scholarly journals Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

2021 ◽  
Author(s):  
Thomas E Walker ◽  
Mehdi Shirzadeh ◽  
He Mirabel Sun ◽  
Jacob W McCabe ◽  
Andrew Roth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Cell Function ◽  
Atp Hydrolysis ◽  
Variable Temperature ◽  
Native Mass Spectrometry ◽  
Solution Temperature ◽  
Temperature Dependent ◽  
Chemical Action ◽  
Native Ms

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature-electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL/GroES complexes. The results show clear evidences for destabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the previously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreport-ed stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry reveals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiometry of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temperature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J Mol Biol 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the util-ity of native MS for investigations of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

scholarly journals Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

2021 ◽  
Author(s):  
Thomas E Walker ◽  
Mehdi Shirzadeh ◽  
He Mirabel Sun ◽  
Jacob W McCabe ◽  
Andrew Roth ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Cell Function ◽  
Atp Hydrolysis ◽  
Variable Temperature ◽  
Native Mass Spectrometry ◽  
Solution Temperature ◽  
Temperature Dependent ◽  
Chemical Action ◽  
Native Ms

Chaperonins are nanomachines that harness ATP hydrolysis to power and catalyze protein folding, chemical action that is directly linked to the maintenance of cell function through protein folding/refolding and assembly. GroEL and the GroEL-GroES complex are archetypal examples of such protein folding machines. Here, variable-temperature-electrospray ionization (vT-ESI) native mass spectrometry is used to delineate the effects of solution temperature and ATP concentrations on the stabilities of GroEL and GroEL/GroES complexes. The results show clear evidences for de-stabilization of both GroEL14 and GroES7 at temperatures of 50 oC and 45 oC, respectively, substantially below the pre-viously reported melting temperature (Tm ~ 70 oC). This destabilization is accompanied by temperature-dependent reaction products that have previously unreported stoichiometries, viz. GroEL14-GroESx-ATPy, where x = 1, 2, 8 and y = 0, 1, 2, that are also dependent on Mg2+ and ATP concentrations. Variable-temperature native mass spectrometry re-veals new insights about the stability of GroEL in response to several environmental effects: (i) temperature-dependent ATP binding to GroEL (ii) effects of temperature as well as Mg2+ and ATP concentrations on the stoichiome-try of the GroEL-GroES complex, with Mg2+ showing greater effects compared to ATP; and, (iii) a change in the temper-ature-dependent stoichiometries of the GroEL-GroES complex (GroEL14-GroES7 vs GroEL14-GroES8) between 24 to 56 oC. The similarities between results obtained using native MS and cryo-EM (Clare et al., An expanded protein folding cage in the GroEL-gp31 complex. J. Mol. Biol. 2006, 358, 905-11; Ranson et al., Allosteric signaling of ATP hydrolysis in GroEL–GroES complexes. Nat. Struct. Mol. Biol. 2006, 13, 147-152.) underscores the utility of native MS for investiga-tions of molecular machines as well as identification of key intermediates involved in the chaperone-assisted protein folding cycle.

scholarly journals Development of a target identification approach using native mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miaomiao Liu ◽  
Wesley C. Van Voorhis ◽  
Ronald J. Quinn
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Target Identification ◽  
Native Mass Spectrometry ◽  
Pharmaceutical Drugs ◽  
Ligand Complex ◽  
Experimental Conditions ◽  
Protein Mixture ◽  
Native Ms ◽  
Drug Target Identification

AbstractA key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein–ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand–protein interactions in a protein mixture under various experimental conditions. A protein–ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.

Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions

2021 ◽  
Vol 51 (1) ◽  
Author(s):  
Arthur Laganowsky ◽  
David E. Clemmer ◽  
David H. Russell
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Noncovalent Interactions ◽  
Variable Temperature ◽  
Regulatory Protein ◽  
Conformational Dynamics ◽  
Native Mass Spectrometry ◽  
Annual Review ◽  
Publication Date

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein–ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

2020 ◽  
Vol 117 (31) ◽  
pp. 18459-18469
Author(s):  
Keith J. Mickolajczyk ◽  
Paul Dominic B. Olinares ◽  
Yiming Niu ◽  
Nan Chen ◽  
Sara E. Warrington ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Protein Function ◽  
Atp Hydrolysis ◽  
Native Mass Spectrometry ◽  
Chemical Probes ◽  
Rich Region ◽  
Aaa Atpase ◽  
Aaa Protein ◽  
Bimolecular Interaction

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

scholarly journals Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

2020 ◽  
Vol 48 (2) ◽  
pp. 547-558 ◽  
Author(s):  
Cagla Sahin ◽  
Deseree J. Reid ◽  
Michael T. Marty ◽  
Michael Landreh
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Vesicles ◽  
Native Mass Spectrometry ◽  
Integral Membrane Proteins ◽  
Structural Basis ◽  
Lipid Interactions ◽  
Native Ms ◽  
Peripheral Membrane Protein

A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein–protein, protein–ligand, and protein–lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.

scholarly journals Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miranda P. Collier ◽  
Karen Betancourt Moreira ◽  
Kathy H. Li ◽  
Yu-Chan Chen ◽  
Daniel Itzhak ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Folding ◽  
Insect Cells ◽  
Native Mass Spectrometry ◽  
Cellular Protein ◽  
Small Population ◽  
Relative Stabilities ◽  
Assembly Mechanism ◽  
And Function ◽  
Insight Into

AbstractThe eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

Membrane Protein–Lipid Interactions Probed Using Mass Spectrometry

2019 ◽  
Vol 88 (1) ◽  
pp. 85-111 ◽  
Author(s):  
Jani Reddy Bolla ◽  
Mark T. Agasid ◽  
Shahid Mehmood ◽  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Native Mass Spectrometry ◽  
Lipid Interactions ◽  
X Ray Crystallography ◽  
Native Ms ◽  
Molecular Aspects ◽  
Lipid Protein Interactions

Membrane proteins that exist in lipid bilayers are not isolated molecular entities. The lipid molecules that surround them play crucial roles in maintaining their full structural and functional integrity. Research directed at investigating these critical lipid–protein interactions is developing rapidly. Advancements in both instrumentation and software, as well as in key biophysical and biochemical techniques, are accelerating the field. In this review, we provide a brief outline of structural techniques used to probe protein–lipid interactions and focus on the molecular aspects of these interactions obtained from native mass spectrometry (native MS). We highlight examples in which lipids have been shown to modulate membrane protein structure and show how native MS has emerged as a complementary technique to X-ray crystallography and cryo–electron microscopy. We conclude with a short perspective on future developments that aim to better understand protein–lipid interactions in the native environment.

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

2021 ◽  
Author(s):  
Yang Yang ◽  
Daniil G. Ivanov ◽  
Igor A. Kaltashov
Keyword(s):  
Mass Spectrometry ◽  
Cell Surface ◽  
Host Cell ◽  
Structural Heterogeneity ◽  
Native Mass Spectrometry ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
Native Ms ◽  
Surface Receptor ◽  
Host Cell Surface

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community - the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of structural heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native mass spectrometry (MS) as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

scholarly journals Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry

Life ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 554
Author(s):  
Hao Yan ◽  
Julia Lockhauserbäumer ◽  
Gergo Peter Szekeres ◽  
Alvaro Mallagaray ◽  
Robert Creutznacher ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dissociation Constants ◽  
Protein Secondary Structure ◽  
Binding Pocket ◽  
Native Mass Spectrometry ◽  
Resonance Spectroscopy ◽  
Blood Group Antigens ◽  
Binding Affinities ◽  
Native Ms ◽  
Capsid Protein Vp1

Infection by the human noroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). The HBGA-binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for the binding of HBGAs to P dimers and, in some cases, disagreed on whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of a reference protein is crucial. We analysed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of β-sheets has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

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