Insight into Coupled Binding and Folding in Insulin Dimer Association from T-Jump Induced Dissociation Experiments

Molecular recognition in protein-protein interactions is a crucial element of the biological functions of proteins, involving a number of remarkable conformational changes. The association of insulin into a homodimer is a particularly interesting example due to the coupled folding and binding processes involved. After initiating insulin dimer dissociation with a nanosecond temperature jump, transient infrared spectra show a fast and a slow response that occurs on sub-µs and sub-ms time scales. Using a rate distribution map analysis reflecting both spectral and time evolution, we assign these processes to the partial melting of α helical structure followed by the interfacial β sheet unfolding. Rate distributions variations are observed by varying the initial temperature, pH, and co-solvent. To extract thermodynamic parameters and kinetic rate constants, the T-jump data is analyzed self-consistently with FTIR, CD, and isothermal titration calorimetry. By analyzing kinetic models, we conclude that insulin dimer dissociation requires significant conformational changes within the monomer in concert with the folding of the interfacial β sheet in order to dimerize.