Soft interactions and volume exclusion by polymeric crowders can stabilize or destabilize transient structure in disordered proteins depending on polymer concentration

Abstract

The effects of macromolecular crowding on the transient structure of intrinsically disordered proteins is not well-understood. Crowding by biological molecules inside cells could modulate transient structure and alter IDP function. Volume exclusion theory and observations of structured proteins suggest that IDP transient structure would be stabilized by macromolecular crowding. Amide hydrogen exchange (HX) of IDPs in highly concentrated polymer solutions would provide valuable insights into IDP transient structure under crowded conditions. Here, we have used mass spectrometry to measure HX by a transiently helical random coil domain of the activator of thyroid and retinoid receptor (ACTR) in solutions containing 300 g L and 400 g L of Ficoll, a synthetic polysaccharide, using a recently-developed strong cation exchange-based cleanup method [Rusinga, et al., Anal Chem 2017;89:1275-1282]. Transiently helical regions of ACTR exchanged faster in 300 g L Ficoll than in dilute buffer. In contrast, one transient helix exchanged more slowly in 400 g L Ficoll. Nonspecific interactions destabilize ACTR helicity in 300 g L Ficoll because ACTR engages with the Ficoll polymer mesh. In contrast, 400 g L Ficoll is a semi-dilute solution where ACTR cannot engage the Ficoll mesh. At this higher concentration, volume exclusion stabilizes ACTR helicity because ACTR is compacted in interstitial spaces between Ficoll molecules. Our results suggest that the interplay between nonspecific interactions and volume exclusion in different cellular compartments could modulate IDP function by altering the stability of IDP transient structures. Proteins 2017; 85:1468-1479. © 2017 Wiley Periodicals, Inc.

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

“…MAP2c, p21 Cip1 , and FlgM show global compaction and local structuring in crowded conditions . The distal helix of calcineurin and transiently helical regions of ACTR are also stabilized when crowded by synthetic polymers . However, conformational compaction induced by crowders is not necessary to promote secondary structure formation for IDPs/ IDRs.…”

“…164,165 The distal helix of calcineurin and transiently helical regions of ACTR are also stabilized when crowded by synthetic polymers. 166,167 However, conformational compaction induced by crowders is not necessary to promote secondary structure formation for IDPs/ IDRs. For example, α-Casein, the C-terminal activation domain of c-Fos, and the kinase-inhibition domain of p27 Kip1 shows little structural changes under crowded conditions.…”

Depletion interactions modulate the binding between disordered proteins in crowded environments

“…The cellular milieu, densely packed with globular and polymeric macromolecules [12][13][14] , is thus also expected to influence the conformational distributions and dynamics of IDPs. Experimental evidence, simulations, and theory suggest that the effects of such macromolecular crowding are moderate but detectable, including the compaction and local structure formation of unfolded and intrinsically disordered proteins 8,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] , which may have important effects on their function. However, a quantitative understanding of the effects of crowding on IDPs is largely lacking.…”

Atomistic Modeling of Intrinsically Disordered Proteins Under Polyethylene Glycol Crowding: Quantitative Comparison with Experimental Data and Implication of Protein–Crowder Attraction

“…Furthermore, NMR relaxation data suggested weak interactions of N- and C-terminal residues with cytoplasmic components in mammalian cells, which were partially recapitulated by protein crowders but not polymer crowders . Recently hydrogen-exchange mass spectrometry (HX-MS) showed faster exchange throughout the binding domain of the activator for thyroid hormone and retinoid receptors (ACTR) at 300 g/L Ficoll but slower exchange for one segment at 400 g/L Ficoll . The former observation was interpreted as indicating ACTR expansion, whereas the latter observation could be due to Ficoll-induced ACTR aggregation, as found for other IDPs. , Techniques such as FRET and SANS report on gross properties (e.g., FRET efficiency and scattering profile) and hence lack the power to resolve conformational ensembles.…”