Gül H. Zerze

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUSs nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.

Free energy surface of an intrinsically disordered protein: comparison between temperature replica exchange molecular dynamics and bias-exchange metadynamics.

Intrinsically disordered proteins (IDPs), which are expected to be largely unstructured under physiological conditions, make up a large fraction of eukaryotic proteins. Molecular dynamics simulations have been utilized to probe structural characteristics of these proteins, which are not always easily accessible to experiments. However, exploration of the conformational space by brute force molecular dynamics simulations is often limited by short time scales. Present literature provides a number of enhanced sampling methods to explore protein conformational space in molecular simulations more efficiently. In this work, we present a comparison of two enhanced sampling methods: temperature replica exchange molecular dynamics and bias exchange metadynamics. By investigating both the free energy landscape as a function of pertinent order parameters and the per-residue secondary structures of an IDP, namely, human islet amyloid polypeptide, we found that the two methods yield similar results as expected. We also highlight the practical difference between the two methods by describing the path that we followed to obtain both sets of data.

Molecular simulations indicate marked differences in the structure of amylin mutants, correlated with known aggregation propensity.

Human islet amyloid polypeptide (hIAPP), a 37-residue protein cosecreted with insulin by β-cells in the pancreas, is known to form amyloid fibrils in type II diabetes patients. This fibril formation is also associated with β-cell death. However, rat IAPP (rIAPP) does not aggregate into fibrils, nor is it associated with β-cell toxicity. Determining solution properties of hIAPP experimentally is difficult because it aggregates quickly. Our study uses molecular dynamics simulation to explore and compare in-solution characteristics of hIAPP and rIAPP, as well as two single-point hIAPP mutants, hIAPP I26P and hIAPP S20G, which exhibit observed differences from hIAPP in aggregation propensities. We find that all four polypeptide monomers sample structured states in solution. More importantly, differences in the helicity over residues 7-16 may play an important role in early aggregation, although this region is outside of commonly assumed amyloidogenic region 20-29. The long-range contacts, though unexpected of IDPs, cause variation in sampled conformations among four polypeptides within same amino acid sequence. Our results also yield evidence that previously determined structures bound to micelles are also transiently sampled in the solution state. In particular, similarities found in region 8-17 together with the helical differences that we observe in region 7-16 lead us to suggest that the region 7-16 is potentially responsible for amyloidogenic behavior of amylin peptides. Our results also provide support for the proposed mechanism of fibril formation based on experimentally observed transient helices in amyloidogenic peptides.

Surface force measurements and simulations of mussel-derived peptide adhesives on wet organic surfaces

Significance The need for bio-inspired wet adhesives has significantly increased in the past few decades (e.g., for dental and medical transplants, coronary artery coatings, cell encapsulants, etc.). However, the molecular basis behind catechol-facilitated adhesion to organic surfaces remains unclear, thus hindering synthesis and optimization of novel underwater adhesives. The present combined experimental and theoretical study reconciles bioadhesion measurements of novel catechol-containing peptides to self-assembled monolayers (SAMs) with all-atom molecular dynamics simulations, yielding a comprehensive framework that explicitly identifies the basis for underwater adhesion. Simulations and surface forces apparatus measurements agree with one another, and both approaches show strong peptide adhesion to hydrophobic SAMs, and weak peptide adhesion to hydrophilic SAMs, providing a starting point for the development of next-generation underwater glues. Translating sticky biological molecules—such as mussel foot proteins (MFPs)—into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue’s molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces.

Sequence- and Temperature-Dependent Properties of Unfolded and Disordered Proteins from Atomistic Simulations.

We use all-atom molecular simulation with explicit solvent to study the properties of selected intrinsically disordered proteins and unfolded states of foldable proteins, which include chain dimensions and shape, secondary structure propensity, solvent accessible surface area, and contact formation. We find that the qualitative scaling behavior of the chains matches expectations from theory under ambient conditions. In particular, unfolded globular proteins tend to be more collapsed under the same conditions than charged disordered sequences of the same length. However, inclusion of explicit solvent in addition naturally captures temperature-dependent solvation effects, which results in an initial collapse of the chains as temperature is increased, in qualitative agreement with experiment. There is a universal origin to the collapse, revealed in the change of hydration of individual residues as a function of temperature: namely, that the initial collapse is driven by unfavorable solvation free energy of individual residues, which in turn has a strong temperature dependence. We also observe that in unfolded globular proteins, increased temperature also initially favors formation of native-like (rather than non-native-like) structure. Our results help to establish how sequence encodes the degree of intrinsic disorder or order as well as its response to changes in environmental conditions.

Modest Influence of FRET Chromophores on the Properties of Unfolded Proteins

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ(2), has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.

A carbon nanotube reporter of microRNA hybridization events in vivo

MicroRNAs and other small oligonucleotides in biofluids are promising disease biomarkers, yet conventional assays require complex processing steps that are unsuitable for point-of-care testing or for implantable or wearable sensors. Single-walled carbon nanotubes are an ideal material for implantable sensors, owing to their emission in the near-infrared spectral region, photostability and exquisite sensitivity. Here, we report an engineered carbon-nanotube-based sensor capable of real-time optical quantification of hybridization events of microRNA and other oligonucleotides. The mechanism of the sensor arises from competitive effects between displacement of both oligonucleotide charge groups and water from the nanotube surface, which result in a solvatochromism-like response. The sensor, which allows for detection via single-molecule sensor elements and for multiplexing by using multiple nanotube chiralities, can monitor toehold-based strand-displacement events, which reverse the sensor response and regenerate the sensor complex. We also show that the sensor functions in whole urine and serum, and can non-invasively measure DNA and microRNA after implantation in live mice.

Mechanistic View of hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Separation Altered by Mutation and Arginine Methylation

hnRNPA2, a component of RNA-processing membraneless organelles, forms inclusions when mutated in a syndrome characterized by the degeneration of neurons (bearing features of amyotrophic lateral sclerosis [ALS] and frontotemporal dementia), muscle, and bone. Here we provide a unified structural view of hnRNPA2 self-assembly, aggregation, and interaction and the distinct effects of small chemical changes-disease mutations and arginine methylation-on these assemblies. The hnRNPA2 low-complexity (LC) domain is compact and intrinsically disordered as a monomer, retaining predominant disorder in a liquid-liquid phase-separated form. Disease mutations D290V and P298L induce aggregation by enhancing and extending, respectively, the aggregation-prone region. Co-aggregating in disease inclusions, hnRNPA2 LC directly interacts with and induces phase separation of TDP-43. Conversely, arginine methylation reduces hnRNPA2 phase separation, disrupting arginine-mediated contacts. These results highlight the mechanistic role of specific LC domain interactions and modifications conserved across many hnRNP family members but altered by aggregation-causing pathological mutations.

Folding thermodynamics of β-hairpins studied by replica-exchange molecular dynamics simulations.

We study the differences in folding stability of β‐hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3‐1, and TrpZip4. By performing replica‐exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3‐1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β‐hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild‐type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state‐of‐the‐art all‐atom protein force fields. Proteins 2015; 83:1307–1315.

Effect of O-Linked Glycosylation on the Equilibrium Structural Ensemble of Intrinsically Disordered Polypeptides

Glycosylation is one of the most common post-translational modifications (PTMs), which provides a large proteome diversity. Previous work on glycosylation of globular proteins has revealed remarkable effects of glycosylation on protein function, altering the folding stability and structure and/or altering the protein surface which affects their binding characteristics. Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) of large proteins are also frequently glycosylated, yet how glycosylation affects their function remains to be elucidated. An important open question is, does glycosylation affect IDP structure or binding characteristics or both? In this work, we particularly address the structural effects of O-linked glycosylation by investigating glycosylated and unglycosylated forms of two different IDPs, tau174-183 and human islet amyloid polypeptide (hIAPP), by all-atom explicit solvent simulations. We simulate these IDPs in aqueous solution for O-linked glycosylated and unglycosylated forms by employing two modern all-atom force fields for which glycan parameters are also available. We find that O-linked glycosylation only has a modest effect on equilibrium structural ensembles of IDPs, for the cases studied here, which suggests that the functional role of glycosylation may be primarily exerted by modulation of the protein binding characteristics rather than structure.