Combining ns temperature-jump pump with X-ray pulse probe: study of structural dynamics in insulin

Biological functions frequently require protein–protein interactions that involve secondary and tertiary structural perturbation. Here we study protein–protein dissociation and reassociation dynamics in insulin, a model system for protein oligomerization. Insulin dimer dissociation into monomers was induced by a nanosecond temperature-jump (T-jump) of ∼8 °C in aqueous solution, and the resulting protein and solvent dynamics were tracked by time-resolved X-ray solution scattering (TRXSS) on time scales of 10 ns to 100 ms. The protein scattering signals revealed the formation of five distinguishable transient species during the association process that deviate from simple two-state kinetics. Our results show that the combination of T-jump pump coupled to TRXSS probe allows for direct tracking of structural dynamics in nonphotoactive proteins.

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Rimmerman, D., Leshchev, D., Hsu, D. J., Hong, J., Kosheleva, I., and Chen, L. X.
Direct Observation of Insulin Association Dynamics with Time-Resolved X-ray Scattering.
J. Phys. Chem. Lett. 4413–4418 (2017)

Free-energy surfaces for insulin dissociation reaction as a result of T-jump. The red lines represent the nonequilibrium population immediately after T-jump. The yellow line shows the ensemble pathway toward dissociation as the system is driven back toward equilibrium.