title: Studying Protein-Protein Binding through T-Jump Induced Dissociation: Transient 2D IR Spectroscopy of Insulin Dimer.
abstract: Insulin homodimer associates through the coupled folding and binding of two partially disordered monomers. We aim to understand this dynamics by observing insulin dimer dissociation initiated with a nanosecond temperature jump using transient two-dimensional infrared spectroscopy (2D IR) of amide I vibrations. With the help of equilibrium FTIR and 2D IR spectra, and through a systematic study of the dependence of dissociation kinetics on temperature and insulin concentration, we are able to decompose and analyze the spectral evolution associated with different secondary structures. We find that the dissociation under all conditions is characterized by two processes whose influence on the kinetics varies with temperature: the unfolding of the β sheet at the dimer interface observed as exponential kinetics between 250 and 1000 μs and nonexponential kinetics between 5 and 150 μs that we attribute to monomer disordering. Microscopic reversibility arguments lead us to conclude that dimer association requires significant conformational changes within the monomer in concert with the folding of the interfacial β sheet. While our data indicates a more complex kinetics, we apply a two-state model to the β-sheet unfolding kinetics to extract thermodynamic parameters and kinetic rate constants. The association rate constant, ka (23 °C) = 8.8 × 10(5) M(-1) s(-1) (pH 0, 20% EtOD), is approximately 3 orders of magnitude slower than the calculated diffusion limited association rate, which is explained by the significant destabilizing effect of ethanol on the dimer state and the highly positive charge of the monomers at this pH.
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, and the James Franck Institute, The University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Xin-Xing
authors.initials: XX
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Jones
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, and the James Franck Institute, The University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Ann
authors.initials: A
authors.lastname: Fitzpatrick
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors.forename: Chunte Sam
authors.initials: CS
authors.lastname: Peng
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, and the James Franck Institute, The University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Chi-Jui
authors.initials: CJ
authors.lastname: Feng
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, and the James Franck Institute, The University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Baiz
authors.affiliation: Department of Chemistry, Institute for Biophysical Dynamics, and the James Franck Institute, The University of Chicago , Chicago, Illinois 60637, United States.
authors.forename: Andrei
authors.initials: A
authors.lastname: Tokmakoff
mesh_terms: D000818:Animals; D002417:Cattle; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
chemical_list: D007328:Insulin; C034329:insulin dimers