Native Mass Spectrometry as a Tool in Structural Biology

Lorenzen, Kristina; Duijn, Esther van

doi:10.1002/0471140864.ps1712s62

Cited by 56 publications

(58 citation statements)

References 56 publications

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“…This stabilizing effect can be explained through enhanced cooling from imidazole evaporation that delays the dissociation. Coulomb repulsion can be minimized due to the reduced net charge state of the protein that stabilizes the complex [52]. Due to the high gas-phase basicity of imidazole (217 kcal mol -1 ), it is able to strip protons from the protonated protein ions in the gas phase [53].…”

Section: K D -Determination Of the Cma Series By Nanoesi-ms Titrationmentioning

confidence: 99%

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Cubrilovic

Biela

Sielaff

et al. 2012

J. Am. Soc. Mass Spectrom.

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NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K D ) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K D values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K D ) measured via nanoESI-MS were compared with kinetic inhibition constants (K i ) in solution.

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Section: K D -Determination Of the Cma Series By Nanoesi-ms Titrationmentioning

confidence: 99%

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Cubrilovic

Biela

Sielaff

et al. 2012

J. Am. Soc. Mass Spectrom.

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“…In native MS, protein complexes are typically sprayed from an aqueous ammonium acetate solution at physiological pH. Addition of a low percentage of organic modifiers to the electrospray solution, generates Cascade intermediates that may be used as indicators of the complex interaction network or topology (38,41,42).…”

Section: Resultsmentioning

confidence: 99%

Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

Duijn

Barbu

Barendregt

et al. 2012

Molecular & Cellular Proteomics

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The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes. Molecular & Cellular

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“…Native mass spectrometry relies on gentle transfer of proteins and their complexes from solution into the gas phase of the mass spectrometer, without major structural rearrangements or disruption of non-covalent interactions [25][26][27][28] . As such it is especially useful for detecting non-covalent ligand binding 29,30 .…”

Section: Introductionmentioning

confidence: 99%

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

et al. 2016

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The intrinsically disordered and amyloidogenic protein -synuclein (AS) has been linked to several neurodegenerative states, including Parkinson's disease. Here, nano-electrospray-ionization mass spectrometry (nano-ESI-MS), ion mobility (IM), and native top-down electron transfer dissociation (ETD) techniques are employed to study AS interaction with small molecules known to modulate its aggregation, such as epigallocatechin-3-gallate (EGCG) and dopamine (DA). The complexes formed by the two ligands under identical conditions reveal peculiar differences. While EGCG engages AS in compact conformations, DA preferentially binds to the protein in partially extended conformations. The two ligands also have different effects on AS structure as assessed by IM, with EGCG leading to protein compaction and DA to its extension. Native top-down ETD on the protein-ligand complexes shows how the different observed modes of binding of the two ligands could be related to their known opposite effects on AS aggregation. The results also show that the protein can bind either ligand in the absence of any covalent modifications, such as e.g. oxidation.

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Native Mass Spectrometry as a Tool in Structural Biology

Cited by 56 publications

References 56 publications

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

Opposite Structural Effects of Epigallocatechin-3-gallate and Dopamine Binding to α-Synuclein

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