| 1. |
- Brodmerkel, Maxim N., et al.
(författare)
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Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles
- 2024
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084.
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Tidskriftsartikel (refereegranskat)abstract
- Collision induced unfolding is method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the collision induced unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation. The dimers have highly similar structures, but the activation reveals differences in the dynamics that arises in response to the activation.
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| 2. |
- Brodmerkel, Maxim N., et al.
(författare)
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Stability and conformational memory of electrosprayed and rehydrated bacteriophage MS2 virus coat proteins
- 2022
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Ingår i: Current Research in Structural Biology. - : Elsevier. - 2665-928X. ; 4, s. 338-348
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Tidskriftsartikel (refereegranskat)abstract
- Proteins are innately dynamic, which is important for their functions, but which also poses significant challenges when studying their structures. Gas-phase techniques can utilise separation and a range of sample manipulations to transcend some of the limitations of conventional techniques for structural biology in crystalline or solution phase, and isolate different states for separate interrogation. However, the transfer from solution to the gas phase risks affecting the structures, and it is unclear to what extent different conformations remain distinct in the gas phase, and if resolution in silico can recover the native conformations and their differences. Here, we use extensive molecular dynamics simulations to study the two distinct conformations of dimeric capsid protein of the MS2 bacteriophage. The protein undergoes notable restructuring of its peripheral parts in the gas phase, but subsequent simulation in solvent largely recovers the native structure. Our results suggest that despite some structural loss due to the experimental conditions, gas-phase structural biology techniques provide meaningful data that inform not only about the structures but also conformational dynamics of proteins.
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| 3. |
- Brodmerkel, Maxim N.
(författare)
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Theoretical and Biochemical : Advancing Protein Structure Investigations with Complementing Computations
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Life as we know it today would not exist without proteins. The functions of proteins for us and other organisms are linked to their three-dimensional structures. As such, protein structure investigations are a crucial contribution for understanding proteins and the molecular basis of life. Some methods probe the structure of proteins in the gas phase, which brings various advantages as well as complications. Amongst them is mass spectrometry, a powerful method that provides a multitude of information on gaseous protein structures. Whilst mass spectrometry shines in obtaining data of the higher-order structures, atomistic details are out of reach. Molecular dynamics simulations on the other hand allow the interrogation of proteins in high-resolution, which makes it an ideal method for their structural research, be it in or out of solution.This thesis aims to advance the understanding of protein structures and the methods for their study utilising classic molecular dynamics simulations. The research presented in this thesis can be divided into two themes, comprising the rehydration of vacuum-exposed structures and the interrogation of the induced unfolding process of proteins. Out of their native environment, proteins undergo structural changes when exposed to vacuum. Investigating the ability to revert those potential vacuum-induced structural changes by means of computational rehydration provided detailed information on the underlying protein dynamics and how much of the structure revert back to their solution norm. We have further shown through rehydration simulations that applying an external electric field for dipole-orientation purposes does not induce irreversible changes to the protein structures. Our investigations on the induced unfolding of protein structures allowed a detailed look into the process of unfolding, accurately pinpointing areas within the proteins that unfolded first. The details provided by our simulations enabled us to describe potential mechanisms of the unfolding processes of different proteins on an atomistic level. The obtained results thus provide a potent theoretical basis for current and future experiments, where it will be very interesting to see MD compared with or complemented to experiments.
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| 4. |
- Broeker, N. K., et al.
(författare)
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Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 23:1, s. 59-68
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.
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| 5. |
- Dhillon, Arun, et al.
(författare)
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Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin
- 2024
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Ingår i: Journal of Virology. - 0022-538X .- 1098-5514. ; 98:3
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Tidskriftsartikel (refereegranskat)abstract
- Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackieadenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon’s hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.
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| 6. |
- Duelfer, Jasmin, et al.
(författare)
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Glycan-Induced Protein Dynamics in Human Norovirus P Dimers Depend on Virus Strain and Deamidation Status
- 2021
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 26:8
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Tidskriftsartikel (refereegranskat)abstract
- Noroviruses are the major cause of viral gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. The structural aspects of glycan binding to fully deamidated GII.4 P dimers have been investigated before. However, considering the high specificity and half-life of N373D under physiological conditions, large fractions of partially deamidated virions with potentially altered dynamics in their P domains are likely to occur. Therefore, we also examined glycan binding to partially deamidated GII.4 Saga and GII.4 MI001 P dimers. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric subpopulation. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a complex role of deamidation in modulating glycan-mediated cell attachment in GII.4 strains.
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| 7. |
- Han, Huijong, et al.
(författare)
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The XBI BioLab for life science experiments at the European XFEL
- 2021
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Ingår i: Journal of applied crystallography. - 0021-8898 .- 1600-5767. ; 54, s. 7-21
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Tidskriftsartikel (refereegranskat)abstract
- The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.
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| 8. |
- Hantke, Max F., et al.
(författare)
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A data set from flash X-ray imaging of carboxysomes
- 2016
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Ingår i: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
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| 9. |
- Kierspel, Thomas, et al.
(författare)
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Coherent diffractive imaging of proteins and viral capsids : simulating MS SPIDOC
- 2023
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Nature. - 1618-2642 .- 1618-2650. ; 415:18 SI, s. 4209-4220
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Tidskriftsartikel (refereegranskat)abstract
- MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm−1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.
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| 10. |
- Nitsche, Julius, et al.
(författare)
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Structural basis for activation of plasma-membrane Ca(2+)-ATPase by calmodulin.
- 2018
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Ingår i: Communications Biology. - : Nature Publishing Group. - 2399-3642. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Plasma-membrane Ca(2+)-ATPases expel Ca(2+) from the cytoplasm and are key regulators of Ca(2+) homeostasis in eukaryotes. They are autoinhibited under low Ca(2+) concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca(2+) pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme.
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| 11. |
- Sobolev, Egor, et al.
(författare)
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Megahertz single-particle imaging at the European XFEL
- 2020
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Ingår i: Communications Physics. - : Springer Science and Business Media LLC. - 2399-3650. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.
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| 12. |
- Zaccheus, Mona V., 1983-, et al.
(författare)
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Structural studies of the O‐antigen polysaccharide from Escherichia coli TD2158 having O18 serogroup specificity and aspects of its interaction with the tailspike endoglycosidase of the infecting bacteriophage HK620
- 2012
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 357, s. 118-125
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Tidskriftsartikel (refereegranskat)abstract
- We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure: α-D-Glcp-(1→6|) →2)-α-L-Rhap-91→6)-α-D-Glcp-(1→4)-α-D-Ga|lp-(1→3)-α-D-GlcpNAc-(1→ β-D-Glcp/β-D-GlcpNAc-(1→3) A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc-substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 μM and disfavored the O18A2-type having a β-D-Glcp-(1→3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9:1 ratio stressing the clear preference of O18A1-type repeats to be cleaved by HK620TSP.
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