| 1. |
- Caleman, Carl, et al.
(författare)
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Nanocrystal imaging using intense and ultrashort X-ray pulses
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Annan publikation (populärvet., debatt m.m.)abstract
- Structural studies of biological macromolecules are severely limited by radiation damage. Traditional crystallography curbs the effects of damage by spreading damage over many copies of the molecule of interest in the crystal. X-ray lasers offer an additional opportunity for limiting damage by out-running damage processes with ultrashort and very intense X-ray pulses. Such pulses may allow the imaging of single molecules, clusters or nanoparticles, but coherent flash imaging will also open up new avenues for structural studies on nano- and micro-crystalline substances. This paper addresses the potentials and limitations of nanocrystallography with extremely intense coherent X-ray pulses. We use urea nanocrystals as a model for generic biological substances, and simulate the primary and secondary ionization dynamics in the crystalline sample. The results establish conditions for diffraction experiments as a function of X-ray fluence, pulse duration, and the size of nanocrystals.
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| 2. |
- Hammar, Petter, et al.
(författare)
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The lac repressor displays facilitated diffusion in living cells
- 2012
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 336:6088, s. 1595-1598
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on the chromosome. It has been proposed that to find these sites fast and accurately, TFs combine one-dimensional (1D) sliding on DNA with 3D diffusion in the cytoplasm. This facilitated diffusion mechanism has been demonstrated in vitro, but it has not been shown experimentally to be exploited in living cells. We have developed a single-molecule assay that allows us to investigate the sliding process in living bacteria. Here we show that the lac repressor slides 45 ± 10 base pairs on chromosomal DNA and that sliding can be obstructed by other DNA-bound proteins near the operator. Furthermore, the repressor frequently (>90%) slides over its natural lacO(1) operator several times before binding. This suggests a trade-off between rapid search on nonspecific sequences and fast binding at the specific sequence.
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| 3. |
- Marklund, Erik G., et al.
(författare)
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Transcription-factor binding and sliding on DNA studied using micro- and macroscopic models
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:49, s. 19796-19801
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Tidskriftsartikel (refereegranskat)abstract
- Transcription factors search for specific operator sequences by alternating rounds of 3D diffusion with rounds of 1D diffusion (sliding) along the DNA. The details of such sliding have largely been beyond direct experimental observation. For this purpose we devised an analytical formulation of umbrella sampling along a helical coordinate, and from extensive and fully atomistic simulations we quantified the free-energy landscapes that underlie the sliding dynamics and dissociation kinetics for the LacI dimer. The resulting potential of mean force distributions show a fine structure with an amplitude of 1 k(B)T for sliding and 12 kBT for dissociation. Based on the free-energy calculations the repressor slides in close contact with DNA for 8 bp on average before making a microscopic dissociation. By combining the microscopic molecular-dynamics calculations with Brownian simulation including rotational diffusion from the microscopically dissociated state we estimate a macroscopic residence time of 48 ms at the same DNA segment and an in vitro sliding distance of 240 bp. The sliding distance is in agreement with previous in vitro sliding-length estimates. The in vitro prediction for the macroscopic residence time also compares favorably to what we measure by single-molecule imaging of nonspecifically bound fluorescently labeled LacI in living cells. The investigation adds to our understanding of transcription-factor search kinetics and connects the macro-/mesoscopic rate constants to the microscopic dynamics.
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| 4. |
- O'Keefe, James H., et al.
(författare)
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Omega-3 Blood Levels and Stroke Risk : A Pooled and Harmonized Analysis of 183 291 Participants From 29 Prospective Studies
- 2024
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Ingår i: Stroke. - : American Heart Association. - 0039-2499 .- 1524-4628. ; 55:1, s. 50-58
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:The effect of marine omega-3 PUFAs on risk of stroke remains unclear.METHODS:We investigated the associations between circulating and tissue omega-3 PUFA levels and incident stroke (total, ischemic, and hemorrhagic) in 29 international prospective cohorts. Each site conducted a de novo individual-level analysis using a prespecified analytical protocol with defined exposures, covariates, analytical methods, and outcomes; the harmonized data from the studies were then centrally pooled. Multivariable-adjusted HRs and 95% CIs across omega-3 PUFA quintiles were computed for each stroke outcome.RESULTS:Among 183 291 study participants, there were 10 561 total strokes, 8220 ischemic strokes, and 1142 hemorrhagic strokes recorded over a median of 14.3 years follow-up. For eicosapentaenoic acid, comparing quintile 5 (Q5, highest) with quintile 1 (Q1, lowest), total stroke incidence was 17% lower (HR, 0.83 [CI, 0.76–0.91]; P<0.0001), and ischemic stroke was 18% lower (HR, 0.82 [CI, 0.74–0.91]; P<0.0001). For docosahexaenoic acid, comparing Q5 with Q1, there was a 12% lower incidence of total stroke (HR, 0.88 [CI, 0.81–0.96]; P=0.0001) and a 14% lower incidence of ischemic stroke (HR, 0.86 [CI, 0.78–0.95]; P=0.0001). Neither eicosapentaenoic acid nor docosahexaenoic acid was associated with a risk for hemorrhagic stroke. These associations were not modified by either baseline history of AF or prevalent CVD.CONCLUSIONS:Higher omega-3 PUFA levels are associated with lower risks of total and ischemic stroke but have no association with hemorrhagic stroke.
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| 5. |
- Sahin, Cagla, et al.
(författare)
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Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures
- 2023
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 145:19, s. 10659-10668
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Tidskriftsartikel (refereegranskat)abstract
- Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.
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| 6. |
- 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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| 7. |
- Alcayde, B., et al.
(författare)
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Fatigue behaviour of glass-fibre-reinforced polymers : Numerical and experimental characterisation
- 2024
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Ingår i: Composite structures. - : Elsevier Ltd. - 0263-8223 .- 1879-1085. ; 337
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a novel numerical methodology to model the degradation and failure of composite materials like GFRP submitted to monotonic and high cycle fatigue loads. This is done by using the Serial–Parallel Rule of Mixtures homogenisation technique together with a proper mechanical characterisation of the constituent materials of the composite. This paper also proposes an efficient way of estimating the fatigue properties of each of the material constituents (fibre or matrix) to comply with the experimental results obtained at composite level; this enables to estimate the fatigue strength of any stacking/orientation of fibres with only one mechanical characterisation of the material properties. A comparison of the results obtained analytically and experimentally for GFRP is presented. The results show the applicability and accuracy of the proposed methodology in this field.
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| 8. |
- Allison, Timothy M., et al.
(författare)
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Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 10872-10880
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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.
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| 9. |
- Allison, Timothy M., et al.
(författare)
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Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society. - 0003-2700 .- 1520-6882. ; 92:16, s. 10881-10890
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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.
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| 10. |
- Brodmerkel, Maxim, et al.
(författare)
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Molecular dynamics simulations reveal barrel opening during the unfolding of the outer membrane protein FhaC
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Many membrane proteins carry out gatekeeping and transport functions across the membrane, which makes them tremendously important for the control of what passes into or out from the cell. Their underlying dynamics can be very challenging to capture for structural biology techniques, for which structural heterogeneity often is problematic. Native ion mobility mass spectrometry (IM-MS) is capable of maintaining non-covalent interactions between biomolecules in vacuo, allowing for intact protein complexes from heterogeneous mixtures to be analysed with respect to their masses and structures, making it a powerful tool for structural biology. Recent collision induced unfolding (CIU) experiments, where IM-MS is used to track the unfolding of proteins after activation, were used to investigate the dynamics of the membrane protein FhaC from Bordetella pertussis. FhaC is a β-barrel transmembrane protein found in the outer membrane, where it secretes virulence factors to the outside of the bacterium, requiring notable changes to its structure. CIU cannot on its own provide detailed information about the structural changes along the unfolding pathway. Here, we use MD simulations to mimic the CIU experiments to see if the unfolding proceeds as expected, with cytoplasm-facing domains leading the unfolding, or if other parts of the structure breaks first. By separating our simulation data according to experimental CIU data from literature, we match the structures in the former to the unfolding states identified in the latter, and find that FhaC instead unfolds from a “seam” in the β-barrel. In a wider context, our investigation provides insights into the structural stability and unfolding dynamics of β-barrel membrane proteins and how they can be studied using a combination of CIU and MD.
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