Browsing by Author "Schafer, Nicholas P."
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Item A structural dynamics model for how CPEB3 binding to SUMO2 can regulate translational control in dendritic spines(Public Library of Science, 2022) Gu, Xinyu; Schafer, Nicholas P.; Bueno, Carlos; Lu, Wei; Wolynes, Peter G.; Center for Theoretical Biological PhysicsA prion-like RNA-binding protein, CPEB3, can regulate local translation in dendritic spines. CPEB3 monomers repress translation, whereas CPEB3 aggregates activate translation of its target mRNAs. However, the CPEB3 aggregates, as long-lasting prions, may raise the problem of unregulated translational activation. Here, we propose a computational model of the complex structure between CPEB3 RNA-binding domain (CPEB3-RBD) and small ubiquitin-like modifier protein 2 (SUMO2). Free energy calculations suggest that the allosteric effect of CPEB3-RBD/SUMO2 interaction can amplify the RNA-binding affinity of CPEB3. Combining with previous experimental observations on the SUMOylation mode of CPEB3, this model suggests an equilibrium shift of mRNA from binding to deSUMOylated CPEB3 aggregates to binding to SUMOylated CPEB3 monomers in basal synapses. This work shows how a burst of local translation in synapses can be silenced following a stimulation pulse, and explores the CPEB3/SUMO2 interplay underlying the structural change of synapses and the formation of long-term memories.Item Assemblies of calcium/calmodulin-dependent kinase II with actin and their dynamic regulation by calmodulin in dendritic spines(National Academy of Sciences, 2019) Wang, Qian; Chen, Mingchen; Schafer, Nicholas P.; Bueno, Carlos; Song, Sarah S.; Hudmon, Andy; Wolynes, Peter G.; Waxham, M. Neal; Cheung, Margaret S.The structural dynamics of the dendritic synapse, arising from the remodeling of actin cytoskeletons, has been widely associated with memory and cognition. The remodeling is regulated by intracellular Ca2+ levels. Under low Ca2+ concentration, actin filaments are bundled by a calcium signaling protein, CaMKII. When the Ca2+ concentration is raised, CaMKII dissociates from actin and opens the window for actin remodeling. At present, the molecular details of the actin bundling and regulation are elusive. Herein we use experimental tools along with molecular simulations to construct a model of how CaMKII bundles actin and how the CaMKII–actin architecture is regulated by Ca2+ signals. In this way, our results explain how Ca2+ signals ultimately change the structure of the dendritic synapse.Item Discrete Kinetic Models from Funneled Energy Landscape Simulations(Public Library of Science, 2012) Schafer, Nicholas P.; Hoffman, Ryan M.B.; Burger, Anat; Craig, Patricio O.; Komives, Elizabeth A.A general method for facilitating the interpretation of computer simulations of protein folding with minimally frustrated energy landscapes is detailed and applied to a designed ankyrin repeat protein (4ANK). In the method, groups of residues are assigned to foldons and these foldons are used to map the conformational space of the protein onto a set of discrete macrobasins. The free energies of the individual macrobasins are then calculated, informing practical kinetic analysis. Two simple assumptions about the universality of the rate for downhill transitions between macrobasins and the natural local connectivity between macrobasins lead to a scheme for predicting overall folding and unfolding rates, generating chevron plots under varying thermodynamic conditions, and inferring dominant kinetic folding pathways. To illustrate the approach, free energies of macrobasins were calculated from biased simulations of a non-additive structure-based model using two structurally motivated foldon definitions at the full and half ankyrin repeat resolutions. The calculated chevrons have features consistent with those measured in stopped flow chemical denaturation experiments. The dominant inferred folding pathway has an “inside-out”, nucleation-propagation like character.Item Energy landscape underlying spontaneous insertion and folding of an alpha-helical transmembrane protein into a bilayer(Springer Nature, 2018) Lu, Wei; Schafer, Nicholas P.; Wolynes, Peter G.Membrane protein folding mechanisms and rates are notoriously hard to determine. A recent force spectroscopy study of the folding of an α-helical membrane protein, GlpG, showed that the folded state has a very high kinetic stability and a relatively low thermodynamic stability. Here, we simulate the spontaneous insertion and folding of GlpG into a bilayer. An energy landscape analysis of the simulations suggests that GlpG folds via sequential insertion of helical hairpins. The rate-limiting step involves simultaneous insertion and folding of the final helical hairpin. The striking features of GlpG's experimentally measured landscape can therefore be explained by a partially inserted metastable state, which leads us to a reinterpretation of the rates measured by force spectroscopy. Our results are consistent with the helical hairpin hypothesis but call into question the two-stage model of membrane protein folding as a general description of folding mechanisms in the presence of bilayers.Item Funneling and frustration in the energy landscapes of some designed and simplified proteins(American Institute of Physics, 2013) Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.; Center for Theoretical Biological PhysicsWe explore the similarities and differences between the energy landscapes of proteins that have been selected by nature and those of some proteins designed by humans. Natural proteins have evolved to function as well as fold, and this is a source of energetic frustration. The sequence of Top7, on the other hand, was designed with architecture alone in mind using only native state stability as the optimization criterion. Its topology had not previously been observed in nature. Experimental studies show that the folding kinetics of Top7 is more complex than the kinetics of folding of otherwise comparable naturally occurring proteins. In this paper, we use structure prediction tools, frustration analysis, and free energy profiles to illustrate the folding landscapes of Top7 and two other proteins designed by Takada. We use both perfectly funneled (structure-based) and predictive (transferable) models to gain insight into the role of topological versus energetic frustration in these systems and show how they differ from those found for natural proteins. We also study how robust the folding of these designs would be to the simplification of the sequences using fewer amino acid types. Simplification using a five amino acid type code results in comparable quality of structure prediction to the full sequence in some cases, while the two-letter simplification scheme dramatically reduces the quality of structure prediction.Item Mass Spectrometry of RNA-Binding Proteins during Liquid–Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures(American Chemical Society, 2023) Sahin, Cagla; Motso, Aikaterini; Gu, Xinyu; Feyrer, Hannes; Lama, Dilraj; Arndt, Tina; Rising, Anna; Gese, Genis Valentin; Hällberg, B. Martin; Marklund, Erik. G.; Schafer, Nicholas P.; Petzold, Katja; Teilum, Kaare; Wolynes, Peter G.; Landreh, Michael; Center for Theoretical Biological PhysicsLiquid–liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid–liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.Item Oleuropein derivatives from olive fruit extracts reduce α-synuclein fibrillation and oligomer toxicity(Elsevier, 2019) Mohammad-Beigi, Hossein; Aliakbari, Farhang; Sahin, Cagla; Lomax, Charlotte; Tawfike, Ahmed; Schafer, Nicholas P.; Amiri-Nowdijeh, Alireza; Eskandari, Hoda; Møller, Ian Max; Hosseini-Mazinani, Mehdi; Christiansen, Gunna; Ward, Jane L.; Morshedi, Dina; Otzen, Daniel E.; Center for Theoretical Biological PhysicsAggregation of α-synuclein (αSN) is implicated in neuronal degeneration in Parkinson's disease and has prompted searches for natural compounds inhibiting αSN aggregation and reducing its tendency to form toxic oligomers. Oil from the olive tree (Olea europaea L.) represents the main source of fat in the Mediterranean diet and contains variable levels of phenolic compounds, many structurally related to the compound oleuropein. Here, using αSN aggregation, fibrillation, size-exclusion chromatography–multiangle light scattering (SEC-MALS)-based assays, and toxicity assays, we systematically screened the fruit extracts of 15 different olive varieties to identify compounds that can inhibit αSN aggregation and oligomer toxicity and also have antioxidant activity. Polyphenol composition differed markedly among varieties. The variety with the most effective antioxidant and aggregation activities, Koroneiki, combined strong inhibition of αSN fibril nucleation and elongation with strong disaggregation activity on preformed fibrils and prevented the formation of toxic αSN oligomers. Fractionation of the Koroneiki extract identified oleuropein aglycone, hydroxyl oleuropein aglycone, and oleuropein as key compounds responsible for the differences in inhibition across the extracts. These phenolic compounds inhibited αSN amyloidogenesis by directing αSN monomers into small αSN oligomers with lower toxicity, thereby suppressing the subsequent fibril growth phase. Our results highlight the molecular consequences of differences in the level of effective phenolic compounds in different olive varieties, insights that have implications for long-term human health.Item OpenAWSEM with Open3SPN2: A fast, flexible, and accessible framework for large-scale coarse-grained biomolecular simulations(Public Library of Science, 2021) Lu, Wei; Bueno, Carlos; Schafer, Nicholas P.; Moller, Joshua; Jin, Shikai; Chen, Xun; Chen, Mingchen; Gu, Xinyu; Davtyan, Aram; Pablo, Juan J. de; Wolynes, Peter G.; Center for Theoretical Biological PhysicsWe present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.Item Predictive energy landscapes for folding α-helical transmembrane proteins(National Academy of Sciences, 2014) Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.; Center for Theoretical Biological PhysicsWe explore the hypothesis that the folding landscapes of membrane proteins are funneled once the proteins' topology within the membrane is established. We extend a protein folding model, the associative memory, water-mediated, structure, and energy model (AWSEM) by adding an implicit membrane potential and reoptimizing the force field to account for the differing nature of the interactions that stabilize proteins within lipid membranes, yielding a model that we call AWSEM-membrane. Once the protein topology is set in the membrane, hydrophobic attractions play a lesser role in finding the native structure, whereas polar-polar attractions are more important than for globular proteins. We examine both the quality of predictions made with AWSEM-membrane when accurate knowledge of the topology and secondary structure is available and the quality of predictions made without such knowledge, instead using bioinformatically inferred topology and secondary structure based on sequence alone. When no major errors are made by the bioinformatic methods used to assign the topology of the transmembrane helices, these two types of structure predictions yield roughly equivalent quality structures. Although the predictive energy landscape is transferable and not structure based, within the correct topological sector we find the landscape is indeed very funneled: Thermodynamic landscape analysis indicates that both the total potential energy and the contact energy decrease as native contacts are formed. Nevertheless the near symmetry of different helical packings with respect to native contact formation can result in multiple packings with nearly equal thermodynamic occupancy, especially at temperatures just below collapse.Item Protein Frustratometer 2: a tool to localize energetic frustration in protein molecules, now with electrostatics(Oxford University Press, 2016) Parra, R. Gonzalo; Schafer, Nicholas P.; Radusky, Leandro G.; Tsai, Min-Yeh; Guzovsky, A. Brenda; Wolynes, Peter G.; Ferreiro, Diego U.; Center for Theoretical Biological PhysicsThe protein frustratometer is an energy landscape theory-inspired algorithm that aims at localizing and quantifying the energetic frustration present in protein molecules. Frustration is a useful concept for analyzing proteins' biological behavior. It compares the energy distributions of the native state with respect to structural decoys. The network of minimally frustrated interactions encompasses the folding core of the molecule. Sites of high local frustration often correlate with functional regions such as binding sites and regions involved in allosteric transitions. We present here an upgraded version of a webserver that measures local frustration. The new implementation that allows the inclusion of electrostatic energy terms, important to the interactions with nucleic acids, is significantly faster than the previous version enabling the analysis of large macromolecular complexes within a user-friendly interface. The webserver is freely available at URL:ᅠhttp://frustratometer.qb.fcen.uba.ar.Item Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level(MDPI, 2019) Lin, Xingcheng; Kulkarni, Prakash; Bocci, Federico; Schafer, Nicholas P.; Roy, Susmita; Tsai, Min-Yeh; He, Yanan; Chen, Yihong; Rajagopalan, Krithika; Mooney, Steven M.; Zeng, Yu; Weninger, Keith; Grishaev, Alex; Onuchic, José Nelson; Levine, Herbert; Wolynes, Peter G.; Salgia, Ravi; Rangarajan, Govindan; Uversky, Vladimir; Orban, John; Jolly, Mohit KumarFolded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.Item Surveying biomolecular frustration at atomic resolution(Springer Nature, 2020) Chen, Mingchen; Chen, Xun; Schafer, Nicholas P.; Clementi, Cecilia; Komives, Elizabeth A.; Ferreiro, Diego U.; Wolynes, Peter G.; Center for Theoretical Biological PhysicsTo function, biomolecules require sufficient specificity of interaction as well as stability to live in the cell while still being able to move. Thermodynamic stability of only a limited number of specific structures is important so as to prevent promiscuous interactions. The individual interactions in proteins, therefore, have evolved collectively to give funneled minimally frustrated landscapes but some strategic parts of biomolecular sequences located at specific sites in the structure have been selected to be frustrated in order to allow both motion and interaction with partners. We describe a framework efficiently to quantify and localize biomolecular frustration at atomic resolution by examining the statistics of the energy changes that occur when the local environment of a site is changed. The location of patches of highly frustrated interactions correlates with key biological locations needed for physiological function. At atomic resolution, it becomes possible to extend frustration analysis to protein-ligand complexes. At this resolution one sees that drug specificity is correlated with there being a minimally frustrated binding pocket leading to a funneled binding landscape. Atomistic frustration analysis provides a route for screening for more specific compounds for drug discovery.Item The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes(National Academy of Sciences, 2024) Jaafari, Hana; Bueno, Carlos; Schafer, Nicholas P.; Martin, Jonathan; Morcos, Faruck; Wolynes, Peter G.; Center for Theoretical BiophysicsProtein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone “devolution.” Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes’ former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.