Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Schennach, Moritz; Schneeberger, Eva-Maria; Breuker, Kathrin

doi:10.1007/s13361-016-1363-7

Cited by 17 publications

(29 citation statements)

References 83 publications

(61 reference statements)

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“…More broadly, through detailed comparisons of CIU fingerprints, quantified using RMSD values, our CIU experiments reveal that the differences in the observed CIU features arise from the altered stabilities of the complexes imparted through peptide binding. We hypothesize that KIX unfolds in a linear α-helix 1-3 fashion in the gas-phase, which correlates well with previous ECD and CID experiments [22, 23]. Such a detailed understanding of the KIX CIU process, linking CIU features to specific regions of the KIX structure, may enable the development of improved PPI inhibitor binding assays.…”

Section: Discussionsupporting

confidence: 86%

“…In particular, ECD of 7+ KIX ions revealed that the observed c and z • fragment ions mainly originated from the backbone near the protein termini, and not from the 3-helix bundle [22]. Further ECD and CID experiments found that the propensity for KIX folding is determined by the intramolecular distribution of charges on the protein surface, rather than its net charge, leading ultimately to produce the surprising stability of KIX in the gas-phase [23]. Together, these experiments showed the gas-phase stabilities of the KIX helices to be α3>α2>α1 [22], and in particular, for the 8+ charge state, α1 appears to be the least stable [23].…”

Section: Resultsmentioning

confidence: 99%

“…Further ECD and CID experiments found that the propensity for KIX folding is determined by the intramolecular distribution of charges on the protein surface, rather than its net charge, leading ultimately to produce the surprising stability of KIX in the gas-phase [23]. Together, these experiments showed the gas-phase stabilities of the KIX helices to be α3>α2>α1 [22], and in particular, for the 8+ charge state, α1 appears to be the least stable [23]. This rank ordering agrees well with our CIU interpretation discussed above, and future work will focus on further assigning the precise origins of the structural transitions observed in our KIX:peptide CIU data.…”

Section: Resultsmentioning

confidence: 99%

“…KIX is particularly amenable to native MS studies given its surprising stability in the gas phase and network of electrostatic interactions [22, 23]. Here, we describe an ion mobility-mass spectrometry (IM-MS) based method capable of detecting peptide binding and assigning the location of that binding uniquely to either of the two available sites within KIX.…”

Section: Introductionmentioning

confidence: 99%

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Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain

Rabuck-Gibbons

Lodge

Mapp

et al. 2018

J. Am. Soc. Mass Spectrom.

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The kinase-inducible domain (KIX) of the transcriptional coactivator CBP binds multiple transcriptional regulators through two allosterically connected sites. Establishing a method for observing activator-specific KIX conformations would facilitate the discovery of drug-like molecules that capture specific conformations and further elucidate how distinct activator-KIX complexes produce differential transcriptional effects. However, the transient and low to moderate affinity interactions between activators and KIX are difficult to capture using traditional biophysical assays. Here, we describe a collision-induced unfolding-based approach that produces unique fingerprints for peptides bound to each of the two available sites within KIX, as well as a third fingerprint for ternary KIX complexes. Furthermore, we evaluate the analytical utility of unfolding fingerprints for KIX complexes using CIUSuite, and conclude by speculating as to the structural origins of the conformational families created from KIX:peptide complexes following collisional activation. Graphical Abstract ᅟ.

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Section: Discussionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain

Rabuck-Gibbons

Lodge

Mapp

et al. 2018

J. Am. Soc. Mass Spectrom.

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“…Since the first correlation of gaseous protein fragment ions to primary sequence in 1990 [9], MS/MS has been widely used to enable mass spectrometry-based analysis of peptides [10, 11, 12] and proteins [13, 14]. The process requires the measurement of the intact mass of the precursor, which is then isolated in the gas phase and activated to break its interresidue bonds.…”

Section: Introductionmentioning

confidence: 99%

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry

Haverland

Skinner

Fellers

et al. 2017

J. Am. Soc. Mass Spectrom.

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Fragmentation of intact proteins in the gas phase is influenced by amino acid composition, the mass and charge of precursor ions, higher order structure, and the dissociation technique used. The likelihood of fragmentation occurring between a pair of residues is referred to as the fragmentation propensity and is calculated by dividing the total number of assigned fragmentation events by the total number of possible fragmentation events for each residue pair. Here, we describe general fragmentation propensities when performing top-down mass spectrometry (TDMS) using denaturing or native electrospray ionization. A total of 5,311 matched fragmentation sites were collected for 131 proteoforms that were analyzed over 165 experiments using native top-down mass spectrometry (nTDMS). These data were used to determine the fragmentation propensities for 399 residue pairs. In comparison to denatured top-down mass spectrometry (dTDMS), the fragmentation pathways occurring either N-terminal to proline or C-terminal to aspartic acid were even more enhanced in nTDMS as compared to other residues. More generally, 257/399 (64%) of the fragmentation propensities were significantly altered (p ≤0.05) when using nTDMS as compared to dTDMS and of these, 123 were altered by 2-fold or greater. The most notable enhancements of fragmentation propensities for TDMS in native vs. denatured mode occurred (1) C-terminal to aspartic acid, (2) between phenylalanine and tryptophan (F|W), and (3) between tryptophan and alanine (W|A). The fragmentation propensities presented here will be of high value in the development of tailored scoring systems used in nTDMS of both intact proteins and protein complexes.

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Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Schneeberger

Breuker

2016

Angewandte Chemie

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Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures.T he characterization of RNAprotein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X-rayc rystallography,orstrategies based on chemical crosslinking,however,can be quite challenging.H erein, we have explored the use of an alternative method, native top-down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single-residue level of RNA. Our data show that the electrostatic interactions between HIV-1 TARR NA and ap eptide comprising the arginine-richb inding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNAb yc ollisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TARRNA by top-down MS.Moreover,the MS data reveal time-dependent 1:2a nd 1:1s toichiometries of the TAR-tat complexes and suggest structural rearrangements of TARRNA induced by binding of tat peptide.Interactions between ribonucleic acids (RNA) and proteins are central to many fundamental biological processes,including gene expression and infection by RNAv iruses.F or athorough understanding of such interactions,RNA-protein complexes are commonly investigated by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, both of which require relatively large quantities of sample material. Moreover,N MR data interpretation can be complicated by unfavorable conformational dynamics, [1] and crystallography can become impossible if ac omplex fails to crystallize properly.S trategies based on (photo)chemical crosslinking [2] have the advantage that they can be performed in vivo [3] but can variously suffer from low crosslinking yields, different crosslinking reactivity of different residues,o rt he formation of intramolecular instead of intermolecular crosslinks.[4] Moreover,c rosslinking reagents generally target specific functional groups such as amines or thiols that may not be present in ab inding region, and efficient crosslinking can require pH values that may not be compatible with RNAprotein complex stability.[5] Although all of the above techniques can provide highly important structural data, each of them requires laborious sample preparation procedures,t hat is,c rystallization, the introduction of heavy isotopes,o ro ptimization of the reaction conditions for the formation of intermolecular crosslinks.As an alternative to these methods,w ee xplore the potential of native top-down mass spectrometry (MS) using electrospray ionization (ESI) [6] fort he characterization of RNA-protein interactions.P revious studies showed that native ESI can produce gaseous RNA-protein or RNAligand complexes, [7] and in top-down MS experiments using collisionally activated dissociation (CAD), Loo [8] and Fabris [7f] observed cleavage of covalent mononucleotide phosphate and RNAp hosphodiester bonds,r espectively,r ather...

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Unfolding and Folding of the Three-Helix Bundle Protein KIX in the Absence of Solvent

Cited by 17 publications

References 83 publications

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain

Defining Gas-Phase Fragmentation Propensities of Intact Proteins During Native Top-Down Mass Spectrometry

Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

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