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.

Insulin dissociates by diverse mechanisms of coupled unfolding and unbinding

“…As mentioned in the introduction, Chen and coworkers found evidence for a partiallysolvated intermediate with dimer-like secondary structure, implying conserved interfacial β sheet character. 29 Although we find no distinct free energy basin that obviously corresponds to this intermediate, the initial partially solvated structures we observe along the α path are consistent with these data, as the interfacial β sheet is conserved. However, even with the loss of the β sheet along the β path, our simulations do not rule out the possibility of structures along the β path also being consistent with the X-ray scattering data.…”

“…Moreover, time resolved X-ray scattering data suggest at least one intermediate in the insulin dimer dissociation that involves quick solvent uptake by a species with dimer-like secondary structure, coincident with a slight increase in molecular volume. 29 To investigate the possible presence of a similar feature in our simulations, we defined three collective variables; in order of increasing specificity, they are (i) the total molecular volume, (ii) the solvent accessible surface area (SASA) of eight residues that make up the hydrophobic core of the interface (Val B12 , Tyr B16 , Phe B24 , Tyr B26 on each monomer), and (iii) the number of native interfacial hydrogen bonds, which only form between Phe B24 and Tyr B26 . The total molecular volume, probed by the X-ray scattering, can reflect solvent uptake, but it can also correspond to large-scale conformational change.…”

“…25 Moreover, this detached state also corresponds to an increase in molecular volume of between 0.6 and 0.8 nm 3 , which is consistent with the evidence from Chen and coworkers for a second intermediate that corresponds to a large increase in molecular volume while maintaining secondary structure. 29 The α and β path correspond to induced fit and conformational selection. In this section, we connect the intramonomeric detachment and core solvation with the intermonomeric rotations discussed earlier to characterize the various mechanisms of the insulin dimer dissociation, making explicit comparisons to the induced fit and conformational selection models of coupled folding and binding.…”

“…By visual analysis, we selected dissociation paths that both led to complete dissociation and did not involve significant unfolding of the monomers since there is limited experimental evidence for extensive loss of secondary structure. 25,29 We also only selected paths that had maximum free energies within the range of previous simulations. 19,30 These were used to initialize string method simulations in the 22-dimensional space of intermonomeric contacts described above (Supplemental Table S1).…”

“…24,25 Time-resolved X-ray scattering data also suggest an intermediate with conserved secondary structure on the timescale of 900 ns. 29 These experiments, although mechanistically suggestive, provide limited structural information; complementary simulations are needed to microscopically interpret these data.…”