanism of export. Using biolayer interferometry (BLI), an optical biosensing method, we have undertaken a survey of protein‐protein interactions among the soluble apparatus proteins (FlhAc, FlhBc, FliH, FliI, FliJ, FliK) and between apparatus proteins and export substrates. Proteins were purified prior to screening for binding partners. One of each potential pair was immobilized and then exposed to others in solution. Interactions identified by yes/no screens were subjected to rigorous characterization to measure affinity and rate constants. Our screen may uncover important interactions heretofore unknown because they are of insufficient affinity for copurification or affinity blotting experiments. Characterization will also allow us to pursue efforts such as identifying export signals in substrates by searching for kinetic changes in variant constructs. We are attempting to observe more‐than‐pairwise interactions. Kinetic characterization of dynamic interactions will allow us develop a better understanding of T3S. This work was supported by grants from the Public Health Service (R15GM080701), NSF (CHE 0922699), the Research Corporation (CC6900).

Using two optical biosensing methods, surface plasmon resonance and biolayer interferometry, a study of the interactions among proteins involved in flagellar type III secretion was undertaken to understand the mechanism of substrate delivery to the membrane‐embedded export complex. An interaction between C‐ring protein FliN and export apparatus protein FliH has been proposed to be a mode of targeting substrates via a complex that is necessarily dynamic as the position of FliN at the base of the C‐ring is too far from the membrane to allow the complex to remain associated during export. Both techniques allow quantitative measurement of association and dissociation. Rate and affinity constants are reported for pairwise interactions; both FliN‐FliH and FliH‐FliI have submicromolar KDs. Experiments investigating the conditions necessary to dissociate FliN from FliH or FliH from FliI are described. Substrates were screened for binding to FliH and FliI in the presence and absence of FliN. Kinetic analysis will allow us to rank affinities, determine how binding changes in the presence of other participants, search for export signals within substrates etc., developing a better understanding of type III secretion. This work was supported by grants from the Public Health Service (R15GM080701), NSF (CHE 0922699), the Research Corporation (CC6900) and a KSU Mentor‐Protégé Grant (to J.L.W.).