Net charge per residue modulates conformational ensembles of intrinsically disordered proteins

Abstract

Intrinsically disordered proteins (IDPs) adopt heterogeneous ensembles of conformations under physiological conditions. Understanding the relationship between amino acid sequence and conformational ensembles of IDPs can help clarify the role of disorder in physiological function. Recent studies revealed that polar IDPs favor collapsed ensembles in water despite the absence of hydrophobic groups-a result that holds for polypeptide backbones as well. By studying highly charged polypeptides, a different archetype of IDPs, we assess how charge content modulates the intrinsic preference of polypeptide backbones for collapsed structures. We characterized conformational ensembles for a set of protamines in aqueous milieus using molecular simulations and fluorescence measurements. Protamines are arginine-rich IDPs involved in the condensation of chromatin during spermatogenesis. Simulations based on the ABSINTH implicit solvation model predict the existence of a globule-to-coil transition, with net charge per residue serving as the discriminating order parameter. The transition is supported by quantitative agreement between simulation and experiment. Local conformational preferences partially explain the observed trends of polymeric properties. Our results lead to the proposal of a schematic protein phase diagram that should enable prediction of polymeric attributes for IDP conformational ensembles using easily calculated physicochemical properties of amino acid sequences. Although sequence composition allows the prediction of polymeric properties, interresidue contact preferences of protamines with similar polymeric attributes suggest that certain details of conformational ensembles depend on the sequence. This provides a plausible mechanism for specificity in the functions of IDPs.Monte Carlo | polyampholyte | polyelectrolyte I ntrinsically disordered proteins (IDPs) are a class of proteins that fail to fold autonomously in aqueous solutions to welldefined three-dimensional structures (1, 2). This "intrinsic disorder" has been implicated in a range of regulatory functions that require IDPs to interact with other macromolecular ligands (3-11). Many of these interactions promote disorder-to-order transitions within IDPs (3, 9), and different mechanistic models (3, 12, 13) have been proposed for coupled folding and binding. To develop a better understanding of how disorder is used in function (2), we have pursued quantitative, polymer-physics-based descriptions (14-16) for conformational ensembles of .Low hydrophobicity is a defining characteristic of IDP sequences (21,22). This suggests that IDPs cannot collapse to form compact, globular conformations in aqueous solutions (21). However, spectroscopic (23-27) and computational investigations (17, 28) have shown that polar tracts form heterogeneous ensembles of collapsed structures in aqueous solutions. These sequences are rich in uncharged, polar amino acids and are devoid of canonical hydrophobic residues. Collapse of polar tracts has been observed for polyglutamine (17...

Single-molecule spectroscopy of the unexpected collapse of an unfolded protein at low pH

“…4,9 In accord with physical intuition, unfolded polypeptide chains have been shown to expand with increasing net charge. 4,14,30,31,33,53 Based on these results, an increase in net charge, |Z|, of unfolded CspTm from 3.8 at pH 7 to 10.2 at pH 2.9 would therefore be expected to lead to an expansion of the chain, in contrast to our experimental observation. In the past, the effect of charges on the dimensions of polymers has often been treated as an additional contribution to the effective excluded volume of the chain, and several theories have been developed based on this concept to describe the behavior of polyelectrolytes and polyampholytes 30,31,54 (all of which are conceptually similar).…”

“…2(b)), indicating a continuous expansion of unfolded CspTm driven by interactions of the two denaturants with the polypeptide, with chain dimensions comparable to those obtained at pH 7. This result is surprising, since recent experiments 4, 9, 14 and Monte-Carlo simulations 53 suggest that the dimensions of unfolded proteins increase with increasing net charge of the chain; according to this effect, the high net charge of unfolded CspTm at pH 2.9 should lead to a pronounced expansion of the chain at low ionic strength. Our results imply that a change in the protonation state of the polypeptide by lowering the pH does not only increase the net charge, but must also alter other properties of the polypeptide chain.…”

“…Our most surprising observation is the lack of expansion of the unfolded protein when transferred from pH 7 to pH 2.9, even though the higher net charge of unfolded CspTm at pH 2.9 (+10.2) will strongly increase charge repulsion within the chain 4,9,14,53 (Fig. 2): unfolded CspTm at pH 2.9 exhibits R G values surprisingly close to those found at pH 7.…”

N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions

“…Indeed, IDPs are more susceptible to undergoing PTMs than folded proteins 1. Because of the inherent flexibility of these proteins, PTMs will have an important impact on their activity, cellular localization, and interaction properties, by modulating their structural dynamics 2, 3, 4, 5, 6, 7, 8. For example, N‐terminal acetylation of α‐synuclein9 increases the helical propensity of the N‐terminal segment7 and enhances the affinity of α‐synuclein for calmodulin by a factor of 10 10.…”

Letter to the Editor: A response to Horne and Lucey (2017)

“…We have repeated the hydrophobicity calculations on caseins using the scale of Kyte and Doolittle (Kyte and Doolittle, 1982). The use of this scale rather than any other is easy to justify because it is widely used in current protein science and, in conjunction with either the fractions of positively and negatively charged residues or normalised net charge, is used quite generally, and with a high success rate, to discriminate between folded proteins and IDPs (Uversky et al, 2000, Mao et al, 2010, Huang et al, 2014. Using the Kyte and Doolittle scale, or an optimised IDP hydropathy scale (Huang et al, 2014), caseins are predicted correctly to be IDPs and to be more hydrophilic, on average, than the great majority of globular proteins (Redwan et al, 2015).…”