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.

Keywords: effective concentration; flexible linker; fluorescent biosensor; intrinsically disordered protein; polymer physics.

Fig. 1.

A FRET biosensor for measuring effective concentrations. (A) Titration with free MBD2 peptide displaces the intramolecular interaction and results in a decrease in the FRET efficiency. (B) Structure of the interaction pair in the fusion protein: the antiparallel coiled coil formed between MBD2 and p66α (PDB ID code 2L2L) (39). (C) SDS/PAGE gel of the purified fusion protein reveals internal cleavage sites in mRuby3. The cleavage products cannot be removed, but do not affect measurements.

Fig. 2.

Measurement of effective concentrations. (A) Titration curves of fusion proteins containing GS-linkers of variable length reveal that the effective concentration scales monotonously with linker length. (B) The correction factor corresponding to the difference in affinity between WT and V227A was determined by titration of fusion protein GS₁₂₀ containing the V227A mutation with both competitor peptides. (C) Effective concentrations follow a power law as revealed by the straight line in a log-log plot. The scaling exponent that is the focus of the remainder of the manuscript is derived from nonlinear fitting as indicated. (D) The expected geometric relation between scaling exponents for protein size, ν, and effective concentration. The linker defines a volume in which tethered ligand can diffuse, and the effective concentration is inversely proportional to this diffusion volume. Error bars represent 95% confidence interval.

Fig. 3.

Net charge per residue dominates the scaling of effective concentration with linker length. (A) Net charge was uniformly distributed into a background of GS-linkers. The same pattern is used for subsequent linker series. (B) Measurement of effective concentration allows determination of scaling exponents for each linker composition. Error bars represent 95% CI. (C) Scaling exponents demonstrate a linker expansion with increasing net charge. Compared to the negatively charged residues, arginine expands the chain less and lysine more. Error bars represent SE.

Fig. 4.

The effect of polyampholyte strength and proline, leucine, and tyrosine residues on scaling exponents. (A) Scaling exponents from neutral polyampholyte linkers with an equal proportion of glutamate residues and either lysine or arginine. The scaling exponents show a strong compaction to a globular chain for polyampholytes containing arginine only. (B) Chain expansion shows a complex dependence on proline content. At low fractions of proline residues, proline residues lead to linker expansion, which may be reversed at high proline fractions. (C) Hydrophobicity was increased by introduction of leucine residues, but led to practically no change in chain compaction. (D) Tyrosine residues led to strong compaction, likely due to π-interactions. Error bars are SE estimated from the fit.

Fig. 5.

Lack of additivity of individual contributions. (A) Proline or leucine residues were introduced in equal proportion to glutamate residues, but the scaling coefficient follows that of the glutamate-only series. This suggests that the expansion caused by net charge and chain stiffness are not additive. (B) Charge expansion caused by glutamate against a constant fraction of 0.1 of leucine residues shows that hydrophobicity has a negligible effect. (C) Increasing fractions of leucine only has a negligible effect on the dimensions of the linkers expanded by charge. Error bars represent SE.