Atomic resolution conformational dynamics of intrinsically disordered proteins from NMR spin relaxation

AbstractIntrinsically disordered proteins (IDPs) constitute a large fraction of the human proteome and are critical in the regulation of cellular processes. A detailed understanding of the conformational dynamics of IDPs could help to elucidate their roles in health and disease. However the inherent flexibility of IDPs makes structural studies and their interpretation challenging. Molecular dynamics (MD) simulations could address this challenge in principle, but inaccuracies in the simulation models and the need for long simulations have stymied progress. To overcome these limitations, we adopt an hierarchical approach that builds on the “flexible meccano” model of Bernadó et al. (J. Am. Chem. Soc. 2005, 127, 17968-17969). First, we exhaustively sample small IDP fragments in all-atom simulations to capture local structure. Then, we assemble the fragments into full-length IDPs to explore the stereochemically possible global structures of IDPs. The resulting ensembles of three-dimensional structures of full-length IDPs are highly diverse, much more so than in standard MD simulation. For the paradigmatic IDP α-synuclein, our ensemble captures both local structure, as probed by nuclear magnetic resonance (NMR) spectroscopy, and its overall dimension, as obtained from small-angle X-ray scattering (SAXS) in solution. By generating representative and meaningful starting ensembles, we can begin to exploit the massive parallelism afforded by current and future high-performance computing resources for atomic-resolution characterization of IDPs.

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Interpretation of biomolecular NMR spin relaxation parametersThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Biochemistry and Cell Biology ◽

10.1139/o09-152 ◽

2010 ◽

Vol 88 (2) ◽

pp. 131-142 ◽

Cited By ~ 15

Author(s):

Tyler Reddy ◽

Jan K. Rainey

Keyword(s):

Spin Relaxation ◽

Intrinsically Disordered Proteins ◽

Nmr Relaxation ◽

Disordered Proteins ◽

Detailed Model ◽

Intrinsically Disordered ◽

Density Mapping ◽

Model Free ◽

Relaxation Experiments ◽

Nmr Spin Relaxation

Biomolecular nuclear magnetic resonance (NMR) spin relaxation experiments provide exquisite information on the picosecond to nanosecond timescale motions of bond vectors. Spin–lattice (T1) and spin–spin (T2) relaxation times and the steady-state nuclear Overhauser effect (NOE) are the first set of parameters extracted from typical 15N or 13C NMR relaxation experiments. Therefore, verifying that T1, T2, and NOE are consistent with theoretical predictions is an important step before carrying out the more detailed model-free and reduced spectral density mapping analyses commonly employed. In this mini-review, we discuss the essential motional parameters used to describe biomolecular dynamics in the context of a variety of examples of folded and intrinsically disordered proteins and peptides in aqueous and membrane mimetic environments. Estimates of these parameters can be used as input for an online interface, introduced herein, allowing plotting of trends of T1, T2, and NOE with magnetic field strength. The plots may serve as a first-check to the spectroscopist preparing to embark on a detailed NMR relaxation analysis.

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From folding to function: complex macromolecular reactions unraveled one-by-one with optical tweezers

Essays in Biochemistry ◽

10.1042/ebc20200024 ◽

2021 ◽

Author(s):

Pétur O. Heidarsson ◽

Ciro Cecconi

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Structural Changes ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Disordered Proteins ◽

Kinase Activation ◽

Intrinsically Disordered ◽

Complex Protein ◽

Methodological Principles

Abstract Single-molecule manipulation with optical tweezers has uncovered macromolecular behaviour hidden to other experimental techniques. Recent instrumental improvements have made it possible to expand the range of systems accessible to optical tweezers. Beyond focusing on the folding and structural changes of isolated single molecules, optical tweezers studies have evolved into unraveling the basic principles of complex molecular processes such as co-translational folding on the ribosome, kinase activation dynamics, ligand–receptor binding, chaperone-assisted protein folding, and even dynamics of intrinsically disordered proteins (IDPs). In this mini-review, we illustrate the methodological principles of optical tweezers before highlighting recent advances in studying complex protein conformational dynamics – from protein synthesis to physiological function – as well as emerging future issues that are beginning to be addressed with novel approaches.

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Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level

Biomolecules ◽

10.3390/biom9020077 ◽

2019 ◽

Vol 9 (2) ◽

pp. 77 ◽

Cited By ~ 6

Author(s):

Xingcheng Lin ◽

Prakash Kulkarni ◽

Federico Bocci ◽

Nicholas Schafer ◽

Susmita Roy ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Structural Plasticity ◽

Disordered Proteins ◽

Dimensional Structure ◽

Related Disorder ◽

Structural Ordering ◽

Intrinsically Disordered ◽

Collective Motions ◽

Folded Proteins

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.

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Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

10.26434/chemrxiv.11537913 ◽

2020 ◽

Author(s):

Franziska Zosel ◽

Andrea Holla ◽

Benjamin Schuler

Keyword(s):

Fluorescence Spectroscopy ◽

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Control Sample ◽

Resonance Energy ◽

Disordered Proteins ◽

Single Molecule Fluorescence ◽

Intrinsically Disordered ◽

Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.

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NSP 11 of SARS-CoV-2 is an Intrinsically Disordered Protein

10.1101/2020.10.07.330068 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kundlik Gadhave ◽

Prateek Kumar ◽

Ankur Kumar ◽

Taniya Bhardwaj ◽

Neha Garg ◽

...

Keyword(s):

Amino Acid ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Alpha Helix ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Helical Conformation ◽

Structural Protein ◽

Intrinsically Disordered ◽

Protein Size

AbstractThe intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of disordered proteome is also proven and are related with its conformational dynamics inside the host. The SARS-CoV-2 virus has a large proteome, in which, structure and functions of many proteins are not known as of yet. Previously, we have investigated the dark proteome of SARS-CoV-2. However, the disorder status of non-structural protein 11 (nsp11) was not possible because of very small in size, just 13 amino acid long, and for most of the IDP predictors, the protein size should be at least 30 amino acid long. Also, the structural dynamics and function status of nsp11 was not known. Hence, we have performed extensive experimentation on nsp11. Our results, based on the Circular dichroism spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behaviour of nsp11 in the presence of membrane mimetic environment, alpha helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an α-helical conformation, suggesting the helical propensity of nsp11. At the end, we again confirmed the IDP behaviour of nsp11 using molecular dynamics simulations.

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Sequence-dependent correlated segments in the intrinsically disordered region of ChiZ

10.1101/2020.04.22.055590 ◽

2020 ◽

Author(s):

Alan Hicks ◽

Cristian A. Escobar ◽

Timothy A. Cross ◽

Huan-Xiang Zhou

Keyword(s):

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Md Simulations ◽

Proton Exchange ◽

Disordered Proteins ◽

Amide Proton ◽

Relaxation Rates ◽

Intrinsically Disordered ◽

Amide Proton Exchange ◽

Sequence Dependent

AbstractIntrinsically disordered proteins (IDPs) account for a significant fraction of any proteome and are central to numerous cellular functions. Yet how sequences of IDPs code for their conformational dynamics is poorly understood. Here we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. This IDP is the N-terminal fragment (residues 1-64) of the transmembrane protein ChiZ, a component of the cell division machinery in Mycobacterium tuberculosis. Its N-half contains most of the prolines and all of the anionic residues while the C-half most of the glycines and cationic residues. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11-29) emerge as “correlated segments”, identified by frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11-14 and 23-28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent: segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5-10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose correlated segment as a defining feature for the conformation and dynamics of IDPs.

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Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

10.1101/2022.01.14.476386 ◽

2022 ◽

Author(s):

Spencer Smyth ◽

Zhenfu Zhang ◽

Alaji Bah ◽

Thomas Tsangaris ◽

Jennifer Dawson ◽

...

Keyword(s):

Conformational Changes ◽

Intrinsically Disordered Proteins ◽

Regulatory Protein ◽

Conformational Dynamics ◽

Correlation Spectroscopy ◽

Terminal Region ◽

Disordered Proteins ◽

Initiation Factor ◽

Dependent Manner ◽

Intrinsically Disordered

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-microsecond timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs were significantly reduced upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

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Cyclic Ion Mobility – Collision Activation Experiments Elucidate Protein Behaviour in the Gas-Phase

10.26434/chemrxiv.11687100.v1 ◽

2020 ◽

Author(s):

Charles Eldrid ◽

Jakub Ujma ◽

Hannah Britt ◽

Tristan Cragnolini ◽

Symeon Kalfas ◽

...

Keyword(s):

Gas Phase ◽

Ion Mobility ◽

Protein Unfolding ◽

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Globular Proteins ◽

Disordered Proteins ◽

Mass Selection ◽

Intrinsically Disordered ◽

Partially Folded States

Elucidating the properties of intrinsically disordered proteins (IDPs) and unfolded and partially folded states of globular proteins is challenging owing to their heterogeneous and dynamic nature. Protein unfolding and misfolding is a key feature of a broad range of debilitating diseases, whilst the conformational propensities of intrinsically disordered proteins can play a significant role in modulating their activity, and the properties of unfolded states of globular proteins modulates their stability and tendency to aggregate. Ion mobility-mass spectrometry (IM-MS) is a powerful method for interrogating these systems, however limits in resolution and the difficulty in probing the energetics of interconversions amongst heterogeneous ensembles are major issues. Herein, using a quadrupole/cyclic-IM/ time-of-flight MS instrument, we show how the combination of precursor mass selection, mobility selection (IMn) and collisional activation (CA) allows the elucidation of complicated gas-phase dynamic behavior. The methodology employed is general and is demonstrated using a classic model globular protein, cytochrome C, and an aggregation-prone IDP, amylin. CA allows investigations of protein conformational dynamics and unfolding in the gas-phase for heterogeneous mixtures, whilst the additional precursor mass selection capability provides high resolution and selectivity, facilitating more in-depth investigation. Understanding protein dynamics in the gas-phase will allow greater insight into protein behaviour and allow application of gas-phase techniques to clinically relevant systems.

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Sequence-Dependent Correlated Segments in the Intrinsically Disordered Region of ChiZ

Biomolecules ◽

10.3390/biom10060946 ◽

2020 ◽

Vol 10 (6) ◽

pp. 946 ◽

Cited By ~ 3

Author(s):

Alan Hicks ◽

Cristian Escobar ◽

Timothy Cross ◽

Huan-Xiang Zhou

Keyword(s):

Intrinsically Disordered Proteins ◽

Conformational Dynamics ◽

Md Simulations ◽

Proton Exchange ◽

Disordered Proteins ◽

Amide Proton ◽

Relaxation Rates ◽

Intrinsically Disordered ◽

Amide Proton Exchange ◽

Sequence Dependent

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5–10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose the correlated segment as a defining feature for the conformations and dynamics of IDPs.

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