Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Abstract

Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.

Flexible linkers in CaMKII control the balance between activating and inhibitory autophosphorylation

“…A key idea behind this model is that we assume that the longer linker in CaMKII-b slows down the rates of trans-phosphorylation reactions within a holoenzyme when compared to CaMKII-Page 12 of 55 a, but that the rates of cis-phosphorylation are the same as in the two isoforms (Sørensen and Kjaergaard, 2019).…”

Linker dependence of avidity in multivalent interactions between disordered proteins

“…Recently, we developed a biosensor that allows measurement of intra-molecular effective concentrations. 15 We adapted this system by introducing mClover3 N-terminally in A/P and mRuby3 26 compete with the intra-molecular complex and force ring opening. To allow the free peptide to compete efficiently, we introduced a mutation in the ACTR domain of A/P (L17A) to weaken the interaction.…”

“…We recently showed that the sequence of such www.biophysics.dk -9 -linkers has a large effect on effective concentrations due to attractive or repulsive interactions. 15 Linker-linker interactions may play important roles in multivalent disordered proteins, but such interactions are undesirable for the purpose of establishing a theoretical baseline. Ideally, we want linkers that solely act as tunable entropic spacers.…”

“…Preparation of DNA constructs. Constructs were based on a previously developed biosensor for effective concentrations, 15 and created by insertion of synthetic genes (Genscript) into either this construct or between the NdeI and BamHI sites of a pET15b vector. The full sequences of the fusion proteins are given in the supplementary materials.…”

“…An advantage of formulating avidity in terms of effective concentrations is that it can be measured independently of the overall binding reaction through competition experiments. 14,15 In disordered proteins, effective concentration are practically never measured experimentally, but are typically estimated from polymer models. For a bivalent interaction, the avidity binding constant has often successfully been expressed as: 16,17 ",$%$&' = "* "+ -..…”