| 1. |
- Abramsson, Mia L, et al.
(författare)
-
Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.
|
|
| 2. |
- Abramsson, Mia L., et al.
(författare)
-
Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
- 2021
-
Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:12, s. 2385-2393
-
Tidskriftsartikel (refereegranskat)abstract
- In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.
|
|
| 3. |
- Sahin, Cagla, et al.
(författare)
-
Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions
- 2021
-
Ingår i: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 57:12, s. 1450-1453
-
Tidskriftsartikel (refereegranskat)abstract
- Although native mass spectrometry is widely applied to monitor chemical or thermal protein denaturation, it is not clear to what extent it can inform about alkali-induced unfolding. Here, we probe the relationship between solution- and gas-phase structures of proteins under alkaline conditions. Native ion mobility-mass spectrometry reveals that globular proteins are destabilized rather than globally unfolded, which is supported by solution studies, providing detailed insights into alkali-induced unfolding events. Our results pave the way for new applications of MS to monitor structures and interactions of proteins at high pH.
|
|
| 4. |
- Sahin, Cagla, et al.
(författare)
-
Ion mobility-mass spectrometry shows stepwise protein unfolding under alkaline conditions
- 2021
-
Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 57:12, s. 1450-1453
-
Tidskriftsartikel (refereegranskat)abstract
- Although native mass spectrometry is widely applied to monitor chemical or thermal protein denaturation, it is not clear to what extent it can inform about alkali-induced unfolding. Here, we probe the relationship between solution- and gas-phase structures of proteins under alkaline conditions. Native ion mobility-mass spectrometry reveals that globular proteins are destabilized rather than globally unfolded, which is supported by solution studies, providing detailed insights into alkali-induced unfolding events. Our results pave the way for new applications of MS to monitor structures and interactions of proteins at high pH.
|
|
| 5. |
- Sendker, Franziska L., et al.
(författare)
-
Emergence of fractal geometries in the evolution of a metabolic enzyme
- 2024
-
Ingår i: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 628:8009, s. 894-900
-
Tidskriftsartikel (refereegranskat)abstract
- Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2,3,4; however, so far, molecular assembly into fractals is restricted to synthetic systems5,6,7,8,9,10,11,12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.
|
|
| 6. |
- Young, Gavin, et al.
(författare)
-
Quantitative mass imaging of single biological macromolecules
- 2018
-
Ingår i: Science. - : American Association for the Advancement of Science. - 0036-8075 .- 1095-9203. ; 360:6387, s. 423-427
-
Tidskriftsartikel (refereegranskat)abstract
- Careful measurements of light scattering can provide information on individual macromolecules and complexes. Young et al. used a light-scattering approach for accurate mass determination of proteins as small as 20 kDa (see the Perspective by Lee and Klenerman). Movies of protein complex association and dissociation were analyzed to extract biophysical parameters from single molecules and assemblies without labeling. Using this approach, the authors determined in vitro kinetics of fibril and aggregate growth and association constants for a complex protein-glycoprotein assembly.Science, this issue p. 423; see also p. 378The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.
|
|
| 7. |
- Allison, Timothy M., et al.
(författare)
-
Complementing machine learning‐based structure predictions with native mass spectrometry
- 2022
-
Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6
-
Tidskriftsartikel (refereegranskat)abstract
- The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
|
|
| 8. |
- Allison, Timothy M., et al.
(författare)
-
Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology
- 2020
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 10872-10880
-
Tidskriftsartikel (refereegranskat)abstract
- Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic—to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.
|
|
| 9. |
- Allison, Timothy M., et al.
(författare)
-
Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data
- 2020
-
Ingår i: Analytical Chemistry. - : American Chemical Society. - 0003-2700 .- 1520-6882. ; 92:16, s. 10881-10890
-
Tidskriftsartikel (refereegranskat)abstract
- The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.
|
|
| 10. |
- Costeira-Paulo, Joana, 1993-, et al.
(författare)
-
Collision induced unfolding coupled with gas-phase hydrogen/deuterium exchange give evidence for highly zwitterionic proteins in the gas phase
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The net charge of a natively folded electrosprayed protein can be accurately predicted from the protein size. How charges are distributed on a protein – the charge configuration – is still a challenge however, both to determine experimentally and to predict from theory and computations, hampering both modelling and interpretation of native mass spectrometry experiments. Here, a combination of molecular dynamics simulations and experiments, including a novel conjunction of collision induced unfolding and gas-phase hydrogen/deuterium exchange, were used on a set of engineered proteins differing in their surface chemistry to general principles underpinning the charge configurations. Testing three charging models against simulations and experiments, only the highly zwitterionic model passed falsification efforts. Our results are consistent with the notion of the proteins being partially kinetically trapped in a charge configuration inherited from solution.
|
|
| 11. |
- Hochberg, Georg K A, et al.
(författare)
-
Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.
- 2018
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 359:6378, s. 930-935
-
Tidskriftsartikel (refereegranskat)abstract
- Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.
|
|
| 12. |
- Landreh, Michael, et al.
(författare)
-
Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
- 2017
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
|
|
| 13. |
- Marklund, Erik, 1979-, et al.
(författare)
-
Controlling Protein Orientation in Vacuum Using Electric Fields
- 2017
-
Ingår i: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 8:18, s. 4540-4544
-
Tidskriftsartikel (refereegranskat)abstract
- Single-particle imaging using X-ray free-electron lasers is an emerging technique that could provide high-resolution structures of macromolecules in the gas phase. One of the largest difficulties in realizing this goal is the unknown orientation of the individual sample molecules at the time of exposure. Preorientation of the molecules has been identified as a possible solution to this problem. Using molecular dynamics simulations, we identify a range of electric field strengths where proteins become oriented without losing their structure. For a number of experimentally relevant cases we show that structure determination is possible only when orientation information is included in the orientation-recovery process. We conclude that nondestructive field orientation of intact proteins is feasible and that it enables a range of new structural investigations with single particle imaging.
|
|
| 14. |
- Marklund, Erik G, et al.
(författare)
-
Collision cross sections for structural proteomics.
- 2015
-
Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 23:4
-
Tidskriftsartikel (refereegranskat)abstract
- Ion mobility mass spectrometry (IM-MS) allows the structural interrogation of biomolecules by reporting their collision cross sections (CCSs). The major bottleneck for exploiting IM-MS in structural proteomics lies in the lack of speed at which structures and models can be related to experimental data. Here we present IMPACT (Ion Mobility Projection Approximation Calculation Tool), which overcomes these twin challenges, providing accurate CCSs up to 10(6) times faster than alternative methods. This allows us to assess the CCS space presented by the entire structural proteome, interrogate ensembles of protein conformers, and monitor molecular dynamics trajectories. Our data demonstrate that the CCS is a highly informative parameter and that IM-MS is of considerable practical value to structural biologists.
|
|
| 15. |
- Marklund, Erik G., Teknologie doktor, 1979-, et al.
(författare)
-
Structural and functional aspects of the interaction partners of the small heat-shock protein in Synechocystis
- 2018
-
Ingår i: Cell stress & chaperones (Print). - : Springer Science and Business Media LLC. - 1355-8145 .- 1466-1268. ; 23:4, s. 723-732
-
Tidskriftsartikel (refereegranskat)abstract
- The canonical function of small heat-shock proteins (sHSPs) is to interact with proteins destabilized under conditions of cellular stress. While the breadth of interactions made by many sHSPs is well-known, there is currently little knowledge about what structural features of the interactors form the basis for their recognition. Here, we have identified 83 in vivo interactors of the sole sHSP in the cyanobacterium Synechocystis sp. PCC 6803, HSP16.6, reflective of stable associations with soluble proteins made under heat-shock conditions. By performing bioinformatic analyses on these interactors, we identify primary and secondary structural elements that are enriched relative to expectations from the cyanobacterial genome. In addition, by examining the Synechocystis interactors and comparing them with those identified to bind sHSPs in other prokaryotes, we show that sHSPs associate with specific proteins and biological processes. Our data are therefore consistent with a picture of sHSPs being broadly specific molecular chaperones that act to protect multiple cellular pathways.
|
|
| 16. |
- Marklund, Erik, Teknologie doktor, 1979-, et al.
(författare)
-
Weighing-up protein dynamics : the combination of native mass spectrometry and molecular dynamics simulations
- 2019
-
Ingår i: Current opinion in structural biology. - : CURRENT BIOLOGY LTD. - 0959-440X .- 1879-033X. ; 54, s. 50-58
-
Forskningsöversikt (refereegranskat)abstract
- Structural dynamics underpin biological function at the molecular level, yet many biophysical and structural biology approaches give only a static or averaged view of proteins. Native mass spectrometry yields spectra of the many states and interactions in the structural ensemble, but its spatial resolution is limited. Conversely, molecular dynamics simulations are innately high-resolution, but have a limited capacity for exploring all structural possibilities. The two techniques hence differ fundamentally in the information they provide, returning data that reflect different length scales and time scales, making them natural bedfellows. Here we discuss how the combination of native mass spectrometry with molecular dynamics simulations is enabling unprecedented insights into a range of biological questions by interrogating the motions of proteins, their assemblies, and interactions.
|
|
| 17. |
- Marty, Michael T, et al.
(författare)
-
Bayesian deconvolution of mass and ion mobility spectra : from binary interactions to polydisperse ensembles.
- 2015
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 87:8
-
Tidskriftsartikel (refereegranskat)abstract
- Interpretation of mass spectra is challenging because they report a ratio of two physical quantities, mass and charge, which may each have multiple components that overlap in m/z. Previous approaches to disentangling the two have focused on peak assignment or fitting. However, the former struggle with complex spectra, and the latter are generally computationally intensive and may require substantial manual intervention. We propose a new data analysis approach that employs a Bayesian framework to separate the mass and charge dimensions. On the basis of this approach, we developed UniDec (Universal Deconvolution), software that provides a rapid, robust, and flexible deconvolution of mass spectra and ion mobility-mass spectra with minimal user intervention. Incorporation of the charge-state distribution in the Bayesian prior probabilities provides separation of the m/z spectrum into its physical mass and charge components. We have evaluated our approach using systems of increasing complexity, enabling us to deduce lipid binding to membrane proteins, to probe the dynamics of subunit exchange reactions, and to characterize polydispersity in both protein assemblies and lipoprotein Nanodiscs. The general utility of our approach will greatly facilitate analysis of ion mobility and mass spectra.
|
|
