Session F06: Physics of Proteins II: Intrinsically Disordered Proteins & Protein Folding

Sequence-Dependent Backbone Dynamics and Membrane Association of Intrinsically Disordered Proteins

Intrinsically 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 ensembles, conformational dynamics, and ultimately, functions is poorly understood. I will report advances from our computational and experimental studies of ChiZ, a membrane protein that contains an intrinsically disordered N-terminal region (NT) and is a component of the cell division machinery in Mycobacterium tuberculosis. NMR data revealed non-uniform backbone dynamics along the sequence of the 64-residue NT [1]. Molecular dynamics (MD) simulations traced the origin to correlated segments, which are stabilized by polyproline II stretches, salt bridges, cation-p interactions, and sidechain-backbone hydrogen bonds. Moreover, the extent of segmental correlation is sequence-dependent: segments (in particular, in the N-half; e.g., residues 11-29) where internal interactions are more prevalent manifest elevated supra-ns “collective” motions and suppressed local motions on the sub-ns timescales. NMR experiments found that NT associates with acidic membranes, but most residues remain dynamic, exception for a subset of Arg residues [2]. MD simulations provided crucial details on the fuzzy conformational ensemble, showing NT anchored to membranes in the midsection, in particular by Arg37. Anionic residues, all the N-half, compete with acid membranes for interacting with Arg residues. Consequently C-half Arg residues have higher propensities to interact with the membrane. This asymmetry between N-half and C-half in membrane interaction is accentuated when NT is tethered to the membrane via the adjacent transmembrane helix. These findings serve as paradigms for sequence-conformation-dynamics-function relations of IDPs.   

Assessing the Ability of Molecular Dynamics Force Fields to Capture Conformational Dynamics of Amino Acid Residues in Water

Molecular dynamics (MD) is a powerful tool for studying intrinsically disordered proteins, however, its reliability depends on the accuracy of the force field.  We here assess Amber ff19SB & ff14SB, OPLS-AA/M, and CHARMM36m with respect to their capacity to capture intrinsic conformational dynamics of a guest amino acid residue x of unblocked GxG in water. Using published spectroscopic data (5 J-coupling constants and amide I' band profiles), MD force fields are evaluated with respect to their ability to reproduce experimental data of 14 residues x (x= G, A, L, V, I, F, Y, D^P, E^P, R, C, N, S, T) in GxG peptides through reduced χ² functions.  As a benchmark, a Ramachandran distribution is created as a linear combination of Gaussian sub distributions to best fit the experimental data.  Our results show that the Gaussian model outperforms all four MD force fields for all 14 guest residues. The major weaknesses of the four MD force fields include insufficient variability of the polyproline II (pPII) population among the guest residues, oversampling of antiparallel β-strand at the expense of transitional β-strand region, inadequate sampling of turn-forming conformations for ionizable and polar residues, and lack of guest residue-specificity in the Ramachandran distributions.  While Amber ff19SB performs worse than the other three force fields upon comparison of χ² values, it best accounts for guest residue-specificity, particularly pPII variability, among residues compared to the other three force fields.   

The Dark Side of the Low and High Temperature Denaturation of Myoglobin

We re-investigate a simple model used in the literature concerning the thermodynamic analysis of protein cold denaturation. We derive an exact thermodynamic expression for cold denaturation and give a better approximation than exists in the literature for predicting cold denaturation temperatures in the two-state model. We discuss the ``dark-side'' implications of this work for previous temperature dependent protein dynamics experiments and discuss micro-fluidic experimental technologies which could explore the thermal stability range of proteins below the bulk freezing point of water.   

Quantification of the molar degree of folding/unfolding using a scaling model

The unfolded states in proteins and nucleic acids remain weakly understood despite their importance in folding processes; misfolding diseases (Parkinson's and Alzheimer's); natively unfolded proteins (as many as 30% of eukaryotic proteins, according to Fink); and the study of ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is discussed to quantify the molar degree of folding and to define the unfolded state. The scaling model can be applied to various analytic methods and to simulations. Examples are given using small-angle X-ray and neutron scattering.  For instance, from pressure-induced unfolding of SNase, from acid-unfolded cytochrome c, and from folding of Azoarcus ribozyme. These examples quantitatively show three characteristic unfolded states for proteins, the statistical nature of a protein folding pathway, and the relationship between the extent of folding and chain size during folding for charge-driven folding in RNA.   

Beyond 3 Å-1: total X-ray scattering and atomic pair distribution function (PDF) analysis to determine the high-resolution structure of protein and polysaccharide in aqueous solutions

Resolving structures and dynamics of biomacromolecules in aqueous solutions at high spatial resolution are experimentally nontrivial due to the high instrumental requirements and challenges in modeling. Small-angle x-ray/neutron scattering, using scattering data below ≈ 1 Å^-1, provides low-resolution structural information of biomacromolecules. While there have been reports of wide-angle scattering characterization (up to ≈ 3 Å^-1) of biomacromolecules, high-quality data suitable for high-resolution modeling providing detailed insights into the structure are unavailable. Here, for the first time, by taking advantage of high-energy wide-angle X-ray scattering (WAXS) at 11-ID-B at the Advanced Photon Source, Argonne National Laboratory, we reliably collected WAXS profiles of several types of biomacromolecules including protein and polysaccharide in aqueous solutions up to ≈ 20 Å^-1. Our atomic pair distribution function analyses shed new light on resolving the conformation states of these biomacromolecules.   

Local interactions and transient secondary structures govern backbone dynamics of intrinsically disordered proteins

The lack of well-defined structures in intrinsically disordered proteins (IDPs) calls for a fundamental reassessment of how their amino-acid sequences code for functions. Some attention has been paid to nascent structures, but a missing link is sequence-dependent backbone dynamics, which we previously showed to arise from the formation of correlated segments stabilized by polyproline II (PPII) helices and salt bridges in the ChiZ disordered N-terminal region.¹ To define general rules governing sequence-dependent backbone dynamics, we performed molecular dynamics simulations on eight IDPs. Nearly all above-average transverse relaxation rates and heteronuclear Overhauser enhancements along the sequence were attributable to interactions between side chains and formation of secondary structures. PPII stretches are the most common form of transient secondary structures, and are found in most IDPs and are often stabilized by local interactions. However, in some IDPs, stable α-helices are also present. These locally rigidified elements may code for nascent structures, whereas segments with fast dynamics may readily adapt to binding partners.²

1. Hicks, A. et al. Biomolecules 10, 946 (2020).

2. Hicks, A. et al. JACS Au 1, 66-78 (2021).   

Exploring SANS as a novel approach to investigate protein stability with Hydrogen-Deuterium Exchange

Hydrogen deuterium exchange, (HDX) is of increasing interest as it provides useful information of protein dynamics in solution. Recently, the rapid development of HDX mass spectrometry (HDX-MS) shows a great potential to understand the protein flexibility and conformational changes by investigating the exchange of amide hydrogens with deuterium. Due to the very large difference of the neutron scattering cross section between H and D, techniques such as small angle neutron scattering (SANS), can offer an alternate approach to monitoring HDX. Unlike HDX-MS, SANS allows for the continual measurement of HDX over time and is a non-invasive technique to investigate the HDX in solution. Even though SANS does not have the sensitivity to study the amino acid sequence specific exchange kinetics, it could provide the spatial distribution information of exchangeable protons in a protein, as a function of the exchange time. With the use of SANS, we probed the HDX of a couple of proteins over the course of a day. Novel analysis approach is designed to extract the exchange kinetics and the spatial distribution of exchangeable protons, from SANS. Additionally, the effect of the sample conditions, such as the temperature and buffer conditions, on the HDX will be presented.   

Aggregation of amphipathic peptides into amyloid-like fibrils

In aqueous solution, amphipathic peptides aggregate into amyloid-like fibrils that are being considered for several biomedical applications due to their mechanical properties and biocompatibility. Here, we perform all-atom molecular dynamics simulations in explicit solvent to study the aggregation of amphipathic peptides. We study systems containing more than 200,000 atoms, which are simulated for up to 14 μs for six different peptide sequences. We show that peptide sequences that do not form fibrils in experiments show a low propensity to form hydrogen bonds and β-structures in our simulations, and vice-versa. Simulations are also performed at different temperatures to highlight the importance of hydrophobic interactions on aggregation. The aggregation rate in our simulations increases with increasing temperature for highly hydrophobic amphipathic peptides. This is related to the strength of hydrophobic interactions that is enhanced with increasing temperature. We also observe coassembly process of peptides composed of L- and D-handed peptides.    

A Transferable Explicit-Solvent Polarizable Coarse-Grained Model for Proteins

Current transferrable coarse-grained (CG) force fields are either limited to only peptides with the environment encoded in an implicit form or cannot capture transitions into secondary/tertiary peptide structures from a primary sequence of amino acids. In this work, we present a transferrable CG forcefield with an explicit representation of the environment for accurate simulations with proteins. The forcefield consists of a set of pseudo-atoms representing different chemical groups, that can be joined/associated together to create different biomolecular systems. This preserves the transferability of the forcefield to multiple environments and simulation conditions. The novelty of this CG model is the addition of electronic polarization that can respond to environmental heterogeneity/fluctuations and couple to protein's structural transitions. We demonstrate the validity of this model with the folding of several benchmark proteins from scratch without adding extra constraints. All the peptides studied can be nicely folded with their signature secondary and tertiary structures at the correct positions. The CG model shows a great potential to aid biomolecular research, particularly the folding-unfolding process in a realistic cell-like crowded heterogeneous environment.   

Characterization of Amyloid Fibril Bundling in Hen Egg White Lysozyme

Amyloids are proteins that, in their misfolded conformations, self-assemble into non-branching rigid fibrils. Growth of these fibrils involves both primary and secondary nucleation processes, resulting in “sigmoidal” growth kinetics. These fibrils can further bundle together and form large sheets known as amyloid plaques. Although these plaques are no longer believed to be the primary pathogenic aggregate in neurodegenerative diseases such as Alzheimer's Disease, they are prominent contributors to non-neuropathic anyloidoses. In cardiac amyloidosis plaques restrict contraction of the ventricles, leading to arrythmia and heart failure. However the mechanisms driving assembly of plaques from individual fibrils have remained elusive. 

This study combined optical microscopy, transmission electron microscopy, and Thioflavin T fluorescence assays to determine if amyloid fibril bundling in hen egg white lysozyme  resulted from equilibrium association of RFs or from fibril-mediated secondary nucleation during growth. The data gathered suggests that amyloid plaques can form independent of fibril growth, with RF length not significantly impacting plaque formation. However, the degree of plaque formation is significantly enhanced during growth, most likely due to secondary nucleation.   

How to probe intrinsically disordered proteins and protein unfolding using advanced FT-IR technology?

A significant number of proteins contain intrinsically disordered regions that play vital roles in protein functions. Structural and spectroscopic techniques have been developed to study folded proteins. Advanced FT-IR technology is an emerging technology with growing applications. Here we report how temperature-resolved and time-resolved Fourier transform infrared (FT-IR) techniques were employed to study intrinsically disordered proteins and protein folding. We found that intrinsically disordered nature of proteins may change across physiological temperatures. In addition, binding of a ligand or drug candidate can alter the properties of protein structure and stability. Consequently, it is applicable to use FT-IR techniques to study drug binding to intrinsically disordered proteins.    

Binding of amyloid-like peptides to lipid bilayers: effects of ions and lipid content

In amyloid diseases, cell toxicity can emerge from interactions of peptides with the cell membrane. Several factors have been shown to modulate the magnitude of these peptide-bilayer interactions, which can enhance or inhibit cell toxicity. These factors include lipid composition, the presence of ions in the solution. Here, we perform all-atom molecular dynamics simulations to provide an understanding at the atomic level of peptide-bilayer interactions and their modulation by Ca²⁺ and selected lipids. We find that both electrostatic and hydrophobic interactions contribute to peptide-bilayer binding. In particular, the binding is induced by positively charged residues interacting with lipid head groups. Hydrophobic interaction sustains this bound state. These mechanisms in detail as well as how they are affected by Ca²⁺ and selected lipid content of the bilayer will be discussed in detail. Additionally, we investigate the sequence dependence of peptide-bilayer interactions, showing that several factors such as net charge, sequence pattern and type of positive residues, can significantly affect the binding strength.    

Hidden Order in an Intrinsically Disordered Protein Region

Previously, we presented evidence of glassy dynamics in a model intrinsically disordered protein region (IDR) system, suggesting that it contains hidden regions of order. This model system is a polyprotein derived from the disordered neurofilament light tail (NFLt) domain, which is part of a large group of IDRs in the neuronal cytoskeleton that are responsible for the structure and mechanics of the axon. Using magnetic tweezers, we showed that NFLt polyproteins exhibit a slow extension change over time, in response to a change in applied tension. These extension changes exhibit a nonexponential (often logarithmic) time dependence and are history-dependent, two characteristic features of a glassy system. Here, we use a combination of coarse-grained molecular dynamics simulations and experimental data on the NFLt polyproteins to determine the microscopic origin of the glassy dynamics. We establish which residues form the hidden ordered regions and develop a model based on these residues that can recapitulate the polyprotein’s glassy behavior. Based on their associated sequence properties, we suspect that hidden ordered regions and glassy behavior are likely to apply broadly to other IDRs found throughout the cellular cytoskeleton.