Disordered proteins mitigate the temperature dependence of site-specific binding free energies

Biophysical characterization of protein-protein interactions involving disordered proteins is challenging. A common simplification is to measure the thermodynamics and kinetics of disordered site binding using peptides containing only the minimum residues necessary. We should not assume, however, that these few residues tell the whole story. Son of sevenless (Sos), a multidomain signaling protein from Drosophila melanogaster, is critical to the mitogen-activated protein kinase pathway, passing an external signal to Ras, which leads to cellular responses. The disordered 55 kDa C-terminal domain of Sos is an auto-inhibitor that blocks guanidine exchange factor activity. Activation requires another protein, Downstream of receptor kinase (Drk), which contains two Src homology 3 (SH3) domains. Here, we utilize nuclear magnetic resonance spectroscopy and isothermal titration calorimetry to quantify the thermodynamics and kinetics of the N-terminal SH3 domain binding to the strongest sites incorporated into the flanking disordered sequences. Comparing these results to those for isolated peptides provides information about how the larger domain affects binding. The affinities of sites on the disordered domain are like those of the peptides at low temperatures but less sensitive to temperature. Our results, combined with observations showing that IDPs become more compact with increasing temperature, suggest a mechanism for this effect

Mechanosensitive Self-Replication Driven by Self-Organization

Self-replicating molecules are likely to have played an important role in the origin of life, and a small number of fully synthetic self-replicators have already been described. Yet it remains an open question which factors most effectively bias the replication toward the far-from-equilibrium distributions characterizing even simple organisms. We report here two self-replicating peptide-derived macrocycles that emerge from a small dynamic combinatorial library and compete for a common feedstock. Replication is driven by nanostructure formation, resulting from the assembly of the peptides into fibers held together by β sheets. Which of the two replicators becomes dominant is influenced by whether the sample is shaken or stirred. These results establish that mechanical forces can act as a selection pressure in the competition between replicators and can determine the outcome of a covalent synthesis.

Nature and regulation of protein folding on the ribosome

Co-translational protein folding is an essential process by which cells ensure the safe and efficient production and assembly of new proteins in their functional native states following biosynthesis on the ribosome. In this review, we describe recent progress in probing the changes during protein synthesis of the free energy landscapes that underlie co-translational folding and discuss the critical coupling between these landscapes and the rate of translation that ultimately determines the success or otherwise of the folding process. Recent developments have revealed a variety of mechanisms by which both folding and translation can be modulated or regulated, and we discuss how these effects are utilised by the cell to optimise the outcome of protein biosynthesis. [Abstract copyright: Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B

Morphology-Dependent Interactions between α-Synuclein Monomers and Fibrils

Amyloid fibrils may adopt different morphologies depending on the solution conditions and the protein sequence. Here, we show that two chemically identical but morphologically distinct α-synuclein fibrils can form under identical conditions. This was observed by nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy, as well as by cryo-transmission electron microscopy (cryo-TEM). The results show different surface properties of the two morphologies, A and B. NMR measurements show that monomers interact differently with the different fibril surfaces. Only a small part of the N-terminus of the monomer interacts with the fibril surface of morphology A, compared to a larger part of the monomer for morphology B. Differences in ThT binding seen by fluorescence titrations, and mesoscopic structures seen by cryo-TEM, support the conclusion of the two morphologies having different surface properties. Fibrils of morphology B were found to have lower solubility than A. This indicates that fibrils of morphology B are thermodynamically more stable, implying a chemical potential of fibrils of morphology B that is lower than that of morphology A. Consequently, at prolonged incubation time, fibrils of morphology B remained B, while an initially monomorphic sample of morphology A gradually transformed to B

From Selection to Instruction and Back: Competing Conformational Selection and Induced Fit Pathways in Abiotic Hosts

Two limiting cases of molecular recognition, induced fit (IF) and conformational selection (CS), play a central role in allosteric regulation of natural systems. The IF paradigm states that a substrate “instructs” the host to change its shape after complexation, while CS asserts that a guest “selects” the optimal fit from an ensemble of preexisting host conformations. With no studies that quantitatively address the interplay of two limiting pathways in abiotic systems, we herein and for the first time describe the way by which twisted capsule M-1, encompassing two conformers M-1(+) and M-1(−), trap CX4 (X=Cl, Br) to give CX4⊂M-1(+) and CX4⊂M-1(−), with all four states being in thermal equilibrium. With the assistance of 2D EXSY, we found that CBr4 would, at its lower concentrations, bind M-1 via a M-1(+)→M-1(−)→CBr4⊂M-1(−) pathway corresponding to conformational selection. For M-1 complexing CCl4 though, data from 2D EXSY measurements and 1D NMR line-shape analysis suggested that lower CCl4 concentrations would favor CS while the IF pathway prevailed at higher proportions of the guest. Since CS and IF are not mutually exclusive, we reason that our work sets the stage for characterizing the dynamics of a wide range of already existing hosts to broaden our fundamental understanding of their action. The objective is to master the way in which encapsulation takes place for designing novel and allosteric sequestering agents, catalysts and chemosensors akin to those found in nature

Probing the dynamic stalk region of the ribosome using solution NMR

Abstract: We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the CTD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome–nascent chain complexes

In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-Synuclein within E. coli cells

α-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson’s disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, α-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution

The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes

Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson’s disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosome-associated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF–RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery