Uncovering the Contributions of Charge Regulation to the Stability of Single Alpha Helices**

Abstract

The single alpha helix (SAH) is a recurring motif in biology. The consensus sequence has a di-block architecture that includes repeats of four consecutive glutamate residues followed by four consecutive lysine residues. Measurements show that the overall helicity of sequences with consensus E 4 K 4 repeats is insensitive to a wide range of pH values. Here, we use the recently introduced q-canonical ensemble, which allows us to decouple measurements of charge state and conformation, to explain the observed insensitivity of SAH helicity to pH. We couple the outputs from separate measurements of charge and conformation with atomistic simulations to derive residuespecific quantifications of preferences for being in an alpha helix and for the ionizable residues to be charged vs. un-charged. We find a clear preference for accommodating uncharged Glu residues within internal positions of SAHforming sequences. The stabilities of alpha helical conformations increase with the number of E 4 K 4 repeats and so do the numbers of accessible charge states that are compatible with forming conformations of high helical content. There is conformational buffering whereby charge state heterogeneity buffers against large-scale conformational changes thus making the overall helicity insensitive to large changes in pH. Further, the results clearly argue against a single, rod-like alpha helical conformation being the only or even dominant conformation in the ensembles of so-called SAH sequences.

Pervasive, conserved secondary structure in highly charged protein regions

“…These particular sequences show the hallmarks of so-called single alpha helices (SAH), helices of length 25–200 residues frequently, though not exclusively, formed by (E 4 K 4 ) n repeats [9,33,34]. Below, we establish the evolutionary conservation of these regions, suggesting their structure, as well as high charge density, are likely to confer a fitness benefit.…”

“…The notion that intrinsically disordered proteins or regions sometimes adopt structure is well-understood [34], particularly in the case of folding upon binding [48]. Because of this distinction between conformations in isolation versus when bound to a partner, structures in the PDB may tell only a portion of the story.…”

“…To answer these questions, we systematically identify highly charged regions proteome-wide in related eukaryotes (budding yeasts) and characterize their sequence properties, predicted structure, and evolution. In contrast to previous studies [3,9,10,34], our work examines the entire proteome, permitting us to quantify the frequency and, with proteome-scale homology, evolutionary conservation of charged regions at the genomic scale. We find that naturally occurring polyampholytes are highly prevalent and, despite being low-complexity with often-poor sequence conservation, these regions are often predicted to form or contain alpha-helices.…”

Emergent microenvironments of nucleoli

“…This is because long D/E tracts are likely to extract entropic penalties associated with organizing water molecules around ionic moieties [ 158 ]. Further, the high linear charge density engenders intramolecular electrostatic repulsions that extract penalties in terms of conformational entropy [ 159 , 160 ]. These entropic penalties can be offset by neutralizing some of the charge, and the most efficient way to achieve this is through proton binding, which leads to upshifts of pK a values of acidic groups – a sequence-encoded emergent property known as charge regulation [ 160 , 161 ].…”

Decoding optimal ligand design for multicomponent condensates

“…We hypothesized that the (PS(GS)4) linker following G75G76 in each Ub is too flexible to elicit change in conformation of M1-Ub4. Therefore, we installed sequences A(EA3K)3A or A(EA3K)6A which are predicted to be helical (Chen et al, 2013;Fossat et al) between the Ub units in a M1 (1-74) construct. SAXS data suggested that the structures of these M1-Ub4 constructs are significantly more extended compared to M1-Ub4 (1-76) as Rg values increased by nearly 10 Å (Table S2).…”