Bound Cations Significantly Stabilize the Structure of Multiprotein Complexes in the Gas Phase

Han, Linjie; Hyung, Suk Joon; Ruotolo, Brandon T.

doi:10.1002/anie.201109127

Cited by 51 publications

(64 citation statements)

References 32 publications

(17 reference statements)

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“…35,37 Although the current work did not examine the behavior of such complexes, our findings are consistent with the view 35, 37 that multidentate contacts within individual subunits and across different chains are a key contributor to this effect.…”

Section: Discussionsupporting

confidence: 93%

“…9, 30-32 4 Factors that influence the structural integrity of electrosprayed proteins are of great interest. [33][34][35][36][37][38][39] Protein binding to low molecular weight ions is of particular importance in this context. 35,37,40,41 These bound ions may originate from specific solution phase interactions.…”

mentioning

confidence: 99%

“…[33][34][35][36][37][38][39] Protein binding to low molecular weight ions is of particular importance in this context. 35,37,40,41 These bound ions may originate from specific solution phase interactions. 4,20,[42][43][44] In addition, native ESI is prone to forming nonspecific adducts.…”

mentioning

confidence: 99%

“…52 Divalent species such as Ca 2+ affect CIU and CID processes via mechanisms that remain to be fully elucidated. 35,37 One possibility is that metal ions have a lower mobility than protons, thereby suppressing charge migration which is an integral part of the CID mechanism for multi-subunit systems. [30][31][32][56][57][58] Alternatively, metal adducts may form multidentate contacts with the protein 35, 37 analogous to structural metal ions in solution.…”

mentioning

confidence: 99%

“…Bound metals cause changes in protein behavior that are more pronounced than those of anionic adducts. 37 Also, metal adduction is more prevalent in the commonly used positive ESI mode. 52,54 Metal-mediated effects during ESI are not limited to multi-subunit systems, but have also been reported for monomeric proteins.…”

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confidence: 99%

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Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

2016

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Much remains to be learned about the way in which bound metal ions modulate the response of electrosprayed proteins and protein complexes to collisional excitation. Nonspecific metal adducts can affect the extent of collision-induced unfolding (CIU) and collision-induced dissociation (CID). Here, we examine how Na(+) and Ca(2+) adducts alter the CIU response of monomeric proteins under native electrospray conditions. Both of these metals are commonly encountered in biological samples. Measured collision cross sections are largely independent of metal adduction as long as in-source excitation is minimized. In contrast, under CIU conditions, the metal-adducted proteins are markedly more compact than their metal-free counterparts. This phenomenon is particularly pronounced for Ca(2+) binding, but Na(+) adducts have significant effects as well. Molecular dynamics simulations reproduce the experimentally observed trends. The simulations show that structural expansion of the collisionally unfolded proteins is limited by multidentate metal contacts that restrict the conformational freedom of the polypeptide chains. Multidentate interactions with carboxylates and other electron-rich moieties are to be anticipated for divalent metals such as Ca(2+). It is surprising that Na(+) also engages in multidentate ligation. Electrostatic mapping reveals that the propensity of both Na(+) and Ca(2+) to interact with multiple electron-rich groups is caused by ineffective charge shielding during ion pairing. Despite their compactness, the CIU structures of metalated proteins do not retain native-like elements. Instead, CIU generates inside-out conformations where previously surface-exposed hydrophilic side chains get buried along with most of the metal ions. Our findings caution that the observation of compact conformers after collisional excitation does not imply the survival of solution-like structural features. We also discuss possible implications of adduct-mediated effects for CIU fingerprinting studies.

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Section: Discussionsupporting

confidence: 93%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

2016

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Hofmeister Salts Recover a Misfolded Multiprotein Complex for Subsequent Structural Measurements in the Gas Phase

Han

Ruotolo

2013

Angewandte Chemie

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Proteins are the workhorses of the cell, the functions of which are largely predicated on their structures and dynamics. Detailed knowledge of these attributes has enabled countless breakthroughs in human health and disease. [1] Many aspects, however, of protein structure remain poorly understood, including their high-order interactions. [2] This knowledge gap drives the development of new tools capable of protein structure characterization, some of which operate by measuring desolvated protein structure. [3] While desolvation enables the application of powerful analytical techniques that cannot be used in a solvated environment, and recent results indicate that many features of native protein structure survive in the gasphase, [4] desolvation may also act to obfuscate critical details of protein conformation. 8 Recently, multiple strategies have emerged for observing labile protein structures in the absence of bulk water and have proven useful in stabilizing protein-small molecule interactions, globular proteins, and their complexes. [5] Here, we report the first evidence that a misfolded protein complex, which exists both in solution and in the gas-phase, can be recovered back to a 'native-like' structure through the addition of salts prior to desorption/ ionization. These data represent the first time that such a solution-phase multiprotein folding equilibrium is captured by gas-phase measurements.Our experiments involve the direct addition of salt additives to proteins in solution, mimicking the well-known Hofmeister series, [6] followed by transfer into the gas phase using nano-electropsray ionization (nESI). We then utilize ion mobility-mass spectrometry (IM-MS) to measure the influence of such additives on both the composition and structure of the resulting gas-phase ions. IM separates proteins and complexes based on their collision cross-section (CCS). Such information can be used, along with computational procedures, to deduce the three-dimensional structures of biomolecules. [7] MS can then be used to analyze the composition of ions that elute from the IM separator. [8] While previous measurements have allowed us to rank the ability of bound anions and cations to stabilize proteins in the gas phase, these experiments started from thermodynamically stable proteins that were natively-folded prior to nESI and did not reflect protein stabilities in solution. [5b-d] The protein system we have chosen to study here is the lectin concanavalin A (ConA), a ~103 kDa homo-tetramer having a dimer-of-dimers arrangement. [9] The ConA tetramer can reversibly self-assemble to form dimers and tetramers in a manner that depends upon solution pH, temperature, and ionic strength. [10] In addition to these properties, IM-MS reveals that the ConA tetramer can generate an alternate quaternary structure, which can be recovered back to a native-like conformation in a salt-dependant manner. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptConA has been long studied for its mitogenic, cell surf...

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Uncovering the Stoichiometry of Pyrococcus furiosus RNase P, a Multi‐Subunit Catalytic Ribonucleoprotein Complex, by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

Lai

et al. 2014

Angewandte Chemie

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We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg2+‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

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Bound Cations Significantly Stabilize the Structure of Multiprotein Complexes in the Gas Phase

Cited by 51 publications

References 32 publications

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

Hofmeister Salts Recover a Misfolded Multiprotein Complex for Subsequent Structural Measurements in the Gas Phase

Uncovering the Stoichiometry of Pyrococcus furiosus RNase P, a Multi‐Subunit Catalytic Ribonucleoprotein Complex, by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

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