A general goal of the Molecular Interactions Research Group (MIRG) is to facilitate implementation of technologies for quantitative characterization of molecular interactions: What is the affinity of a given interaction? How is the affinity regulated by cofactors or other conditions?
The MIRG has completed the experimental leg of their first study (MIRG 2002 Study), and the results were presented in summary form at ABRF 2002 in Austin. The goals of the MIRG 2002 study were (1) to compare the methods used by core facilities to characterize biomolecular interactions (i.e., differences between technologies, analytical methods, sample preparation, instrumentation variability within each technology, etc.); (2) to identify areas that need improvement; and (3) to define the extent to which two or more capabilities are available in one core or location. The model interaction system sent out by the MIRG and studied by more than 50 academic, government, and industry labs was the interaction of a small molecule inhibitor, carboxybenzenesulfonamide (CBS), with the protein carbonic anhydrase. The system was studied by isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and analytical ultracentrifugation (AUC) technologies. Participants were asked to measure one or more of the following: (1) affinity, molar binding ratio, and apparent binding enthalpy change by ITC; (2) affinity and kinetics by SPR; and (3) molecular mass, homogeneity, and assembly state by AUC, using equilibrium and velocity approaches. A summary of the results for the MIRG 2002 study is available at www.abrf.org. A manuscript giving a more comprehensive analysis of the results is in preparation by the MIRG.
The MIRG is now developing a study that we plan to conduct later this year. The ideal system we are developing is a protein–protein interaction whose affinity can be varied over a few of orders of magnitude by changes in conditions. There are a couple of scientific reasons for having a variable-affinity protein–protein system. First, it will allow testing of specific limitations of different technologies with one protein–protein pair. For example, a suitable Kd value that can be readily measured by AUC might be in the 100 nM range, whereas a low nanomolar or subnanomolar Kd value might be just what is needed for testing the limits of ITC and SPR capabilities. Second, a variable-affinity protein–protein system would allow different questions about a given technology to be answered with the same protein–protein system. What is the weakest affinity that can be measured by a given technology? The tightest? Having a single protein–protein interacting pair for the MIRG studies reduces our workload and costs by keeping the number of expression, purification, and formulation conditions to a minimum.