scholarly journals Analysis of the impact of molecular motions on the efficiency of XL-MS and the distance restraints in hybrid structural biology

2018 ◽  
Author(s):  
Vladimir Svetlov ◽  
Evgeny Nudler
Keyword(s):  
Structural Biology ◽  
Protein Complexes ◽  
Protein Docking ◽  
Protein Fold ◽  
Molecular Motions ◽  
Cross Link ◽  
Distance Restraints ◽  
Cross Links ◽  
Cross Linker ◽  
The Impact

AbstractCovalent cross-link mapping by mass spectrometry (XL-MS) is rapidly becoming the most widely used method of hybrid structural biology. We investigated the impact of incremental variations of cross-linker length have on the depth of XL-MS interrogation of protein-protein complexes, and assessed the role molecular motions in solution play in generation of cross-link-derived distance restraints. Supplementation of a popular NHS-ester cross-linker, DSS, with 2 reagents shorter or longer by CH2-CH2, increased the number of non-reductant cross-links by ~50%. Molecular dynamics simulations of these cross-linkers revealed 3 individual, partially overlapping ranges of motion, consistent with partially overlapping sets of cross-links formed by each reagent. Similar simulations elucidated protein fold-specific ranges of motions for the reactive and backbone atoms from rigid and flexible target domains. Together these findings create a quantitative framework for generation of cross-linker- and protein fold-specific distance restraints for XL-MS-guided protein-protein docking.

scholarly journals Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

2020 ◽  
Author(s):  
Jan Fiala ◽  
Zdeněk Kukačka ◽  
Petr Novák
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Water Soluble ◽  
Cross Linking ◽  
Side Chains ◽  
Model Protein ◽  
Cross Linker ◽  
Progressive Technology ◽  
The Impact ◽  
Chemical Cross Linking

The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

scholarly journals Differentiation of physical and chemical cross-linking in gelatin methacryloyl hydrogels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lisa Rebers ◽  
Raffael Reichsöllner ◽  
Sophia Regett ◽  
Günter E. M. Tovar ◽  
Kirsten Borchers ◽  
...  
Keyword(s):  
Near Infrared ◽  
Cross Linking ◽  
Cross Link ◽  
Scanning Calorimetry ◽  
Link Density ◽  
Double Bond Conversion ◽  
Cross Links ◽  
Physical And Chemical ◽  
The Impact ◽  
Chemical Cross Linking

AbstractGelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.

scholarly journals A quantitative mapping approach to identify direct interactions within complexomes

2021 ◽  
Author(s):  
Philipp Trepte ◽  
Christopher Secker ◽  
Soon Gang Choi ◽  
Julien Olivet ◽  
Eduardo Silva Ramos ◽  
...  
Keyword(s):  
Structural Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
3D Structure ◽  
Binary Interaction ◽  
Live Cells ◽  
Distance Measurements ◽  
Polyglutamine Expansion ◽  
Multiprotein Complexes ◽  
The Impact

ABSTRACTComplementary methods are required to fully characterize all protein complexes, or the complexome, of a cell. Affinity purification coupled to mass-spectrometry (AP-MS) can identify the composition of complexes at proteome-scale. However, information on direct contacts between subunits is often lacking. In contrast, solving the 3D structure of protein complexes can provide this information, but structural biology techniques are not yet scalable for systematic, proteome-wide efforts. Here, we optimally combine two orthogonal high-throughput binary interaction assays, LuTHy and N2H, and demonstrate that their quantitative readouts can be used to differentiate direct interactions from indirect associations within multiprotein complexes. We also show that LuTHy allows accurate distance measurements between proteins in live cells and apply these findings to study the impact of the polyglutamine expansion mutation on the structurally unresolved N-terminal domain of Huntingtin. Thus, we present a new framework based on quantitative interaction assays to complement structural biology and AP-MS techniques, which should help to provide first-approximation contact maps of multiprotein complexes at proteome-scale.Graphical Abstract

scholarly journals Integrative Modeling of a Sin3/HDAC Complex Sub-structure

2019 ◽  
Author(s):  
Charles A. S. Banks ◽  
Ying Zhang ◽  
Sayem Miah ◽  
Yan Hao ◽  
Mark K. Adams ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Spatial Arrangement ◽  
Scaffolding Protein ◽  
Cross Link ◽  
High Confidence ◽  
Affinity Capture ◽  
Existing Structures ◽  
Distance Restraints ◽  
Cross Links ◽  
Sin3 Complex

AbstractSin3/HDAC complexes function by deacetylating histones, which makes chromatin more compact and modulates gene expression. Although components used to build these complexes have been well defined, we still have only a limited understanding of the structure of the Sin3/HDAC subunits as they are assembled around the scaffolding protein SIN3A. To characterize the spatial arrangement of Sin3 subunits, we combined Halo affinity capture, chemical cross-linking and high-resolution mass spectrometry (XL-MS) to determine intersubunit distance constraints, identifying 66 high-confidence interprotein and 63 high-confidence self cross-links for 13 Sin3 subunits. To validate our XL-MS data, we first mapped self cross-links onto existing structures to verify that cross-link distances were consistent with cross-linker length and subsequently deleted crosslink hotspot regions within the SIN3A scaffolding protein which then failed to capture crosslinked partners. Having assessed cross-link authenticity, we next used distance restraints from interprotein cross-links to guide assembly of a Sin3 complex substructure. We identified the relative positions of subunits SAP30L, HDAC1, SUDS3, HDAC2, and ING1 around the SIN3A scaffold. The architecture of this subassembly suggests that multiple factors have space to assemble to collectively influence the behavior of the catalytic subunit HDAC1.

scholarly journals A structural biology community assessment of AlphaFold 2 applications

2021 ◽  
Author(s):  
Mehmet Akdel ◽  
Douglas EV Pires ◽  
Eduard Porta-Pardo ◽  
Jurgen Janes ◽  
Arthur O Zalevsky ◽  
...  
Keyword(s):  
Structural Biology ◽  
Protein Complexes ◽  
Homology Modelling ◽  
Structural Data ◽  
Science Research ◽  
Structural Features ◽  
Structural Elements ◽  
Recent Developments ◽  
Transformative Impact ◽  
The Impact

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods have led to protein structure predictions that have reached the accuracy of experimentally determined models. While this has been independently verified, the implementation of these methods across structural biology applications remains to be tested. Here, we evaluate the use of AlphaFold 2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modelling of interactions; and modelling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modelled when compared to homology modelling, identifying structural features rarely seen in the PDB. AF2-based predictions of protein disorder and protein complexes surpass state-of-the-art tools and AF2 models can be used across diverse applications equally well compared to experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life science research.

scholarly journals SARS-CoV-2 Variants, RBD Mutations, Binding Affinity, and Antibody Escape

2021 ◽  
Vol 22 (22) ◽  
pp. 12114
Author(s):  
Lin Yang ◽  
Jiacheng Li ◽  
Shuai Guo ◽  
Chengyu Hou ◽  
Chenchen Liao ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Protein Complexes ◽  
Amino Acid Position ◽  
Protein Docking ◽  
Binding Energies ◽  
Missense Mutations ◽  
Vaccination Rates ◽  
Almost All ◽  
The Impact ◽  
Compensation Rule

Since 2020, the receptor-binding domain (RBD) of the spike protein of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been constantly mutating, producing most of the notable missense mutations in the context of “variants of concern”, probably in response to the vaccine-driven alteration of immune profiles of the human population. The Delta variant, in particular, has become the most prevalent variant of the epidemic, and it is spreading in countries with the highest vaccination rates, causing the world to face the risk of a new wave of the contagion. Understanding the physical mechanism responsible for the mutation-induced changes in the RBD’s binding affinity, its transmissibility, and its capacity to escape vaccine-induced immunity is the “urgent challenge” in the development of preventive measures, vaccines, and therapeutic antibodies against the coronavirus disease 2019 (COVID-19) pandemic. In this study, entropy–enthalpy compensation and the Gibbs free energy change were used to analyze the impact of the RBD mutations on the binding affinity of SARS-CoV-2 variants with the receptor angiotensin converting enzyme 2 (ACE2) and existing antibodies. Through the analysis, we found that the existing mutations have already covered almost all possible detrimental mutations that could result in an increase of transmissibility, and that a possible mutation in amino-acid position 498 of the RBD can potentially enhance its binding affinity. A new calculation method for the binding energies of protein–protein complexes is proposed based on the entropy–enthalpy compensation rule. All known structures of RBD–antibody complexes and the RBD–ACE2 complex comply with the entropy–enthalpy compensation rule in providing the driving force behind the spontaneous protein–protein docking. The variant-induced risk of breakthrough infections in vaccinated people is attributed to the L452R mutation’s reduction of the binding affinity of many antibodies. Mutations reversing the hydrophobic or hydrophilic performance of residues in the spike RBD potentially cause breakthrough infections of coronaviruses due to the changes in geometric complementarity in the entropy–enthalpy compensations between antibodies and the virus at the binding sites.

scholarly journals Advances in integrative structural biology: Towards understanding protein complexes in their cellular context

2021 ◽  
Vol 19 ◽  
pp. 214-225
Author(s):  
Samantha J. Ziegler ◽  
Sam J.B. Mallinson ◽  
Peter C. St. John ◽  
Yannick J. Bomble
Keyword(s):  
Structural Biology ◽  
Protein Complexes ◽  
Cellular Context ◽  
Integrative Structural Biology

scholarly journals Linear Dynamic Viscoelasticity of Dual Cross-Link Poly(Vinyl Alcohol) Hydrogel with Determined Borate Ion Concentration

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 71
Author(s):  
Takuro Taniguchi ◽  
Kenji Urayama
Keyword(s):  
Vinyl Alcohol ◽  
Ion Concentration ◽  
Poly Vinyl Alcohol ◽  
Relaxation Dynamics ◽  
Cross Link ◽  
Dynamic Viscoelasticity ◽  
Linear Dynamic ◽  
Cross Links ◽  
Pva Gel ◽  
Pva Hydrogel

We investigated the linear dynamic viscoelasticity of dual cross-link (DC) poly(vinyl alcohol) (PVA) (DC-PVA) hydrogels with permanent and transient cross-links. The concentrations of incorporated borate ions to form transient cross-links in the DC-PVA hydrogels (CBIN) were determined by the azomethine-H method. The dynamic viscoelasticity of the DC-PVA hydrogel cannot be described by a simple sum of the dynamic viscoelasticity of the PVA gel with the same permanent cross-link concentration and the PVA aqueous solution with the same borate ion concentration (CB = CBIN) as in the DC-PVA gel. The DC-PVA hydrogel exhibited a considerably higher relaxation strength, indicating that the introduction of permanent cross-links into temporary networks increases the number of viscoelastic chains with finite relaxation times. In contrast, the relaxation frequency (ωc) (given by the frequency at the maximum of loss modulus) for the DC-PVA hydrogel was slightly lower but comparable to that for a dilute PVA solution with the same CB. This signifies that the relaxation dynamics of the DC-PVA hydrogels is essentially governed by the lifetime of an interchain transient cross-link (di-diol complex of boron). The effect of permanent cross-linking on the relaxation dynamics was observed in the finite broadening of the relaxation-time distribution in the long time region.

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

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