ABSTRACT
Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.
Subject(s)
Mass Spectrometry , Mass Spectrometry/methods , Spectrum Analysis , IonsABSTRACT
Eye lens α- and ß-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (â¼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of ß-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe ß-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order ß-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed ßB1, ßB2, and ßA3 isoforms are found to have different homodimerization propensities, and only ßA3 forms larger homooligomers. Heterodimerization of ßB2 with ßA3 occurs â¼3 times as fast as that of ßB1 with ßA3, and ßB1 and ßB2 heterodimerize less readily. Ion mobility experiments, molecular dynamics simulations, and PISA analysis together reveal that observed oligomers are consistent with predominantly compact, ring-like topologies.
Subject(s)
Lens, Crystalline , gamma-Crystallins , beta-Crystallins , Lens, Crystalline/chemistry , Dimerization , Mass SpectrometryABSTRACT
LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2-4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2-4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2-4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2-4, with long and short linkers, forms heterogeneous complexes, whereas QT4-6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.
Subject(s)
Gene Expression Regulation , Transcription Factors , Animals , Drosophila melanogaster , Protein Binding , Signal Transduction , Transcription Factors/metabolismABSTRACT
Ion mobility (IM) mass spectrometry provides structural information about protein shape and size in the form of an orientationally-averaged collision cross-section (CCSIM). While IM data have been used with various computational methods, they have not yet been utilized to predict monomeric protein structure from sequence. Here, we show that IM data can significantly improve protein structure determination using the modelling suite Rosetta. We develop the Rosetta Projection Approximation using Rough Circular Shapes (PARCS) algorithm that allows for fast and accurate prediction of CCSIM from structure. Following successful testing of the PARCS algorithm, we use an integrative modelling approach to utilize IM data for protein structure prediction. Additionally, we propose a confidence metric that identifies near native models in the absence of a known structure. The results of this study demonstrate the ability of IM data to consistently improve protein structure prediction.
Subject(s)
Ion Mobility Spectrometry , Proteins , Algorithms , Ion Mobility Spectrometry/methods , Mass Spectrometry/methods , Proteins/chemistryABSTRACT
The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge-dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: ß-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term "native-like" to describe results from native ion mobility-mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.
Subject(s)
Concanavalin A/chemistry , Gases/chemistry , Glutamate Dehydrogenase/chemistry , Ion Mobility Spectrometry/methods , Lactoglobulins/chemistry , Ions/chemistry , Molecular Dynamics Simulation , Protein ConformationABSTRACT
Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein-ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein-lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function. However, sample heterogeneity can make assignment of ion mass, charge, composition, and structure very challenging due to the overlap of tens or even hundreds of peaks in the mass spectrum. In this review, we cover data analysis, experimental, and instrumental advances and strategies aimed at solving this problem, with an in-depth discussion of theoretical and practical aspects of the use of available deconvolution algorithms and tools. We also reflect upon current challenges and provide a view of the future of this exciting field.
Subject(s)
Algorithms , Proteins , Ligands , Mass Spectrometry/methods , Membrane Lipids , Proteins/chemistryABSTRACT
Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best-characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane (TM) domain of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled the measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.
Subject(s)
Influenza, Human , Amantadine/pharmacology , Antiviral Agents/pharmacology , Humans , Lipid Bilayers , Viral Matrix ProteinsABSTRACT
The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the â¼100-kDa dimeric protein identify a specific domain that binds 1-1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1-1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.
Subject(s)
COVID-19 , SARS-CoV-2 , Coronavirus Nucleocapsid Proteins , Humans , Nucleocapsid/metabolism , Phosphoproteins , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.
Subject(s)
Protein Interaction Domains and Motifs , WW Domains , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Humans , Protein Binding , ThermodynamicsABSTRACT
Dynein light chain 8 (LC8) interacts with intrinsically disordered proteins (IDPs) and influences a wide range of biological processes. It is becoming apparent that among the numerous IDPs that interact with LC8, many contain multiple LC8-binding sites. Although it is established that LC8 forms parallel IDP duplexes with some partners, such as nucleoporin Nup159 and dynein intermediate chain, the molecular details of these interactions and LC8's interactions with other diverse partners remain largely uncharacterized. LC8 dimers could bind in either a paired "in-register" or a heterogeneous off-register manner to any of the available sites on a multivalent partner. Here, using NMR chemical shift perturbation, analytical ultracentrifugation, and native electrospray ionization MS, we show that LC8 forms a predominantly in-register complex when bound to an IDP domain of the multivalent regulatory protein ASCIZ. Using saturation transfer difference NMR, we demonstrate that at substoichiometric LC8 concentrations, the IDP domain preferentially binds to one of the three LC8 recognition motifs. Further, the differential dynamic behavior for the three sites and the size of the fully bound complex confirmed an in-register complex. Dynamics measurements also revealed that coupling between sites depends on the linker length separating these sites. These results identify linker length and motif specificity as drivers of in-register binding in the multivalent LC8-IDP complex assembly and the degree of compositional and conformational heterogeneity as a promising emerging mechanism for tuning of binding and regulation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Dyneins/metabolism , Intrinsically Disordered Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Dyneins/chemistry , Dyneins/genetics , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Models, Molecular , Protein Conformation , Sequence Homology , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Many soluble and membrane proteins form symmetrical homooligomeric complexes. However, determining the oligomeric state of protein complexes can be difficult. Alpha-hemolysin (αHL) from Staphylococcus aureus is a symmetrical homooligomeric protein toxin that forms transmembrane ß-barrel pores in host cell membranes. The stable pore structure of αHL has also been exploited in vitro as a nanopore tool. Early structural experiments suggested αHL forms a hexameric pore, while more recent X-ray crystal structure and solution studies have identified a heptameric pore structure. Here, using native ion mobility-mass spectrometry (IM-MS) we find that αHL simultaneously forms hexameric and heptameric oligomers in both tetraethylene glycol monooctyl ether (C8E4) and tetradecylphosphocholine (FOS-14) detergent solutions. We also analyze intact detergent micelle-embedded αHL porelike complexes by native IM-MS without the need to fully strip the detergent micelle, which can cause significant gas-phase unfolding. The highly congested native mass spectra are deconvolved using Fourier- and Gábor-transform (FT and GT) methods to determine charge states and detergent stoichiometry distributions. The intact αHL micelle complexes are found to contain oligomeric state-proportional numbers of detergent molecules. This evidence, combined with IM data and results from vacuum molecular dynamics simulations, is consistent with both the hexamer and the heptamer forming porelike complexes. The ability of αHL to form both oligomeric states simultaneously has implications for its use as a nanopore tool and its pore formation mechanism in vivo. This study also demonstrates more generally the power of FT and GT to deconvolve the charge state and stoichiometry distributions of polydisperse ions.
Subject(s)
Detergents/chemistry , Hemolysin Proteins/analysis , Hemolysin Proteins/chemistry , Mass Spectrometry/methods , Micelles , Staphylococcus aureus/metabolism , Crystallography, X-Ray , Ion Mobility Spectrometry , Models, Molecular , Nanopores , Protein Conformation , Protein FoldingABSTRACT
Native ion mobility-mass spectrometry (IM-MS) is a rapidly growing field for studying the composition and structure of biomolecules and biomolecular complexes using gas-phase methods. Typically, ions are formed in native IM-MS using gentle nanoelectrospray ionization conditions, which in many cases can preserve condensed-phase stoichiometry. Although much evidence shows that large-scale condensed-phase structure, such as quaternary structure and topology, can also be preserved, it is less clear to what extent smaller-scale structure is preserved in native IM-MS. This review surveys computational and experimental efforts aimed at characterizing compaction and structural rearrangements of protein and protein complex ions upon transfer to the gas phase. A brief summary of gas-phase compaction results from molecular dynamics simulations using multiple common force fields and a wide variety of protein ions is presented and compared to literature IM-MS data.