Characterizing the Folding Transition-State Ensembles in the Energy Landscape of an RNA Tetraloop.

Molecular dynamics (MD) simulations have become increasingly powerful and can now describe the folding/unfolding of small biomolecules in atomic detail. However, a major challenge in MD simulations is to represent the complex energy landscape of biomolecules using a small number of reaction coordinates. In this study, we investigate the folding pathways of an RNA tetraloop, gcGCAAgc, using five classical MD simulations with a combined simulation time of approximately 120 µs. Our approach involves analyzing the tetraloop dynamics, including the folding transition state ensembles, using the energy landscape visualization method (ELViM). The ELViM is an approach that uses internal distances to compare any two conformations, allowing for a detailed description of the folding process without requiring root mean square alignment of structures. This method has previously been applied to describe the energy landscape of disordered ß-amyloid peptides and other proteins. The ELViM results in a non-linear projection of the multidimensional space, providing a comprehensive representation of the tetraloop's energy landscape. Our results reveal four distinct transition-state regions and establish the paths that lead to the folded tetraloop structure. This detailed analysis of the tetraloop's folding process has important implications for understanding RNA folding, and the ELViM approach can be used to study other biomolecules.

Ocular and Palpebral Manifestations of Facial Palsy: An Epidemiologic Descriptive Study.

Purpose: We describe the ocular and periocular clinical features in patients with a facial palsy diagnosis of any etiology and to report the demographics, relevant medical history and treatment modalities in these patients. Patients and Methods: Retrospective and descriptive observational study. A total of 60 patients with a facial palsy diagnosis in the last 5 years were recruited from an ophthalmological clinic in northeastern Mexico. Demographic data, such as age, sex, disease evolution and etiology, visual acuity, ocular symptoms and ocular and periocular clinical features were obtained. Personal history of previous ophthalmologic surgery, as well as ocular and systemic diseases, were also recorded. Finally, a comparative analysis was done to determine association between signs, symptoms and treatment modalities. Results: A prevalence of 0.14% was reported, 56.7% of patients were female, and mean age of presentation was 55.63±17.2 years. 76.7% of facial palsy was idiopathic in origin, followed by vascular disease in 8.30% and iatrogenic in 6.70%. 40% of patients had a history of arterial hypertension, 36.3% were diabetic, and 6.70% had cerebrovascular disease. Conclusion: Early diagnosis of facial palsy is crucial in establishing an effective treatment plan and avoiding complications. The impact of this disease in patients' quality of life cannot be overlooked, and steps should be taken to address the different impairments that this ailment entails.

Millisecond molecular dynamics simulations of KRas-dimer formation and interfaces.

Ras dimers have been proposed as building blocks for initiating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cellular signaling pathway. To better examine the structure of possible dimer interfaces, the dynamics of Ras dimerization, and its potential signaling consequences, we performed molecular dynamics simulations totaling 1 ms of sampling, using an all-atom model of two full-length, farnesylated, guanosine triphosphate (GTP)-bound, wild-type KRas4b proteins diffusing on 29%POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine)-mixed POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membranes. Our simulations unveil an ensemble of thermodynamically weak KRas dimers spanning multiple conformations. The most stable conformations, having the largest interface areas, involve helix α2 and a hypervariable region (HVR). Among the dimer conformations, we found that the HVR of each KRas has frequent interactions with various parts of the dimer, thus potentially mediating the dimerization. Some dimer configurations have one KRas G-domain elevated above the lipid bilayer surface by residing on top of the other G-domain, thus likely contributing to the recruitment of cytosolic Raf kinases in the context of a stably formed multi-protein complex. We identified a variant of the α4-α5 KRas-dimer interface that is similar to the interfaces obtained with fluorescence resonance energy transfer (FRET) data of HRas on lipid bilayers. Interestingly, we found two arginine fingers, R68 and R149, that directly interact with the beta-phosphate of the GTP bound in KRas, in a manner similar to what is observed in a crystal structure of GAP-HRas complex, which can facilitate the GTP hydrolysis via the arginine finger of GTPase-activating protein (GAP).

From Protein Design to the Energy Landscape of a Cold Unfolding Protein.

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

Characterization of Heparin's Conformational Ensemble by Molecular Dynamics Simulations and Nuclear Magnetic Resonance Spectroscopy.

Heparin is a highly charged, polysulfated polysaccharide and serves as an anticoagulant. Heparin binds to multiple proteins throughout the body, suggesting a large range of potential therapeutic applications. Although its function has been characterized in multiple physiological contexts, heparin's solution conformational dynamics and structure-function relationships are not fully understood. Molecular dynamics (MD) simulations facilitate the analysis of a molecule's underlying conformational ensemble, which then provides important information necessary for understanding structure-function relationships. However, for MD simulations to afford meaningful results, they must both provide adequate sampling and accurately represent the energy properties of a molecule. The aim of this study is to compare heparin's conformational ensemble using two well-developed force fields for carbohydrates, known as GLYCAM06 and CHARMM36, using replica exchange molecular dynamics (REMD) simulations, and to validate these results with NMR experiments. The anticoagulant sequence, an ultra-low-molecular-weight heparin, known as Arixtra (fondaparinux, sodium), was simulated with both parameter sets. The results suggest that GLYCAM06 matches experimental nuclear magnetic resonance three-bond J-coupling values measured for Arixtra better than CHARMM36. In addition, NOESY and ROESY experiments suggest that Arixtra is very flexible in the sub-millisecond time scale and does not adopt a unique structure at 25 C. Moreover, GLYCAM06 affords a much more dynamic conformational ensemble for Arixtra than CHARMM36.

Atomistic Simulations of Thermal Unfolding.

This tutorial will provide a practical overview of the use of atomistic simulations to study thermal unfolding of biomolecules, in particular small proteins and RNA oligomers. The tutorial focuses on the use of atomistic, all atom simulations of biomolecules in explicit solvent, to study (reversible) thermal unfolding. The simulation methods described here have also been applied to study biomolecules using implicit solvent and coarse-grained models. We do not intend to provide an up-to-date review of the vast literature of biomolecular dynamics, enhanced sampling methods, force field developments, and applications of these methods. The purpose of this tutorial is to provide basic guidelines into the use of these methods to the starting scientist.

Modeling of Peptides with Classical and Novel Machine Learning Force Fields: A Comparison.

The replacement of classical force fields (FFs) with novel neural-network-based frameworks is an emergent topic in molecular dynamics (MD) simulations. In contrast to classical FFs, which have proven their capability to provide insights into complex soft matter systems at an atomistic resolution, the machine learning (ML) potentials have yet to demonstrate their applicability for soft materials. However, the underlying philosophy, which is learning the energy of an atom in its surrounding chemical environment, makes this approach a promising tool. In particular for the exploration of novel chemical compounds, which have not been considered in the original parametrization of classical FFs. In this article, we study the performance of the ANI-2x ML model and compare the results with those of two classical FFs, namely, CHARMM27 and the GROMOS96 43a1 FF. We explore the performance of these FFs for bulk water and two model peptides, trialanine and a 9-mer of the α-aminoisobutyric acid, in vacuum and water. The results for water describe a highly ordered water structure, with a structure similar to those using ab initio molecular dynamics simulations. The energy landscape of the peptides described by Ramachandran maps show secondary structure basins similar to those of the classical FFs but differ in the position and relative stability of the basins. Details of the sampled structures show a divergent performance of the different models, which can be related either to the short-ranged nature of the ML potentials or to shortcomings of the underlying data set used for training. These findings highlight the current state of the applicability of ANI-2x ML potential for MD simulations of soft matter systems. Simultaneously, they provide insights for future improvements of current ML potentials.

Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD.

CRAF activation requires binding to membrane-anchored and active GTP-bound RAS. Whereas its RAS-binding domain (RBD) contains the main binding interface to the RAS G domain, its cysteine-rich domain (CRD) is responsible for association to anionic lipid-rich membranes. Both RAF domains are connected by a short linker, and it remains unclear if the two domains act independently or if one domain can impact the function of the other. Here, we used a combination of coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct to investigate the dynamics of the RBD when it is tethered to CRD that is anchored to a POPC:POPS model membrane. First, we show that the RBD positioning is very dynamic with a preferential localization near the membrane surface. Next, we show that membrane-localized RBD has its RAS-binding interface mostly inaccessible because of its proximity to the membrane. Several positively charged residues in this interface were identified from simulations as important for driving RBD association to the membrane. Surface plasmon resonance (SPR) measurements confirmed that mutations of these RBD residues reduced the liposome partitioning of RBD-CRD. Last, simulations indicated that the presence of RBD near the membrane led to a local enrichment of anionic lipids that could potentially enhance the membrane affinity of the entire RBD-CRD construct. This was supported by SPR measurements that showed stronger liposome partitioning of RBD-CRD relative to CRD alone. These findings thus suggest that the RBD and CRD have synergistic effects on their membrane dynamics, with CRD bringing RBD closer to the membrane that impacts its accessibility to RAS and with RBD causing local anionic lipid enrichment that enhances the overall affinity between the membrane and RBD-CRD. These mechanisms have potential implications on the order of events of the interactions between RAS and CRAF at the membrane.

How Anionic Lipids Affect Spatiotemporal Properties of KRAS4B on Model Membranes.

RAS proteins are small membrane-anchored GTPases that regulate key cellular signaling networks. It has been recently shown that different anionic lipid types can affect the spatiotemporal properties of RAS through dimerization/clustering and signaling fidelity. To understand the effects of anionic lipids on key spatiotemporal properties of RAS, we dissected 1 ms of data from all-atom molecular dynamics simulations for KRAS4B on two model anionic lipid membranes that have 30% of POPS mixed with neutral POPC and 8% of PIP2 mixed with POPC. We unveiled the orientation space of KRAS4B, whose kinetics were slower and more distinguishable on the membrane containing PIP2 than the membrane containing POPS. Particularly, the PIP2-mixed membrane can differentiate a third kinetic orientation state from the other two known orientation states. We observed that each orientation state may yield different binding modes with an RAF kinase, which is required for activating the MAPK/ERK signaling pathway. However, an overall occluded probability, for which RAF kinases cannot bind KRAS4B, remains unchanged on the two different membranes. We identified rare fast diffusion modes of KRAS4B that appear coupled with orientations exposed to cytosolic RAF. Particularly, on the membrane having PIP2, we found nonlinear correlations between the orientation states and the conformations of the cationic farnesylated hypervariable region, which acts as an anchor in the membrane. Using diffusion coefficients estimated from the all-atom simulations, we quantified the effect of PIP2 and POPS on the KRAS4B dimerization via Green's function reaction dynamics simulations, in which the averaged dimerization rate is 12.5% slower on PIP2-mixed membranes.

Presence or Absence of Ras Dimerization Shows Distinct Kinetic Signature in Ras-Raf Interaction.

Initiations of cell signaling pathways often occur through the formation of multiprotein complexes that form through protein-protein interactions. Therefore, detecting their presence is central to understanding the function of a cell signaling pathway, aberration of which often leads to fatal diseases, including cancers. However, the multiprotein complexes are often difficult to detect using microscopes due to their small sizes. Therefore, currently, their presence can be only detected through indirect means. In this article, we propose to investigate the presence or absence of protein complexes through some easily measurable kinetic parameters, such as activation rates. As a proof of concept, we investigate the Ras-Raf system, a well-characterized cell signaling system. It has been hypothesized that Ras dimerization is necessary to create activated Raf dimers. Although there are circumstantial evidences supporting the Ras dimerization hypothesis, direct proof of Ras dimerization is still inconclusive. In the absence of conclusive direct experimental proof, this hypothesis can only be examined through indirect evidences of Ras dimerization. In this article, using a multiscale simulation technique, we provide multiple criteria that distinguishes an activation mechanism involving Ras dimerization from another mechanism that does not involve Ras dimerization. The provided criteria will be useful in the investigation of not only Ras-Raf interaction but also other two-protein interactions.

The Plasma Membrane as a Competitive Inhibitor and Positive Allosteric Modulator of KRas4B Signaling.

Mutant Ras proteins are important drivers of human cancers, yet no approved drugs act directly on this difficult target. Over the last decade, the idea has emerged that oncogenic signaling can be diminished by molecules that drive Ras into orientations in which effector-binding interfaces are occluded by the cell membrane. To support this approach to drug discovery, we characterize the orientational preferences of membrane-bound K-Ras4B in 1.45-ms aggregate time of atomistic molecular dynamics simulations. Individual simulations probe active or inactive states of Ras on membranes with or without anionic lipids. We find that the membrane orientation of Ras is relatively insensitive to its bound guanine nucleotide and activation state but depends strongly on interactions with anionic phosphatidylserine lipids. These lipids slow Ras' translational and orientational diffusion and promote a discrete population in which small changes in orientation control Ras' competence to bind multiple regulator and effector proteins. Our results suggest that compound-directed conversion of constitutively active mutant Ras into functionally inactive forms may be accessible via subtle perturbations of Ras' orientational preferences at the membrane surface.

Methionine 170 is an Environmentally Sensitive Membrane Anchor in the Disordered HVR of K-Ras4B.

Ras protein colocalization at the plasma membrane is implicated in the activation of signaling cascades that promote cell growth, survival, and motility. However, the mechanisms that underpin Ras self-association remain unclear. We use molecular dynamics simulations to show how basic and hydrophobic components of the disordered C-terminal membrane tether of K-Ras4B combine to regulate its membrane interactions. Specifically, anionic lipids attract lysine residues to the membrane surface, thereby splitting the peptide population into two states that exchange on the microsecond time scale. These states differ in the membrane insertion of a methionine residue, which is influenced by local membrane composition. As a result, these states may impose context-dependent biases on the disposition of Ras' signaling domain, with possible implications for the accessibility of its effector binding surfaces. We investigate Ras' ability to nanocluster by fly-casting for patches of anionic lipids and find that while anionic lipids promote the intermolecular association of K-Ras4B membrane tethers, at short range this appears to be a passive process in which anionic lipids electrostatically screen these cationic peptides to mitigate their natural repulsion. Together with the sub-microsecond stability of interpeptide contacts, this result suggests that experimentally observed K-Ras4B nanoclustering is not driven by direct intermolecular contact of its membrane tethers.

High pressure NMR reveals conformational perturbations by disease-causing mutations in amyloid ß-peptide.

Here we present the high pressure NMR characterization of Aß42 and two Aß40 variants with Alzheimer-causing mutations E22G and D23N. While chemical shifts only identified localized changes at ambient pressure compared with Aß40, high pressure NMR revealed a common site with heightened pressure sensitivity at Q15, K16 and L17 in all three variants, which correlates to higher ß-propensity at central hydrophobic cluster (CHC) and faster aggregation.

Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein.

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini-mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this study, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization of the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔGf°) of -4.4 and -6.9 kcal mol-1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔVf) was significantly larger for V67 (71 ± 2 mL mol-1) than for L67 (58 ± 3 mL mol-1) inteins. The measured difference in ΔVf (13 ± 3 mL mol-1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. It is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.

Equilibrium Denaturation and Preferential Interactions of an RNA Tetraloop with Urea.

Urea is an important organic cosolute with implications in maintaining osmotic stress in cells and differentially stabilizing ensembles of folded biomolecules. We report an equilibrium study of urea-induced denaturation of a hyperstable RNA tetraloop through unbiased replica exchange molecular dynamics. We find that, in addition to destabilizing the folded state, urea smooths the RNA free energy landscape by destabilizing specific configurations, and forming favorable interactions with RNA nucleobases. A linear concentration-dependence of the free energy (m-value) is observed, in agreement with the results of other RNA hairpins and proteins. Additionally, analysis of the hydrogen-bonding and stacking interactions within RNA primarily show temperature-dependence, while interactions between RNA and urea primarily show concentration-dependence. Our findings provide valuable insight into the effects of urea on RNA folding and describe the thermodynamics of a basic RNA hairpin as a function of solution chemistry.

NMR and Computation Reveal a Pressure-Sensitive Folded Conformation of Trp-Cage.

Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, Tc5b. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of Tc5b were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of Tc5b, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on Tc5b displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

Free-energy landscape of a hyperstable RNA tetraloop.

We report the characterization of the energy landscape and the folding/unfolding thermodynamics of a hyperstable RNA tetraloop obtained through high-performance molecular dynamics simulations at microsecond timescales. Sampling of the configurational landscape is conducted using temperature replica exchange molecular dynamics over three isochores at high, ambient, and negative pressures to determine the thermodynamic stability and the free-energy landscape of the tetraloop. The simulations reveal reversible folding/unfolding transitions of the tetraloop into the canonical A-RNA conformation and the presence of two alternative configurations, including a left-handed Z-RNA conformation and a compact purine Triplet. Increasing hydrostatic pressure shows a stabilizing effect on the A-RNA conformation and a destabilization of the left-handed Z-RNA. Our results provide a comprehensive description of the folded free-energy landscape of a hyperstable RNA tetraloop and highlight the significant advances of all-atom molecular dynamics in describing the unbiased folding of a simple RNA secondary structure motif.

Water Determines the Structure and Dynamics of Proteins.

Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous environment affect the stability of biomolecules. Structurally, water participates chemically in the catalytic function of proteins and nucleic acids and physically in the collapse of the protein chain during folding through hydrophobic collapse and mediates binding through the hydrogen bond in complex formation. Water is a partner that slaves the dynamics of proteins, and water interaction with proteins affect their dynamics. Here we provide a review of the experimental and computational advances over the past decade in understanding the role of water in the dynamics, structure, and function of proteins. We focus on the combination of X-ray and neutron crystallography, NMR, terahertz spectroscopy, mass spectroscopy, thermodynamics, and computer simulations to reveal how water assist proteins in their function. The recent advances in computer simulations and the enhanced sensitivity of experimental tools promise major advances in the understanding of protein dynamics, and water surely will be a protagonist.