| 1. |
- Abramsson, Mia L, et al.
(författare)
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Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry
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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.
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| 2. |
- Abramsson, Mia L., et al.
(författare)
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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry
- 2021
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:12, s. 2385-2393
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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.
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| 3. |
- Costeira-Paulo, Joana, 1993-
(författare)
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Advanced computations and mass-spectrometric techniques to unravel the dynamics and interactions of proteins
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are an important class of biomolecules, involved in the metabolism, regulation, structure and transport in cells. Unfortunately, many diseases are caused by protein dysfunction. When proteins perform their normal function, they may interact with each other or change their conformation. Protein structure is therefore not static, that is the structure should instead be regarded as dynamic. Further understanding of proteins can in turn lead to new means to manipulate them for therapeutic purposes as well as for basic research.To address the challenges posed by the dynamic nature of protein structure and interactions, we combine molecular modelling and mass spectrometric techniques. This has shown to be very powerful, more so than using the techniques individually. With mass spectrometric techniques, proteins can be separated by their mass and shape. This allows for the selection of conformational and binding states. In contrast, computations, namely molecular dynamic simulations, take into account time, which is fundamental for a dynamical analysis.In this thesis, we investigated dihydroorotate dehydrogenase (DHODH) and alpha-synuclein (alphaSyn), two drug targets for cancer and Parkinson's disease, respectively. We also researched the electrospray process necessary for native mass spectrometry. The understanding of this process gives insights into mass spectrometry of native protein conformations and subsequently allows for further studies of disease related systems. Regarding the electrospray process, our findings confirm that charge states are not dependent on the protein surface chemistry, but instead on the surface area and is best described by a zwitterionic configuration model. We also proposed models for the electrospray process of protein associated with lipids and evaluated the lipid-protein interaction in the gas-phase. Regarding the disease related proteins, we show how DHODH can interact with ubiquinone-10 and this architecture is similar to respiratory chain complex I; intramolecular dityrosine crosslinks can prevent alphaSyn aggregation. These findings can help develop new drug strategies.
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| 4. |
- Costeira-Paulo, Joana, 1993-, et al.
(författare)
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Collision induced unfolding coupled with gas-phase hydrogen/deuterium exchange give evidence for highly zwitterionic proteins in the gas phase
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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.
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| 5. |
- Costeira-Paulo, Joana, et al.
(författare)
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Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase
- 2018
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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| 6. |
- Landreh, Michael, et al.
(författare)
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Effects of Detergent Micelles on Lipid Binding to Proteins in Electrospray Ionization Mass Spectrometry
- 2017
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:14, s. 7425-7430
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Tidskriftsartikel (refereegranskat)abstract
- A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESIMS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.
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| 7. |
- Sahin, Cagla, et al.
(författare)
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Structural basis for dityrosine-mediated inhibition of alpha-synuclein fibrillation
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- α-synuclein (aSyn) is a small intrinsically disordered protein which can self-assemble into highly organized β-sheet structures that are found to accumulate in plaques in the brain of Parkinson’s Disease patients. Oxidative stress has been shown to be important for aSyn and its self-assembly. Here we characterize the molecular and structural effects that mild oxidation has on aSyn monomer and its aggregation. Using a combination of biophysical methods, SAXS and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages that reduce aSyn’s size by a factor of √2. MD simulations support our experimental results showing a stable and compact aSyn conformation that prevents self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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| 8. |
- Sahin, Cagla, et al.
(författare)
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Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization
- 2022
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 144:27, s. 11949-11954
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Tidskriftsartikel (refereegranskat)abstract
- α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
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| 9. |
- Salamanova, Evdokiya, et al.
(författare)
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A subset of functional adaptation mutations alter propensity for alpha-helical conformation in the intrinsically disordered glucocorticoid receptor taulcore activation domain
- 2018
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Ingår i: Biochimica et Biophysica Acta - General Subjects. - : ELSEVIER SCIENCE BV. - 0304-4165 .- 1872-8006. ; 1862:6, s. 1452-1461
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Tidskriftsartikel (refereegranskat)abstract
- Background: Adaptive mutations that alter protein functionality are enriched within intrinsically disordered protein regions (IDRs), thus conformational flexibility correlates with evolvability. Pre-structured motifs (PreSMos) with transient propensity for secondary structure conformation are believed to be important for IDR function. The glucocorticoid receptor taulcore transcriptional activation domain (GR taulcore) domain contains three a-helical PreSMos in physiological buffer conditions.Methods: Sixty change-of-function mutants affecting the intrinsically disordered 58-residue GR taulcore were studied using disorder prediction and molecular dynamics simulations.Results: Change-of-function mutations were partitioned into seven clusters based on their effect on IDR predictions and gene activation activity. Some mutations selected from clusters characterized by mutations altering the IDR prediction score, altered the apparent stability of the a-helical form of one of the PreSMos in molecular dynamics simulations, suggesting PreSMo stabilization or destabilization as strategies for functional adaptation. Indeed all tested gain-of-function mutations affecting this PreSMo were associated with increased stability of the a-helical PreSMo conformation, suggesting that PreSMo stabilization may be the main mechanism by which adaptive mutations can increase the activity of this IDR type. Some mutations did not appear to affect PreSMo stability.Conclusions: Changes in PreSMo stability account for the effects of a subset of change-of-function mutants affecting the GR taulcore IDR. General significance: Long IDRs occur in about 50% of human proteins. They are poorly characterized despite much recent attention. Our results suggest the importance of a subtle balance between PreSMo stability and IDR activity, which may provide a novel target for future pharmaceutical intervention.
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