Direct Observation of Insulin Association Dynamics with Time-Resolved X-ray Scattering

Abstract

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.

Figure 1

(A) Protein sample TRXSS signal at 5 µs time delay fit with the signal from T-jump in pure buffer. (B) Temperature (red) and density (black) hydrodynamic response of the buffer derived from fitting of pure buffer TRXSS signal to protein sample TRXSS data as a function of time from an initial temperature of 35 °C.

Figure 2

(A) Characteristic TRXSS difference curves at various time delays after T-jump, presented as time series. (B) (Top) Normalized integrated signal in the region of q < 0.04 Å⁻¹ (magenta) and 0.04 Å⁻¹ < q < 0.2 Å⁻¹ (black). (Bottom) Population dynamics derived from kinetic analysis for associated species along with the scaled temperature derived from fitting of pure buffer TRXSS signal to the protein TRXSS signal. (C) Species-associated TRXSS difference patterns from kinetic analysis; the signal at q > 0.2 Å⁻¹ is multiplied by a factor of 10 to magnify the observed WAXS features. The legend is shared for both B and C panels.

Figure 3

Comparison of 2M state TRXSS signal with signal calculated from steady-state scattering between 45 and 35 °C and theoretical calculation from crystal structure. The signals are magnified ×70 in the q > 0.2 Å region for clarity.

Figure 4

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.