Folding Kinetics Research Articles

A General Framework to Interpret Hydrogen-Deuterium Exchange Native Mass Spectrometry of G-Quadruplex DNA.

G-quadruplexes (G4s) are secondary structures formed by guanine-rich oligonucleotides involved in various biological processes. However, characterizing G4s is challenging, because of their structural polymorphism. Here, we establish how hydrogen-deuterium exchange native mass spectrometry (HDX/MS) can help to characterize the G4 structures and dynamics in solution. We correlated the time range of G4 exchange to the number of guanines involved in the inner and outer tetrads. We also established relationships among exchange rates, numbers of tetrads and bound cations, and stability. The use of HDX/native MS allows for the determination of tetrads formed and assessment of G4 stability at a constant temperature. A key finding is that stable G4s exchange through local fluctuations (EX2 exchange), whereas less stable G4s also undergo exchange through partial or complete unfolding (EX1 exchange). Deconvolution of the bimodal isotope distributions resulting from EX1 exchange provides valuable insight into the kinetics of folding and unfolding processes and allows one to detect and characterize transiently unfolded intermediates, even if scarcely populated. HDX/native MS thus represents a powerful tool for a more comprehensive exploration of the folding landscapes of G4s.

Molecular bases for strong phenotypic effects of single synonymous codon substitutions in the E. coli ccdB toxin gene

BackgroundSingle synonymous codon mutations typically have only minor or no effects on gene function. Here, we estimate the effects on cell growth of ~ 200 single synonymous codon mutations in an operonic context by mutating almost all positions of ccdB, the 101-residue long cytotoxin of the ccdAB Toxin-Antitoxin (TA) operon to most degenerate codons. Phenotypes were assayed by transforming the mutant library into CcdB sensitive and resistant E. coli strains, isolating plasmid pools, and subjecting them to deep sequencing. Since autoregulation is a hallmark of TA operons, phenotypes obtained for ccdB synonymous mutants after transformation in a RelE toxin reporter strain followed by deep sequencing provided information on the amount of CcdAB complex formed.ResultsSynonymous mutations in the N-terminal region involved in translation initiation showed the strongest non-neutral phenotypic effects. We observe an interplay of numerous factors, namely, location of the codon, codon usage, t-RNA abundance, formation of anti-Shine Dalgarno sequences, predicted transcript secondary structure, and evolutionary conservation in determining phenotypic effects of ccdB synonymous mutations. Incorporation of an N-terminal, hyperactive synonymous mutation, in the background of the single synonymous codon mutant library sufficiently increased translation initiation, such that mutational effects on either folding or termination of translation became more apparent. Introduction of putative pause sites not only affects the translational rate, but might also alter the folding kinetics of the protein in vivo.ConclusionIn summary, the study provides novel insights into diverse mechanisms by which synonymous mutations modulate gene function. This information is useful in optimizing heterologous gene expression in E. coli and understanding the molecular bases for alteration in gene expression that arise due to synonymous mutations.

Modeling the Effect on a Novel Fungal Peptaibol Placed in an All-Atom Bacterial Membrane Mimicking System via Accelerated Molecular Dynamics Simulations.

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

Line Shape Analysis of 19F NMR-Monitored Chemical Denaturation of a Fold-Switching Protein RfaH Reveals Its Slow Folding Dynamics.

The recent discovery of metamorphic proteins, which can switch between multiple conformations under native conditions, has challenged the well-established one sequence-one structure paradigm of protein folding. This is exemplified in the C-terminal domain of the multidomain transcription factor RfaH, which converts from an α-helical coiled-coil conformation in its autoinhibited state to a β-barrel conformation upon activation. Here, we use multisite line shape analysis of 19F NMR-monitored equilibrium chemical denaturation measurements of two 19F-labeled variants of full-length RfaH, to show that it folds/unfolds slowly on the NMR time scale, in an apparent all-or-none fashion at physiological pH and room temperature in the 3.3-4.8 M urea concentration range. The significant thermodynamic stability and slow unfolding rate (kinetic stability) are likely responsible for maintaining the closed autoinhibited state of RfaH, preventing spurious interactions with RNA polymerase (RNAP) when not functional. Our results provide a quantitative understanding of the folding-function relationship in the model fold-switching protein RfaH.

RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.

Folding kinetics of an entangled protein.

The possibility of the protein backbone adopting lasso-like entangled motifs has attracted increasing attention. After discovering the surprising abundance of natively entangled protein domain structures, it was shown that misfolded entangled subpopulations might become thermosensitive or escape the homeostasis network just after translation. To investigate the role of entanglement in shaping folding kinetics, we introduce a novel indicator and analyze simulations of a coarse-grained, structure-based model for two small single-domain proteins. The model recapitulates the well-known two-state folding mechanism of a non-entangled SH3 domain. However, despite its small size, a natively entangled antifreeze RD1 protein displays a rich refolding behavior, populating two distinct kinetic intermediates: a short-lived, entangled, near-unfolded state and a longer-lived, non-entangled, near-native state. The former directs refolding along a fast pathway, whereas the latter is a kinetic trap, consistently with known experimental evidence of two different characteristic times. Upon trapping, the natively entangled loop folds without being threaded by the N-terminal residues. After trapping, the native entangled structure emerges by either backtracking to the unfolded state or threading through the already formed but not yet entangled loop. Along the fast pathway, trapping does not occur because the native contacts at the closure of the lasso-like loop fold after those involved in the N-terminal thread, confirming previous predictions. Despite this, entanglement may appear already in unfolded configurations. Remarkably, a longer-lived, near-native intermediate, with non-native entanglement properties, recalls what was observed in cotranslational folding.

Interconnections between m6A RNA modification, RNA structure, and protein-RNA complex assembly.

Protein-RNA complexes exist in many forms within the cell, from stable machines such as the ribosome to transient assemblies like the spliceosome. All protein-RNA assemblies rely on spatially and temporally coordinated interactions between specific proteins and RNAs to achieve a functional form. RNA folding and structure are often critical for successful protein binding and protein-RNA complex formation. RNA modifications change the chemical nature of a given RNA and often alter its folding kinetics. Both these alterations can affect how and if proteins or other RNAs can interact with the modified RNA and assemble into complexes. N6-methyladenosine (m6A) is the most common base modification on mRNAs and regulatory noncoding RNAs and has been shown to impact RNA structure and directly modulate protein-RNA interactions. In this review, focusing on the mechanisms and available quantitative information, we discuss first how the METTL3/14 m6A writer complex is specifically targeted to RNA assisted by protein-RNA and other interactions to enable site-specific and co-transcriptional RNA modification and, once introduced, how the m6A modification affects RNA folding and protein-RNA interactions.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs.

Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST.

The accurate modeling of energetic contributions to protein structure is a fundamental challenge in computational approaches to protein analysis and design. We describe a general computational method, EmCAST (empirical Cα stabilization), to score and optimize the sequence to the structure in proteins. The method relies on an empirical potential derived from the database of the Cα dihedral angle preferences for all possible four-residue sequences, using the data available in the Protein Data Bank. Our method produces stability predictions that naturally correlate one-to-one with the experimental results for solvent-exposed mutation sites. EmCAST predicted four mutations that increased the stability of a three-helix bundle, UBA(1), from 2.4 to 4.8 kcal/mol by optimizing residues in both helices and turns. For a set of eight variants, the predicted and experimental stabilizations correlate very well (R2 = 0.97) with a slope near 1 and with a 0.16 kcal/mol standard error for EmCAST predictions. Tests against literature data for the stability effects of surface-exposed mutations show that EmCAST outperforms the existing stability prediction methods. UBA(1) variants were crystallized to verify and analyze their structures at an atomic resolution. Thermodynamic and kinetic folding experiments were performed to determine the magnitude and mechanism of stabilization. Our method has the potential to enable the rapid, rational optimization of natural proteins, expand the analysis of the sequence/structure relationship, and supplement the existing protein design strategies.

Appraisal of blob-Based Approaches in the Prediction of Protein Folding Times.

A series of reports published in the last 3 years has illustrated that a blob-based model (BBM) can predict the folding time of proteins from their primary amino acid (aa) sequence based on three simple rules established to characterize the long-range backbone dynamics (LRBD) of racemic polypeptides. The sole use of LRBD to predict protein folding times with the BBM represents a radical departure from all other prediction methods currently applied to determine protein folding times, which rely instead on parameters such as the structure content, folding kinetics, chain length, amino acid properties, or contact topography of proteins. Furthermore, the built-in modularity of the BBM enables the parametrization and inclusion of new phenomena affecting the LRBD of polypeptides, while its conceptual simplicity makes it an interesting new mathematical tool for studying protein folding. However, its novelty implies that its relationship with many other methods used to predict protein folding times has not been well researched. Consequently, the purpose of this report is to uncover the physical phenomena encountered during protein folding that are best described by the BBM through the identification of parameters that have been recognized over the years as being strong predictors for protein folding, such as protein size, topology, structural class, and folding kinetics. This was accomplished by determining the parameters most strongly correlated with the folding times predicted by the BBM. While the BBM in its present form appears to be a good indicator of the folding times of the vast majority of the 195 proteins considered so far, this report finds that it excels for moderately large proteins that are primarily composed of locally formed structural motifs such as α-helices or for proteins that fold in multiple steps. Altogether, these observations based on the use of the BBM support the notion that proteins fold the way they do because the LRBD of polypeptides is mostly driven by the local interactions experienced between aa's within reach of one another.

Unveiling Medin Folding and Dimerization Dynamics and Conformations via Atomistic Discrete Molecular Dynamics Simulations.

Medin is a principal component of localized amyloid found in the vasculature of individuals over 50 years old. Its amyloid aggregation has been linked to endothelial dysfunction and vascular inflammation, contributing to the pathogenesis of various vascular diseases. Despite its significance, the structures of the medin monomer, oligomer, and fibril remain elusive, and the dynamic processes of medin aggregation are not fully understood. In this study, we comprehensively investigated the medin folding and dimerization dynamics and conformations using atomistic discrete molecular dynamics simulations. Our simulation results suggested that the folding initiation of the medin involved the formation of β-sheets around medin30-41 and medin42-50, with subsequent capping of other segments to their β-sheet edges. Medin monomers typically consisted of three or four β-strands, along with a dynamic N-terminal helix. Two isolated medin peptides readily aggregated into a β-sheet-rich dimer, displaying a strong aggregation propensity. Dimerization of medin not only enhanced the β-sheet conformations but also led to the formation of β-barrel oligomers. The aggregation tendencies of medin1-18 and medin19-29 were relatively weak. However, the segments of medin30-41 and medin42-50 played a crucial role as they primarily formed a β-sheet core and facilitated medin1-18 and medin19-29 to form intra- and interpeptide β-sheets. The findings highlight the critical role of the medin30-41 and medin42-50 regions in stabilizing the monomer structure and driving the medin amyloid aggregation. These regions could potentially serve as promising targets for designing antiamyloid inhibitors against amyloid aggregation of medin. Additionally, our study provides a full picture of the monomer conformations and dimerization dynamics for medin, which will help better understand the pathology of medin aggregation.

335 Cryogenic electron microscopy studies of human CFTR folding and conformational dynamics elucidate a novel potentiator binding site in the nucleotide binding domains that supports an alternative channelgating mechanism

335 Cryogenic electron microscopy studies of human CFTR folding and conformational dynamics elucidate a novel potentiator binding site in the nucleotide binding domains that supports an alternative channelgating mechanism

Watching ion-driven kinetics of ribozyme folding and misfolding caused by energetic and topological frustration one molecule at a time.

Folding of ribozymes into well-defined tertiary structures usually requires divalent cations. How Mg2+ ions direct the folding kinetics has been a long-standing unsolved problem because experiments cannot detect the positions and dynamics of ions. To address this problem, we used molecular simulations to dissect the folding kinetics of the Azoarcus ribozyme by monitoring the path each molecule takes to reach the folded state. We quantitatively establish that Mg2+ binding to specific sites, coupled with counter-ion release of monovalent cations, stimulate the formation of secondary and tertiary structures, leading to diverse pathways that include direct rapid folding and trapping in misfolded structures. In some molecules, key tertiary structural elements form when Mg2+ ions bind to specific RNA sites at the earliest stages of the folding, leading to specific collapse and rapid folding. In others, the formation of non-native base pairs, whose rearrangement is needed to reach the folded state, is the rate-limiting step. Escape from energetic traps, driven by thermal fluctuations, occurs readily. In contrast, the transition to the native state from long-lived topologically trapped native-like metastable states is extremely slow. Specific collapse and formation of energetically or topologically frustrated states occur early in the assembly process.

Einstein Model of a Graph to Characterize Protein Folded/Unfolded States.

The folded structures of proteins can be accurately predicted by deep learning algorithms from their amino-acid sequences. By contrast, in spite of decades of research studies, the prediction of folding pathways and the unfolded and misfolded states of proteins, which are intimately related to diseases, remains challenging. A two-state (folded/unfolded) description of protein folding dynamics hides the complexity of the unfolded and misfolded microstates. Here, we focus on the development of simplified order parameters to decipher the complexity of disordered protein structures. First, we show that any connected, undirected, and simple graph can be associated with a linear chain of atoms in thermal equilibrium. This analogy provides an interpretation of the usual topological descriptors of a graph, namely the Kirchhoff index and Randić resistance, in terms of effective force constants of a linear chain. We derive an exact relation between the Kirchhoff index and the average shortest path length for a linear graph and define the free energies of a graph using an Einstein model. Second, we represent the three-dimensional protein structures by connected, undirected, and simple graphs. As a proof of concept, we compute the topological descriptors and the graph free energies for an all-atom molecular dynamics trajectory of folding/unfolding events of the proteins Trp-cage and HP-36 and for the ensemble of experimental NMR models of Trp-cage. The present work shows that the local, nonlocal, and global force constants and free energies of a graph are promising tools to quantify unfolded/disordered protein states and folding/unfolding dynamics. In particular, they allow the detection of transient misfolded rigid states.

Protein–lipid charge interactions control the folding of outer membrane proteins into asymmetric membranes

Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known ‘positive-outside’ rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.

Structural Modifications and Novel Protein-Binding Sites in Pre-miR-675-Explaining Its Regulatory Mechanism in Carcinogenesis.

Pre-miR-675 is a microRNA expressed from the exon 1 of H19 long noncoding RNA, and the atypical expression of pre-miR-675 has been linked with several diseases and disorders including cancer. To execute its function inside the cell, pre-miR-675 is folded into a particular conformation, which aids in its interaction with several other biological molecules. However, the exact folding dynamics of pre-miR-675 and its protein-binding motifs are currently unknown. Moreover, how H19 lncRNA and pre-miR-675 crosstalk and modulate each other's activities is also unclear. The detailed structural analysis of pre-miR-675 in this study determines its earlier unknown conformation and identifies novel protein-binding sites on pre-miR-675, thus making it an excellent therapeutic target against cancer. Co-folding analysis between H19 lncRNA and pre-miR-675 determine structural transformations in pre-miR-675, thus describing the earlier unknown mechanism of interaction between these two molecules. Comprehensively, this study details the conformation of pre-miR-675 and its protein-binding sites and explains its relationship with H19 lncRNA, which can be interpreted to understand the role of pre-miR-675 in the development and progression of tumorigenesis and designing new therapeutics against cancers.

Effects of sequence-dependent non-native interactions in equilibrium and kinetic folding properties of knotted proteins.

Determining the role of non-native interactions in folding dynamics, kinetics, and mechanisms is a classic problem in protein folding. More recently, this question has witnessed a renewed interest in light of the hypothesis that knotted proteins require the assistance of non-native interactions to fold efficiently. Here, we conduct extensive equilibrium and kinetic Monte Carlo simulations of a simple off-lattice C-alpha model to explore the role of non-native interactions in the thermodynamics and kinetics of three proteins embedding a trefoil knot in their native structure. We find that equilibrium knotted conformations are stabilized by non-native interactions that are non-local, and proximal to native ones, thus enhancing them. Additionally, non-native interactions increase the knotting frequency at high temperatures, and in partially folded conformations below the transition temperatures. Although non-native interactions clearly enhance the efficiency of transition from an unfolded conformation to a partially folded knotted one, they are not required to efficiently fold a knotted protein. Indeed, a native-centric interaction potential drives the most efficient folding transition, provided that the simulation temperature is well below the transition temperature of the considered model system.

Programmable Dual‐Responsive Actuation of Single‐Hydrogel‐Based Bilayer Actuators by Photothermal and Skin Layer Effects with Graphene Oxides

AbstractShape morphing of stimuli‐responsive composite hydrogels has received considerable attention in different research fields. Although various multilayer structures with dissimilar materials are studied to achieve shape morphing, combining swellable hydrogel layers with non‐swellable layers results in issues with interface adhesion and structural integrity. In this study, single‐hydrogel‐based bilayer actuators comprising poly(N‐isopropylacrylamide) (PNIPAM) matrices and graphene oxide (GO)–PNIPAM hinges are presented. Upon temperature rising, the PNIPAM hydrogel acts as the passive layer due to the formation of dense microstructures near the surface (i.e., the skin layer effect), whereas the GO‐PNIPAM hydrogel functions as the active layer, maintaining porous due to structural modification by the presence of GO. Under light exposure, the GO‐PNIPAM hinges experience selective heating due to the photothermal effect of GO. Consequently, the resulting bilayer structures exhibit programmable dual‐responsive 3D shape morphing. Additionally, the folding kinetics of these actuators can be adjusted based on the applied stimulus (temperature changes or light), as they are driven by different mechanisms, the skin layer, or photothermal effects, respectively. Furthermore, the hinge‐based bilayer structures demonstrate walking and steering locomotion by light exposure. This approach can lead to advances in soft robotics, biomimetic systems, and autonomous soft actuators in hydrogel‐based systems.

Characterization of the folding and binding properties of the PTB domain of FRS2 with phosphorylated and unphosphorylated ligands

PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems.