Search Results (27)

Proinsulin has three distinct regions: the well-folded A- and B-chains and the dynamic disordered C-peptide. The highly conserved B-chain is a hotspot for diabetes-associated mutations, including the severe loss-of-function R(B22)Q mutation linked to childhood-onset diabetes. Here, we explore R(B22)’s role in proinsulin stability using AlphaFold-predicted structures and metadynamics simulations to achieve enhanced sampling of the free energy landscape. Our results show that R(B22) stabilizes proinsulin by interacting with N86. Substituting R(B22) with E or Q disrupts this interaction, increasing conformational flexibility. The R(B22)Q variant exhibits a flattened free energy landscape, favoring unfolded states. Additional substitutions, including Gly, Ala, Lys, Tyr, Asp, and Phe, destabilize proinsulin to varying extents by weakening hydrogen bonding. Disrupting the R(B22)–N86 interaction broadly reduces inter-chain contacts, raising the risk of aggregation-prone states. Given the link between R(B22) mutations and diabetes, our study provides crucial molecular insights into proinsulin instability. These findings highlight the role of key inter-domain (A-Chain–B-chain, B-Chain–C-peptide, and A-Chain–C-peptide) interactions in maintaining protein structures and the implications this has for understanding disease-associated proinsulin variants. Full article

Prolyl cis/trans isomerization is a rate-limiting step in protein folding, often coupling directly to the acquisition of native structure. Here, we investigated the interplay between folding and prolyl isomerization in the N2 domain of the gene-3-protein from filamentous phage fd, which adopts a native-state cis/trans equilibrium at Pro161. Using mutational and Φ-value analysis, we identified a discrete folding nucleus encompassing the β-strands surrounding Pro161. These native-like interactions form early in the folding pathway and provide the energy to shift the cis/trans equilibrium toward the cis form. Variations distant from the Pro161-loop have minimal impact on the cis/trans ratio, underscoring the spatial specificity and localized control of the isomerization process. Using NMR spectroscopy, we determined the structures for both native N2 forms. The cis- and trans-Pro161 conformations are overall identical and exhibit only slight differences around the Pro161-loop. The cis-conformation adopts a more compact structure with improved backbone hydrogen bonding, explaining the approximately 10 kJ·mol⁻¹ stability increase of the cis state. Our findings highlight that prolyl isomerization in the N2 domain is governed by a localized folding nucleus rather than global stability changes. This localized energetic coupling ensures that proline isomerization is not simply a passive, slow step but an integral component of the folding landscape, optimizing both the formation of native structure and the establishment of the cis-conformation. Full article

Protein folding is a process in which a polypeptide must undergo folding process to obtain its three-dimensional structure. Thermodynamically, it is a process of enthalpy to overcome the loss of conformational entropy in folding. Folding is primarily related to hydrophobic interactions and intramolecular hydrogen bondings. During folding, hydrophobic interactions are regarded to be the driving forces, especially in the initial structural collapse of a protein. Additionally, folding is guided by the strong interactions within proteins, such as intramolecular hydrogen bondings related to the α-helices and β-sheets of proteins. Therefore, a protein is divided into the folding key (FK) regions related to intramolecular hydrogen bondings and the non-folding key (non-FK) regions. Various conformations are expected for FK and non-FK regions. Different from non-FK regions, it is necessary for FK regions to form the specific conformations in folding, which are regarded as the necessary folding pathways (or “beacons”). Additionally, sequential folding is expected for the FK regions, and the intermediate state is found during folding. They are reflected on the local basins in the free energy landscape (FEL) of folding. To demonstrate the structural model, molecular dynamics (MD) simulations are conducted on the folding pathway of the TRP-cage in water. Full article

Protein folding is the most fundamental and universal example of biomolecular self-organization and is characterized as an intramolecular process. In contrast, amyloidogenic proteins can interact with one another, leading to protein aggregation. The energy landscape of amyloid fibril formation is characterized by many minima for different competing low-energy structures and, therefore, is much more enigmatic than that of multiple folding pathways. Thus, to understand the entire energy landscape of protein aggregation, it is important to elucidate the full picture of conformational changes and polymorphisms of amyloidogenic proteins. This review provides an overview of the conformational diversity of amyloid-β (Aβ) characterized from experimental and theoretical approaches. Aβ exhibits a high degree of conformational variability upon transiently interacting with various binding molecules in an unstructured conformation in a solution, forming an α-helical intermediate conformation on the membrane and undergoing a structural transition to the β-conformation of amyloid fibrils. This review also outlines the structural polymorphism of Aβ amyloid fibrils depending on environmental factors. A comprehensive understanding of the energy landscape of amyloid formation considering various environmental factors will promote drug discovery and therapeutic strategies by controlling the fibril formation pathway and targeting the consequent morphology of aggregated structures. Full article

RNA/DNA difference (RDD) is a post-transcriptional modification playing a crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genomes, RDDs in plant nuclear genomes are not well studied at present. Here, we investigated the RDDs associated with fusarium head blight (FHB) through a novel method by comparing the RNA-seq data between Fusarium-infected and control samples of four wheat genotypes. A total of 187 high-confidence unique RDDs in 36 genes were identified, representing the first landscape of the FHB-responsive RDD in wheat. The majority (26) of these 36 RDD genes were correlated either positively or negatively with FHB levels. Effects of these RDDs on RNA and protein sequences have been identified, their editing frequency and the expression level of the corresponding genes provided, and the prediction of the effect on the minimum folding free energy of mRNA, miRNA binding, and colocation of RDDs with conserved domains presented. RDDs were predicted to induce modifications in the mRNA and protein structures of the corresponding genes. In two genes, TraesCS1B02G294300 and TraesCS3A02G263900, editing was predicted to enhance their affinity with tae-miR9661-5p and tae-miR9664-3p, respectively. To our knowledge, this study is the first report of the association between RDD and FHB in wheat; this will contribute to a better understanding of the molecular basis underlying FHB resistance, and potentially lead to novel strategies to improve wheat FHB resistance through epigenetic methods. Full article

Despite the recent advances in the prediction of protein structures by deep neutral networks, the elucidation of protein-folding mechanisms remains challenging. A promising theory for describing protein folding is a coarse-grained statistical mechanical model called the Wako-Saitô-Muñoz-Eaton (WSME) model. The model can calculate the free-energy landscapes of proteins based on a three-dimensional structure with low computational complexity, thereby providing a comprehensive understanding of the folding pathways and the structure and stability of the intermediates and transition states involved in the folding reaction. In this review, we summarize previous and recent studies on protein folding and dynamics performed using the WSME model and discuss future challenges and prospects. The WSME model successfully predicted the folding mechanisms of small single-domain proteins and the effects of amino-acid substitutions on protein stability and folding in a manner that was consistent with experimental results. Furthermore, extended versions of the WSME model were applied to predict the folding mechanisms of multi-domain proteins and the conformational changes associated with protein function. Thus, the WSME model may contribute significantly to solving the protein-folding problem and is expected to be useful for predicting protein folding, stability, and dynamics in basic research and in industrial and medical applications. Full article

The spliceosome protein U1A is a prototype case of the RNA recognition motif (RRM) ubiquitous in biological systems. The in vitro kinetics of the chemical denaturation of U1A indicate that the unfolding of U1A is a two-state process but takes place via high energy channeling and a malleable transition state, an interesting variation of typical two-state behavior. Molecular dynamics (MD) simulations have been applied extensively to the study of two-state unfolding and folding of proteins and provide an opportunity to obtain a theoretical account of the experimental results and a molecular model for the transition state ensemble. We describe herein all-atom MD studies including explicit solvent of up to 100 ns on the thermal unfolding (UF) of U1A and 13 mutants. Multiple MD UF trajectories are carried out to ensure accuracy and reproducibility. A vector representation of the MD unfolding process in RMSD space is obtained and used to calculate a free energy landscape for U1A unfolding. A corresponding MD simulation and free energy landscape for the protein CI2, well known to follow a simple two state folding/unfolding model, is provided as a control. The results indicate that the unfolding pathway on the MD calculated free energy landscape of U1A shows a markedly extended transition state compared with that of CI2. The MD results support the interpretation of the observed chevron plots for U1A in terms of a high energy, channel-like transition state. Analysis of the MDUF structures shows that the transition state ensemble involves microstates with most of the RRM secondary structure intact but expanded by ~14% with respect to the radius of gyration. Comparison with results on a prototype system indicates that the transition state involves an ensemble of molten globule structures and extends over the region of ~1–35 ns in the trajectories. Additional MDUF simulations were carried out for 13 U1A mutants, and the calculated φ-values show close accord with observed results and serve to validate our methodology. Full article

Tyrosine-protein kinase Yes (YES1) belongs to the Tyrosine-protein kinase family and is involved in several biological activities, including cell survival, cell–cell adhesion, cell differentiation, and cytoskeleton remodeling. It is highly expressed in esophageal, lung, and bladder cancers, and thus considered as an attractive drug target for cancer therapy. In this study, we performed a virtual screening of phytoconstituents from the IMPPAT database to identify potential inhibitors of YES1. Initially, the molecules were retrieved on their physicochemical properties following the Lipinski rule of five. Then binding affinities calculation, PAINS filter, ADMET, and PASS analyses followed by an interaction analysis to select safe and clinically better hits. Finally, two compounds, Glabrene and Lupinisoflavone C (LIC), with appreciable affinities and a specific interaction towards the AlphaFold predicted structure of YES1, were identified. Their time-evolution analyses were carried out using an all-atom molecular dynamics (MD) simulation, principal component analysis, and free energy landscapes. Altogether, we propose that Glabrene and LIC can be further explored in clinical settings to develop anticancer therapeutics targeting YES1 kinase. Full article

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process. Full article

Single-molecule force spectroscopy has opened a new field of research in molecular biophysics and biochemistry. Pulling experiments on individual proteins permit us to monitor conformational transitions with high temporal resolution and measure their free energy landscape. The force–extension curves of single proteins often present large hysteresis, with unfolding forces that are higher than refolding ones. Therefore, the high energy of the transition state (TS) in these molecules precludes kinetic rates measurements in equilibrium hopping experiments. In irreversible pulling experiments, force-dependent kinetic rates measurements show a systematic discrepancy between the sum of the folding and unfolding TS distances derived by the kinetic Bell–Evans model and the full molecular extension predicted by elastic models. Here, we show that this discrepancy originates from the force-induced movement of TS. Specifically, we investigate the highly kinetically stable protein barnase, using pulling experiments and the Bell–Evans model to characterize the position of its kinetic barrier. Experimental results show that while the TS stays at a roughly constant distance relative to the native state, it shifts with force relative to the unfolded state. Interestingly, a conversion of the protein extension into amino acid units shows that the TS position follows the Leffler–Hammond postulate: the higher the force, the lower the number of unzipped amino acids relative to the native state. The results are compared with the quasi-reversible unfolding–folding of a short DNA hairpin. Full article

The kinase insert domain (KID) of RTK KIT is the key recruitment region for downstream signalling proteins. KID, studied by molecular dynamics simulations as a cleaved polypeptide and as a native domain fused to KIT, showed intrinsic disorder represented by a set of heterogeneous conformations. The accurate atomistic models showed that the helical fold of KID is mainly sequence dependent. However, the reduced fold of the native KID suggests that its folding is allosterically controlled by the kinase domain. The tertiary structure of KID represents a compact array of highly variable α- and 3₁₀-helices linked by flexible loops playing a principal role in the conformational diversity. The helically folded KID retains a collapsed globule-like shape due to non-covalent interactions associated in a ternary hydrophobic core. The free energy landscapes constructed from first principles—the size, the measure of the average distance between the conformations, the amount of helices and the solvent-accessible surface area—describe the KID disorder through a collection of minima (wells), providing a direct evaluation of conformational ensembles. We found that the cleaved KID simulated with restricted N- and C-ends better reproduces the native KID than the isolated polypeptide. We suggest that a cyclic, generic KID would be best suited for future studies of KID f post-transduction effects. Full article

Understanding the energy landscape and the conformational dynamics is crucial for studying many biological or chemical processes, such as protein–protein interaction and RNA folding. Molecular Dynamics (MD) simulations have been a major source of dynamic structure. Although many methods were proposed for learning metastable states from MD data, some key problems are still in need of further investigation. Here, we give a brief review on recent progresses in this field, with an emphasis on some popular methods belonging to a two-step clustering framework, and hope to draw more researchers to contribute to this area. Full article

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207. Full article

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway. Full article

Homochirality of DNA and prevalent chirality of free and protein-bound amino acids in a living organism represents the challenge for modern biochemistry and neuroscience. The idea of an association between age-related disease, neurodegeneration, and racemization originated from the studies of fossils and cataract disease. Under the pressure of new results, this concept has a broader significance linking protein folding, aggregation, and disfunction to an organism’s cognitive and behavioral functions. The integrity of cognitive function is provided by a delicate balance between the evolutionarily imposed molecular homo-chirality and the epigenetic/developmental impact of spontaneous and enzymatic racemization. The chirality of amino acids is the crucial player in the modulation the structure and function of proteins, lipids, and DNA. The collapse of homochirality by racemization is the result of the conformational phase transition. The racemization of protein-bound amino acids (spontaneous and enzymatic) occurs through thermal activation over the energy barrier or by the tunnel transfer effect under the energy barrier. The phase transition is achieved through the intermediate state, where the chirality of alpha carbon vanished. From a thermodynamic consideration, the system in the homo-chiral (single enantiomeric) state is characterized by a decreased level of entropy. The oscillating protein chirality is suggesting its distinct significance in the neurotransmission and flow of perceptual information, adaptive associative learning, and cognitive laterality. The common pathological hallmarks of neurodegenerative disorders include protein misfolding, aging, and the deposition of protease-resistant protein aggregates. Each of the landmarks is influenced by racemization. The brain region, cell type, and age-dependent racemization critically influence the functions of many intracellular, membrane-bound, and extracellular proteins including amyloid precursor protein (APP), TAU, PrP, Huntingtin, α-synuclein, myelin basic protein (MBP), and collagen. The amyloid cascade hypothesis in Alzheimer’s disease (AD) coexists with the failure of amyloid beta (Aβ) targeting drug therapy. According to our view, racemization should be considered as a critical factor of protein conformation with the potential for inducing order, disorder, misfolding, aggregation, toxicity, and malfunctions. Full article