Browsing by Subject "Molecular dynamics"
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Item 3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase(Springer Nature, 2024-12-03) Coricello, Adriana; Nardone, Alanya J.; Lupia, Antonio; Gratteri, Carmen; Vos, Matthijn; Chaptal, Vincent; Alcaro, Stefano; Zhu, Wen; Takagi, Yuichiro; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of MedicineAdvances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.Item A λ-dynamics investigation of insulin Wakayama and other A3 variant binding affinities to the insulin receptor(bioRxiv, 2024-03-17) Barron, Monica P.; Vilseck, Jonah Z.; Biochemistry and Molecular Biology, School of MedicineInsulin Wakayama is a clinical insulin variant where a conserved valine at the third residue on insulin’s A chain (ValA3) is replaced with a leucine (LeuA3), impairing insulin receptor (IR) binding by 140-500 fold. This severe impact on binding from such a subtle modification has posed an intriguing problem for decades. Although experimental investigations of natural and unnatural A3 mutations have highlighted the sensitivity of insulin-IR binding to minor changes at this site, an atomistic explanation of these binding trends has remained elusive. We investigate this problem computationally using λ-dynamics free energy calculations to model structural changes in response to perturbations of the ValA3 side chain and to calculate associated relative changes in binding free energy (ΔΔGbind). The Wakayama LeuA3 mutation and seven other A3 substitutions were studied in this work. The calculated ΔΔGbind results showed high agreement compared to experimental binding potencies with a Pearson correlation of 0.88 and a mean unsigned error of 0.68 kcal/mol. Extensive structural analyses of λ-dynamics trajectories revealed that critical interactions were disrupted between insulin and the insulin receptor as a result of the A3 mutations. This investigation also quantifies the effect that adding an A3 Cδ atom or losing an A3 Cγ atom has on insulin’s binding affinity to the IR. Thus, λ-dynamics was able to successfully model the effects of subtle modifications to insulin’s A3 side chain on its protein-protein interactions with the IR and shed new light on a decades-old mystery: the exquisite sensitivity of hormone-receptor binding to a subtle modification of an invariant insulin residue.Item Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with λ-dynamics(bioRxiv, 2024-12-11) Angelo, Murphy; Bhargava, Yash; Kierzek, Elzbieta; Kierzek, Ryszard; Hayes, Ryan L.; Zhang, Wen; Vilseck, Jonah Z.; Aoki, Scott Takeo; Biochemistry and Molecular Biology, School of MedicineRNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA binding preferences of human Pumilio, a prototypical RNA binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.Item Application of Multiple Length Crosslinkers to the Characterization of Gaseous Protein Structure(American Chemical Society, 2022-09-13) Kit, Melanie Cheung See; Webb, Ian K.; Chemistry and Chemical Biology, School of ScienceThe speed, sensitivity, and tolerance of heterogeneity of native mass spectrometry, as well as the kinetic trapping of solution-like states during electrospray, makes mass spectrometry an attractive method to study protein structure. Increasing resolution of ion mobility measurements and mass resolving power and range are leading to the increase of the information content of intact protein measurements, and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) crosslinkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of crosslinked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase crosslinking to be structurally specific. The gas-phase lengths of the crosslinkers were determined by conformational searches and density functional theory, allowing for the interpretation of the crosslinks as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.Item A Computational Study of the Mechanism for F1-ATPase Inhibition by the Epsilon Subunit(2013) Thomson, Karen J.; Pu, Jingzhi; Ge, Haibo; Sardar, Rajesh; Long, Eric C. (Eric Charles)The multi-protein complex of F0F1 ATP synthase has been of great interest in the fields of microbiology and biochemistry, due to the ubiquitous use of ATP as a biological energy source. Efforts to better understand this complex have been made through structural determination of segments based on NMR and crystallographic data. Some experiments have provided useful data, while others have brought up more questions, especially when structures and functions are compared between bacteria and species with chloroplasts or mitochondria. The epsilon subunit is thought to play a signi cant role in the regulation of ATP synthesis and hydrolysis, yet the exact pathway is unknown due to the experimental difficulty in obtaining data along the transition pathway. Given starting and end point protein crystal structures, the transition pathway of the epsilon subunit was examined through computer simulation.The purpose of this investigation is to determine the likelihood of one such proposed mechanism for the involvement of the epsilon subunit in ATP regulation in bacterial species such as E. coli.Item Conformational Changes in Two Inter-Helical Loops of Mhp1 Membrane Transporter(PLOS, 2015-07-17) Song, Hyun Deok; Zhu, Fangqiang; Department of Physics, School of ScienceMhp1 is a bacterial secondary transporter with high-resolution crystal structures available for both the outward- and inward-facing conformations. Through molecular dynamics simulations of the ligand-free Mhp1 as well as analysis of its crystal structures, here we show that two inter-helical loops, respectively located at the extra- and intracellular ends of the “hash motif” in the protein, play important roles in the conformational transition. In the outward- and inward-facing states of the protein, the loops adopt different secondary structures, either wrapped to the end of an alpha-helix, or unwrapped to extended conformations. In equilibrium simulations of 100 ns with Mhp1 in explicit lipids and water, the loop conformations remain largely stable. In targeted molecular dynamics simulations with the protein structure driven from one state to the other, the loops exhibit resistance and only undergo abrupt changes when other parts of the protein already approach the target conformation. Free energy calculations on the isolated loops further confirm that the wrapping/unwrapping transitions are associated with substantial energetic barriers, and consist of multiple sequential steps involving the rotation of certain backbone torsion angles. Furthermore, in simulations with the loops driven from one state to the other, a large part of the protein follows the loops to the target conformation. Taken together, our simulations suggest that changes of the loop secondary structures would be among the slow degrees of freedom in the conformational transition of the entire protein. Incorporation of detailed loop structures into the reaction coordinate, therefore, should improve the convergence and relevance of the resulting conformational free energy.Item CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy(Sage, 2012-07-24) Piekarz, Andrew D.; Due, Michael R.; Khanna, May; Wang, Bo; Ripsch, Matthew S.; Wang, Ruizhong; Meroueh, Samy O.; Vasko, Michael R.; White, Fletcher A.; Khanna, Rajesh; Anesthesia, School of MedicineBackground: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)]. Results and discussion: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca²⁺ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca²⁺ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. Conclusions: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.Item DCMS: A data analytics and management system for molecular simulation(SpringerOpen, 2014-11-26) Kumar, Anand; Grupcev, Vladimir; Berrada, Meryem; Fogarty, Joseph C.; Tu, Yi-Cheng; Zhu, Xingquan; Pandit, Sagar A.; Xia, Yuni; Department of Computer and Information Science, School of ScienceMolecular Simulation (MS) is a powerful tool for studying physical/chemical features of large systems and has seen applications in many scientific and engineering domains. During the simulation process, the experiments generate a very large number of atoms and intend to observe their spatial and temporal relationships for scientific analysis. The sheer data volumes and their intensive interactions impose significant challenges for data accessing, managing, and analysis. To date, existing MS software systems fall short on storage and handling of MS data, mainly because of the missing of a platform to support applications that involve intensive data access and analytical process. In this paper, we present the database-centric molecular simulation (DCMS) system our team developed in the past few years. The main idea behind DCMS is to store MS data in a relational database management system (DBMS) to take advantage of the declarative query interface (i.e., SQL), data access methods, query processing, and optimization mechanisms of modern DBMSs. A unique challenge is to handle the analytical queries that are often compute-intensive. For that, we developed novel indexing and query processing strategies (including algorithms running on modern co-processors) as integrated components of the DBMS. As a result, researchers can upload and analyze their data using efficient functions implemented inside the DBMS. Index structures are generated to store analysis results that may be interesting to other users, so that the results are readily available without duplicating the analysis. We have developed a prototype of DCMS based on the PostgreSQL system and experiments using real MS data and workload show that DCMS significantly outperforms existing MS software systems. We also used it as a platform to test other data management issues such as security and compression.Item Effects of TRPC1’s Lysines on Heteromeric TRPC5-TRPC1 Channel Function(MDPI, 2024-12-06) Demaree, Isaac S.; Kumar, Sanjay; Tennessen, Kayla; Hoang, Quyen Q.; White, Fletcher A.; Obukhov, Alexander G.; Anatomy, Cell Biology and Physiology, School of MedicineBackground: TRPC5 proteins form plasma membrane cation channels and are expressed in the nervous and cardiovascular systems. TRPC5 activation leads to cell depolarization and increases neuronal excitability, whereas a homologous TRPC1 inhibits TRPC5 function via heteromerization. The mechanism underlying the inhibitory effect of TRPC1 in TRPC5/TRPC1 heteromers remains unknown. Methods: We used electrophysiological techniques to examine the roles of subunit stoichiometry and positively charged luminal residues of TRPC1 on TRPC5/TRPC1 function. We also performed molecular dynamics simulations. Results: We found that increasing the relative amount of TRPC1 in TRPC5/TRPC1 heteromers reduced histamine-induced cation influx through the heteromeric channels. Consistently, histamine-induced cation influx was small in cells co-expressing TRPC5-TRPC1 concatemers and TRPC1, and large in cells co-expressing TRPC5-TRPC1 concatemers and TRPC5. Molecular dynamics simulations revealed that the TRPC1 protein has two positively charged lysine residues that are facing the heteromeric channel pore lumen. Substitution of these lysines with asparagines decreased TRPC1's inhibitory effect on TRPC5/TRPC1 function, indicating that these lysines may regulate cation influx through TRPC5/TRPC1 heteromers. Additionally, we established that extracellular Mg2+ inhibits cation influx through TRPC5/TRPC1, contributing to channel regulation. Conclusions: We revealed that the inhibitory effect of TRPC1 on heteromeric TRPC5/TRPC1 function likely involves luminal lysines of TRPC1.Item Elastic deformation and area per lipid of membranes: atomistic view from solid-state deuterium NMR spectroscopy(Elsevier, 2015-01) Kinnun, Jacob A.; Mallikarjunaiah, K. J.; Petrache, Horia I.; Brown, Michael F.; Department of Physics, School of ScienceThis article reviews the application of solid-state ²H nuclear magnetic resonance (NMR) spectroscopy for investigating the deformation of lipid bilayers at the atomistic level. For liquid-crystalline membranes, the average structure is manifested by the segmental order parameters (SCD) of the lipids. Solid-state ²H NMR yields observables directly related to the stress field of the lipid bilayer. The extent to which lipid bilayers are deformed by osmotic pressure is integral to how lipid-protein interactions affect membrane functions. Calculations of the average area per lipid and related structural properties are pertinent to bilayer remodeling and molecular dynamics (MD) simulations of membranes. To establish structural quantities, such as area per lipid and volumetric bilayer thickness, a mean-torque analysis of ²H NMR order parameters is applied. Osmotic stress is introduced by adding polymer solutions or by gravimetric dehydration, which are thermodynamically equivalent. Solid-state NMR studies of lipids under osmotic stress probe membrane interactions involving collective bilayer undulations, order-director fluctuations, and lipid molecular protrusions. Removal of water yields a reduction of the mean area per lipid, with a corresponding increase in volumetric bilayer thickness, by up to 20% in the liquid-crystalline state. Hydrophobic mismatch can shift protein states involving mechanosensation, transport, and molecular recognition by G-protein-coupled receptors. Measurements of the order parameters versus osmotic pressure yield the elastic area compressibility modulus and the corresponding bilayer thickness at an atomistic level. Solid-state ²H NMR thus reveals how membrane deformation can affect protein conformational changes within the stress field of the lipid bilayer.