Does liquid-liquid phase separation drive peptide folding?

Abstract

Proline-arginine (PR) dipeptide repeats have been shown to undergo liquid-liquid phase separation and are an example of a growing number of intrinsically disordered proteins that can assemble into membraneless organelles. These structures have been posited as nucleation sites for pathogenic protein aggregation. As such, a better understanding of the effects that the increased local concentration and volumetric crowding within droplets have on peptide secondary structure is necessary. Herein we use Fourier transform infrared (FTIR) and two-dimensional infrared (2DIR) spectroscopy to show that formation of droplets by PR₂₀ accompanies changes in the amide-I spectra consistent with folding into poly-proline helical structures.

Fig. 1. (a) A cartoon of flexible protein segments undergoing phase separation through volumetric exclusion induced by a crowding agent, polyethylene glycol (PEG). (b) PR₂₀ phase separated droplets can be seen after the addition of PEG 300 using phase contrast microscopy. Inset shows a PR₂₀ sample before addition of PEG 300. Scale bars represent 10 μm.

Fig. 2. The FTIR spectra of the PR₂₀ peptide taken in deuterated potassium phosphate buffer, with and without 30% PEG, as indicated. Peaks at 1585 and 1610 cm⁻¹ comprise the arginine side-chains while a peak near 1650 cm⁻¹ originates from the amide-I stretch of randomly coiled peptide. Upon addition of PEG, the peak in the amide-I region undergoes a change corresponding to folding from a random coil state.

Fig. 3. (a) The 2DIR spectrum of PR₂₀ without PEG shows a prominent peak corresponding to random-coils as well as the two arginine peaks. (b) After adding PEG, the spectrum broadens along the diagonal. (c) The difference spectrum of the first two spectra. The large peak at 1600 cm⁻¹ originates from PEG. The two peaks at pump frequencies 1630 and 1665 cm⁻¹ of the amide-I region matches the peak locations for polyproline helices. For all spectra, the dotted line indicates where the diagonal cuts (panels to the right) are taken.

Fig. 4. Comparison of the experimental difference 2DIR spectrum (top) and the sum of simulated 2DIR spectra for PPI and PPII helices (bottom), demonstrating the amide-I region of the former is well described by the latter.