“…(2017) [48] Crosslink data from the literature [35] and simulated crosslinks Crosslink data from the literature [35] and simulated crosslinks Crosslink modeling using shortest solvent-accessible distance and explicit modeling of protein flexibility (DynamXL) Brodie et al . (2017) [64] Several zero-length and short-range crosslinkers. Liquid chromatography–MS Isotopically Coded Cleavable Cross-Linking Analysis Software Suite and Kojak [69] Replica exchange discrete molecular dynamics Crosslinking/Mass Spectrometry A crosslinking/mass spectrometry experiment has at least three experimental steps [8] : (i) incubating the protein or protein mixture with a crosslinking reagent, (ii) digesting the protein into peptides, and (iii) mass spectrometric analysis of the resulting peptide mix.…”
Section: A New Age Of Protein Structure Analysismentioning
Observing the structures of proteins within the cell and tracking structural changes under different cellular conditions are the ultimate challenges for structural biology. This, however, requires an experimental technique that can generate sufficient data for structure determination and is applicable in the native environment of proteins. Crosslinking/mass spectrometry (CLMS) and protein structure determination have recently advanced to meet these requirements and crosslinking-driven de novo structure determination in native environments is now possible. In this opinion article, we highlight recent successes in the field of CLMS with protein structure modeling and challenges it still holds.
“…(2017) [48] Crosslink data from the literature [35] and simulated crosslinks Crosslink data from the literature [35] and simulated crosslinks Crosslink modeling using shortest solvent-accessible distance and explicit modeling of protein flexibility (DynamXL) Brodie et al . (2017) [64] Several zero-length and short-range crosslinkers. Liquid chromatography–MS Isotopically Coded Cleavable Cross-Linking Analysis Software Suite and Kojak [69] Replica exchange discrete molecular dynamics Crosslinking/Mass Spectrometry A crosslinking/mass spectrometry experiment has at least three experimental steps [8] : (i) incubating the protein or protein mixture with a crosslinking reagent, (ii) digesting the protein into peptides, and (iii) mass spectrometric analysis of the resulting peptide mix.…”
Section: A New Age Of Protein Structure Analysismentioning
Observing the structures of proteins within the cell and tracking structural changes under different cellular conditions are the ultimate challenges for structural biology. This, however, requires an experimental technique that can generate sufficient data for structure determination and is applicable in the native environment of proteins. Crosslinking/mass spectrometry (CLMS) and protein structure determination have recently advanced to meet these requirements and crosslinking-driven de novo structure determination in native environments is now possible. In this opinion article, we highlight recent successes in the field of CLMS with protein structure modeling and challenges it still holds.
“…Since steric clashes are common in modeled and low-resolution structures, we employ Chiron 44 to optimize the structure of the spike protein. Chiron resolves atomic clashes by performing short-DMD [38][39][40][41]45,46 simulations on protein structures with minimal or no perturbation to the backbone.…”
A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has emerged as a human pathogen, causing global pandemic and resulting in over 400,000 deaths worldwide. The surface spike protein of SARS-CoV-2 mediates the process of coronavirus entry into human cells by binding angiotensin-converting enzyme 2 (ACE2). Due to the critical role in viral-host interaction and the exposure of spike protein, it has been a focus of most vaccines' developments. However, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable 1 . Here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein.Our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. We then utilize our previously developed protein design tool, Eris, to predict thermodynamically stabilizing mutations in proteins. We validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. We finally utilize the pipeline to identify new prefusion spike protein stabilization mutants.
“…The gel was stained with Coomassie blue and different sections were excised for subsequent analysis. Mass spectrometric analysis was then performed using a nano-HPLC system (Easy-nLC II, Thermo Fisher Scientific), coupled to the ESI-source of an LTQ Orbitrap Velos (Thermo Fisher Scientific), using conditions described previously (79). Briefly, samples were injected onto a 100 μm ID, 360 μm OD trap column packed with Magic C18AQ (Bruker-Michrom), 100 Å, 5 μm pore size (prepared in-house) and desalted by washing with solvent A [2% acetonitrile:98% water, 0.1% formic acid (FA)].…”
MeCP2 -a chromatin-binding protein associated with Rett syndrome -has two main isoforms, MeCP2-E1 and MeCP2-E2, with 96% amino acid identity differing in a few N-terminal amino acid residues. Previous studies have shown brain region-specific expression of these isoforms which, in addition to their different cellular localization and differential expression during brain development, suggest they may also have nonoverlapping molecular mechanisms. However, differential functions of MeCP2-E1 and E2 remain largely unexplored. Here, we show that the N-terminal domains (NTD) of MeCP2-E1 and E2 modulate the ability of the methyl binding domain (MBD) to interact with DNA as well as influencing the turnover rates, binding dynamics, response to nuclear depolarization, and circadian oscillations of the two isoforms. Our proteomics data indicate that both isoforms exhibit unique interacting protein partners. Moreover, genome-wide analysis using ChIP-seq provide evidence for a shared as well as a specific regulation of different sets of genes. Our findings provide insight into the functional complexity of MeCP2 by dissecting differential aspects of its two isoforms.All rights reserved. No reuse allowed without permission.
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