69 research outputs found
Hofmeister Salts Recover a Misfolded Multiprotein Complex for Subsequent Structural Measurements in the Gas Phase
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99628/1/8329_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/99628/2/anie_201301893_sm_miscellaneous_information.pd
Collisional unfolding of multiprotein complexes reveals cooperative stabilization upon ligand binding
Cooperative binding mechanisms are a common feature in biology, enabling a diverse range of proteinâbased molecular machines to regulate activities ranging from oxygen uptake to cellular membrane transport. Much, however, is not known about such cooperative binding mechanisms, including how such events typically add to the overall stability of such protein systems. Measurements of such cooperative stabilization events are challenging, as they require the separation and resolution of individual protein complex bound states within a mixture of potential stoichiometries to individually assess protein stabilities. Here, we report ion mobilityâmass spectrometry results for the concanavalin A tetramer bound to a range of polysaccharide ligands. We use collision induced unfolding, a relatively new methodology that functions as a gasâphase analog of calorimetry experiments in solution, to individually assess the stabilities of concanavalin A bound states. By comparing the differences in activation voltage required to unfold different concanavalin Aâligand stoichiometries, we find evidence suggesting a cooperative stabilization of concanavalin A occurs upon binding most carbohydrate ligands. We critically evaluate this observation by assessing a broad range of ligands, evaluating the unfolding properties of multiple protein charge states, and by comparing our gasâphase results with those obtained from calorimetry experiments carried out in solution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112216/1/pro2699.pd
Bound Cations Significantly Stabilize the Structure of Multiprotein Complexes in the Gas Phase
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91320/1/5790_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/91320/2/ange_201109127_sm_miscellaneous_information.pd
Symmetryâ Directed Selfâ Assembly of a Tetrahedral Protein Cage Mediated by de Novoâ Designed Coiled Coils
The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of proteinâ protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiledâ coil domains to assemble proteins into higherâ order structures. Here, we demonstrate the assembly of a trimeric enzyme into a wellâ defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiledâ coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiledâ coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.Geometry lesson: A modular approach for assembling proteins into largeâ scale geometric structures was developed in which coiledâ coil domains acted as â twist tiesâ to facilitate assembly. The geometry of the cage was specified primarily by the rotational symmetries of the coiled coil and building block protein and was largely independent of protein structural details.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/1/cbic201700406_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/2/cbic201700406.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138862/3/cbic201700406-sup-0001-misc_information.pd
Separating and visualising protein assemblies by means of preparative mass spectrometry and microscopy
a b s t r a c t Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to 'soft-land' these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained
Integrating Ion Mobility Mass Spectrometry with Molecular Modelling to Determine the Architecture of Multiprotein Complexes
Current challenges in the field of structural genomics point to the need for new tools and technologies for obtaining structures of macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) and incomplete atomic structures, usually from X-ray crystallography, to generate models of the subunit architecture of protein complexes. We begin by analyzing protein complexes using IM-MS, and by taking measurements of both intact complexes and sub-complexes that are generated in solution. We then examine available high resolution structural data and use a suite of computational methods to account for missing residues at the subunit and/or domain level. High-order complexes and sub-complexes are then constructed that conform to distance and connectivity constraints imposed by IM-MS data. We illustrate our method by applying it to multimeric protein complexes within the Escherichia coli replisome: the sliding clamp, (β2), the Îł complex (Îł3δδâ˛), the DnaB helicase (DnaB6) and the Single-Stranded Binding Protein (SSB4)
Collisional and Coulombic Unfolding of GasâPhase Proteins: High Correlation to Their Domain Structures in Solution
The threeâdimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higherâorder assemblies. While important coursedâgrained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gasâphase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobilityâmass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8â78â
kDa. This correlation and its potential uses for structural biology is discussed. Gasâphase unfolding is used as a means to determine the number of autonomously folded domains within monomeric proteins. Ionâmobility mass spectrometry data show a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8â78â
kDa. CCS=collision crossâsection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108349/1/anie_201403784_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/108349/2/9209_ftp.pd
Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets
Electrospray ionization coupled to
mass spectrometry is a key technology for determining the stoichiometries
of multiprotein complexes. Despite highly accurate results for many
assemblies, challenging samples can generate signals for artifact
proteinâprotein binding born of the crowding forces present
within drying electrospray droplets. Here, for the first time, we
study the formation of preferred protein quaternary structures within
such rapidly evaporating nanodroplets. We use ion mobility and tandem
mass spectrometry to investigate glutamate dehydrogenase dodecamers
and serum amyloid P decamers as a function of protein concentration,
along with control experiments using carefully chosen protein analogues,
to both establish the formation of operative mechanisms and assign
the bimodal conformer populations observed. Further, we identify an
unprecedented symmetric collision-induced dissociation pathway that
we link directly to the quaternary structures of the precursor ions
selected
Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase
AbstractIn this paper, we present an investigation of the gas-phase structural differences between cyclic and linear peptide ions by matrix-assisted laser desorption ionization-ion mobility-mass spectrometry. Specifically, data is shown for gramicidin S (cyclo-VOLFPVOLFP where phenylalanines are D rather than L-type amino acids and the O designates the non-standard amino acid ornithine) and five linear gramicidin S analogues. Results are interpreted as evidence for a β-sheet (or β-hairpin) conformational preference in both linear-protonated and sodiated-cyclic gramicidin S gas-phase peptides, and a preference for the protonated-cyclic peptide to adopt a collapsed, random coil-type conformation. A comparison with solution-phase circular dicrhoism measurements is performed, and structures similar to those observed in the gas phase appear to be favored in low-dielectric solvents such as 2,2,2-triflouroethanol. The utility of ion mobility-mass spectrometry (IM-MS) as a means of rapidly distinguishing between linear and cyclic peptide forms in also discussed
Robotically Assisted Titration Coupled to Ion Mobility-Mass Spectrometry Reveals the Interface Structures and Analysis Parameters Critical for Multiprotein Topology Mapping
Multiprotein complexes have three-dimensional
shapes and dynamic
functions that impact almost every aspect of biochemistry. Despite
this, our ability to rapidly assess the structures of such macromolecules
lags significantly behind high-throughput efforts to identify their
function, especially in the context of human disease. Here, we describe
results obtained by coupling ion mobility-mass spectrometry with automated
robotic sampling of different solvent compositions. This combination
of technologies has allowed us to explore an extensive set of solution
conditions for a group of eight protein homotetramers, representing
a broad sample of protein structure and stability space. We find that
altering solution ionic strength in concert with dimethylsulfoxide
content is sufficient to disrupt the proteinâprotein interfaces
of all of the complexes studied here. Ion mobility measurements captured
for both intact assemblies and subcomplexes match expected values
from available X-ray structures in all cases save two. For these exceptions,
we find that distorted subcomplexes result from extreme disruption
conditions, and are accompanied by small shifts in intact tetramers
size, thus enabling the removal of distorted subcomplex data in downstream
models. Furthermore, we find strong correlations between the relative
intensities of disrupted protein tetramers and the relative number
and type of interactions present at interfaces as a function of disrupting
agent added. In most cases, this correlation appears strong enough
to quantify various types of protein interfacial interactions within
unknown proteins following appropriate calibration
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