scholarly journals 4096 Refined structure of human ferroportin using restraints from mass spectrometry

2020 ◽  
Vol 4 (s1) ◽  
pp. 101-102
Author(s):  
Christian S. Parry ◽  
Andrey Ivanov ◽  
Guelaguetza Vazquez-meves ◽  
Fatemah A. Alhakami ◽  
Jessika Agyepong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Model ◽  
Structural Information ◽  
Comparative Modeling ◽  
Mass Spectrometry Data ◽  
Peptide Transporter ◽  
Transmembrane Helices ◽  
Intracellular Loop ◽  
Water Solvent ◽  
Dynamics Simulations

OBJECTIVES/GOALS: Mammals require iron for hemoglobin, respiration, immunity and as cofactor in enzymes. But free iron is toxic from the production of reactive oxygen species. Ferroportin is the sole exporter of cellular iron and it crucially determines cellular and systemic iron levels. Labile iron must be tightly regulated. This requires structural understanding. METHODS/STUDY POPULATION: We built structure of human ferroportin (FPN1) using the ab ignition prediction approaches of Rosetta/Robetta and by comparative modeling with distance restraints in MODELLER. Templates selected were from solute carrier protein families of distantly related orthologs and homologs including a proton coupled peptide transporter (PDB ID: 4IKV) and the bacterial iron transporter in outward-open and inward-open states, (PDB ID: 5AYM, 5AYO). Each model was validated by experimental mass spectrometry data. The energy minimized structural model was inserted into a lipid bilayer, placed in a rectangular simulation box, covered with TIP3P water solvent balanced with counterions and conditioned. Finally, we carried out 350 nanoseconds molecular dynamics simulations. RESULTS/ANTICIPATED RESULTS: Our first model of FPN1 (571aa), using Rosetta/Robetta ab initio approach, resembles the structure of the proton-dependent transporter, POT and consists of 12 transmembrane helices. The membrane spanning helices veer away from the orientation in the structure of 4IKV. The alternate model using MODELLER and the method of satisfaction of constraints, returned one template, the structure of Bdellovibrio bacteriovorus iron (Fe2+) transporter homolog (5AYN, 440aa) with sequence identity of 19%. Aligning FPN1 on the template sequence incorporating structural information revealed better conservation (29%). This model also comprises 12 transmembrane helices in two bundles separated by a large intracellular loop. The iron binding site predicted in both models match the structures of distant bacterial homologs. DISCUSSION/SIGNIFICANCE OF IMPACT: We are using these experimentally verified structures and functional data to answer questions about the mechanism of ferroportin iron transport, structural dynamics and the significance of mutations in ferroportin seen in different populations, especially the Q248H mutation found in Africans and black Americans with moderate to high prevalence.

scholarly journals The Extreme C-Terminal Region of Gαs Differentially Couples to the Luteinizing Hormone and β2-Adrenergic Receptors

2011 ◽  
Vol 25 (8) ◽  
pp. 1416-1430 ◽  
Author(s):  
Geneva DeMars ◽  
Francesca Fanelli ◽  
David Puett
Keyword(s):  
G Protein ◽  
Comparative Modeling ◽  
Cell System ◽  
Amino Acid Residues ◽  
Intracellular Loop ◽  
C Terminus ◽  
Model Cell ◽  
Rigid Body Docking ◽  
Molecular Landscape ◽  
Dynamics Simulations

The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein α-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of Gαs is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and β2-adrenergic receptor (β2-AR), a model cell system was created for the expression and manipulation of Gαs subunits in LHR+ s49 ck cells that lack endogenous Gαs. On the basis of studies involving truncations, mutations, and chain extensions of Gαs, the CT was found to be necessary for LHR and β2-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the Gαs CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of Gαs with residues in LHR and β2-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of Gαs recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of β2-AR are expected to contribute significantly to differences in Gαs recognition by the two receptors.

scholarly journals Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

2021 ◽  
Author(s):  
Stella Prins ◽  
Valentina Corradi ◽  
David N. Sheppard ◽  
D. Peter Tieleman ◽  
Paola Vergani
Keyword(s):  
Cystic Fibrosis ◽  
Hydrogen Bonds ◽  
Ion Channel ◽  
Hydrophobic Interactions ◽  
Anion Channel ◽  
Transmembrane Helices ◽  
Aromatic Rings ◽  
Intracellular Loop ◽  
Dynamic Interface ◽  
Dynamics Simulations

AbstractDeletion of phenylalanine 508 (F508del), in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, is the most common cause of cystic fibrosis (CF). F508 is located on nucleotide-binding domain 1 (NBD1) in contact with cytosolic extensions of transmembrane helices, in particular intracellular loop 4 (ICL4). We carried out a mutagenesis scan of ICL4 by introducing five or six second-site mutations at eleven positions in cis with F508del, and quantifying changes in membrane proximity and ion-channel function of CFTR. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains, (F1068, R1070W, and F1074, mimicking F1068, F508 and F1074 in wild-type CFTR). F508del-CFTR had a distorted aromatic stack, with F1068 displaced towards space vacated by F508. In F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds, and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR are F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. Finally, two mutations identified in a yeast scan (A141S and R1097T, on adjacent transmembrane helices linked to ICL1 and ICL4) also partially rescued F508del-CFTR function. These studies highlight the importance of hydrophobic interactions and conformational flexibility at the ICL4/NBD1 interface, advancing understanding of the structural underpinning of F508del dysfunction.

scholarly journals PhoX – an IMAC-enrichable Crosslinking Reagent

2019 ◽  
Author(s):  
Barbara A. Steigenberger ◽  
Roland J. Pieters ◽  
Albert J.R. Heck ◽  
Richard A. Scheltema
Keyword(s):  
Mass Spectrometry ◽  
Ribosomal Proteins ◽  
Structural Model ◽  
Structural Information ◽  
Protein Complexes ◽  
Protein Structures ◽  
Measurement Time ◽  
Close Proximity ◽  
Stoichiometric Reaction ◽  
Crosslinking Reagent

AbstractChemical crosslinking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, sub-stoichiometric reaction efficiencies render routine detection of crosslinked peptides problematic. Here we present a new tri-functional crosslinking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the crosslinked peptides amenable to the well-established IMAC enrichment. The handle allows for 300x enrichment efficiency and 97% specificity, dramatically reducing measurement time and improving data quality. We exemplify the approach on various model proteins and protein complexes, e.g. resulting in a structural model of the LRP1/RAP complex. PhoX is also applicable to whole cell lysates. When focusing the database search on ribosomal proteins, our first attempt resulted in 355 crosslinks, out-performing current efforts in less measurement time.

scholarly journals Processing Extremely Large Powder Diffraction Datasets Using The Rietveld Method

2014 ◽  
Vol 70 (a1) ◽  
pp. C502-C502
Author(s):  
David Wragg ◽  
Matthew O'Brien ◽  
Marco Di Michiel ◽  
Francesca Lønstad Bleken ◽  
Helmer Fjellvåg ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Powder Diffraction ◽  
Structural Information ◽  
Rietveld Method ◽  
Structural Parameters ◽  
Parametric Method ◽  
Mass Spectrometry Data ◽  
High Time Resolution ◽  
Synchrotron Powder Diffraction

Modern in situ synchrotron powder diffraction experiments can produce massive volumes of data which are of suitable quality for real structural information to be extracted. Parametric Rietveld refinement (Stinton, 2007) is an ideal method for dealing with such datasets as a huge number of diffraction patterns can be processed in parallel while the number of refined parameters is reduced by linking between scans. The time saving compared to earlier sequential methods of refinement using batch files is very significant with processing times being reduced from weeks to a few hours. The stability of the parametric method allows not only extraction of information from data with very weak trends but also refinement of entire slices of a tomographic map including the regions with zero diffraction. The power of the technique will be illustrated by examples from reactor scanning experiments with high time resolution (Wragg, 2012, 2013) as well as more conventional in situ powder diffraction and operando experiments combining diffraction with mass spectrometry. The extraction of structural information from complete tomographic datasets and reconstructions with real structural parameters will also be demonstrated. The figure shows time and space resolved c-axis data for the SAPO-34 catalyst during methanol to olefin conversion, together with mass spectrometry data collected during the experiment.

Applications of Time-OF-Flight Secondary Ion Mass Spectrometry (TOF-SIMS)

1990 ◽  
Vol 48 (2) ◽  
pp. 308-309
Author(s):  
Bruno Schueler ◽  
Robert W. Odom
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Structural Information ◽  
Time Of Flight ◽  
Biological Tissues ◽  
Polymer Surfaces ◽  
Tof Sims ◽  
Secondary Ions ◽  
Ion Mass Spectrometry ◽  
Secondary Ion

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) provides unique capabilities for elemental and molecular compositional analysis of a wide variety of surfaces. This relatively new technique is finding increasing applications in analyses concerned with determining the chemical composition of various polymer surfaces, identifying the composition of organic and inorganic residues on surfaces and the localization of molecular or structurally significant secondary ions signals from biological tissues. TOF-SIMS analyses are typically performed under low primary ion dose (static SIMS) conditions and hence the secondary ions formed often contain significant structural information.This paper will present an overview of current TOF-SIMS instrumentation with particular emphasis on the stigmatic imaging ion microscope developed in the authors’ laboratory. This discussion will be followed by a presentation of several useful applications of the technique for the characterization of polymer surfaces and biological tissues specimens. Particular attention in these applications will focus on how the analytical problem impacts the performance requirements of the mass spectrometer and vice-versa.

154: Integration of TPSA and High-Throughput Mass Spectrometry Data Improves Prostate Cancer Prediction

2007 ◽  
Vol 177 (4S) ◽  
pp. 52-53
Author(s):  
Stefano Ongarello ◽  
Eberhard Steiner ◽  
Regina Achleitner ◽  
Isabel Feuerstein ◽  
Birgit Stenzel ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Mass Spectrometry ◽  
High Throughput ◽  
Mass Spectrometry Data ◽  
Cancer Prediction

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Use of Molecular Dynamics Simulations in Structure-Based Drug Discovery

2019 ◽  
Vol 25 (31) ◽  
pp. 3339-3349 ◽  
Author(s):  
Indrani Bera ◽  
Pavan V. Payghan
Keyword(s):  
Molecular Dynamics ◽  
Drug Discovery ◽  
Molecular Dynamics Simulations ◽  
Drug Target ◽  
Structural Information ◽  
Drug Binding ◽  
Structure Based Drug Design ◽  
Drug Designing ◽  
Target Binding ◽  
Dynamics Simulations

Background: Traditional drug discovery is a lengthy process which involves a huge amount of resources. Modern-day drug discovers various multidisciplinary approaches amongst which, computational ligand and structure-based drug designing methods contribute significantly. Structure-based drug designing techniques require the knowledge of structural information of drug target and drug-target complexes. Proper understanding of drug-target binding requires the flexibility of both ligand and receptor to be incorporated. Molecular docking refers to the static picture of the drug-target complex(es). Molecular dynamics, on the other hand, introduces flexibility to understand the drug binding process. Objective: The aim of the present study is to provide a systematic review on the usage of molecular dynamics simulations to aid the process of structure-based drug design. Method: This review discussed findings from various research articles and review papers on the use of molecular dynamics in drug discovery. All efforts highlight the practical grounds for which molecular dynamics simulations are used in drug designing program. In summary, various aspects of the use of molecular dynamics simulations that underline the basis of studying drug-target complexes were thoroughly explained. Results: This review is the result of reviewing more than a hundred papers. It summarizes various problems that use molecular dynamics simulations. Conclusion: The findings of this review highlight how molecular dynamics simulations have been successfully implemented to study the structure-function details of specific drug-target complexes. It also identifies the key areas such as stability of drug-target complexes, ligand binding kinetics and identification of allosteric sites which have been elucidated using molecular dynamics simulations.

Nonparametric Pre-Processing Methods and Inference Tools for Analyzing Time-of-Flight Mass Spectrometry Data.

2007 ◽  
Vol 3 (2) ◽  
pp. 127-147 ◽  
Author(s):  
Anestis Antoniadis ◽  
Jeremie Bigot ◽  
Sophie Lambert-Lacroix ◽  
Frederique Letue
Keyword(s):  
Mass Spectrometry ◽  
Time Of Flight ◽  
Mass Spectrometry Data ◽  
Processing Methods ◽  
Flight Mass Spectrometry

scholarly journals Model Analysis and Optimization of BIM Technology in High-rise Shear Wall Residential Structure

2019 ◽  
Vol 267 ◽  
pp. 02001
Author(s):  
Liangli Xiao ◽  
Yan Liu ◽  
Zhuang Du ◽  
Zhao Yang ◽  
Kai Xu
Keyword(s):  
Structural Analysis ◽  
Collaborative Design ◽  
Structural Model ◽  
Structural Information ◽  
Shear Wall ◽  
Structure Design ◽  
Secondary Development ◽  
Optimized Design ◽  
Analysis Model ◽  
High Rise

This study combines specific high-rise shear wall residential projects with the Revit to demonstrate BIM application processes. The use of R-Star CAD may help to realize the link barrier of the building information model and the structural analysis software PKPM. Sequentially, the information supplement of the structural analysis model is completed by extracting the structural information with the Revit secondary development. By the collaborative design platform based on BIM technology, the paper examines the collision check of structural model, conducts collision analysis on other professional models and modifies the design scheme for conflict points. After the statistics of material usage, an optimized design is proposed. The findings of this paper could contribute to provide some reference for the specific application of BIM in structural design and realize the application of BIM technology in the process of building structure design.

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