| 1. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Sumaila, U. Rashid, et al.
(författare)
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WTO must ban harmful fisheries subsidies
- 2021
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 374:6567, s. 544-544
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Satizabal, Claudia L., et al.
(författare)
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Genetic architecture of subcortical brain structures in 38,851 individuals
- 2019
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Ingår i: Nature Genetics. - : Nature Publishing Group. - 1061-4036 .- 1546-1718. ; 51:11, s. 1624-
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Tidskriftsartikel (refereegranskat)abstract
- Subcortical brain structures are integral to motion, consciousness, emotions and learning. We identified common genetic variation related to the volumes of the nucleus accumbens, amygdala, brainstem, caudate nucleus, globus pallidus, putamen and thalamus, using genome-wide association analyses in almost 40,000 individuals from CHARGE, ENIGMA and UK Biobank. We show that variability in subcortical volumes is heritable, and identify 48 significantly associated loci (40 novel at the time of analysis). Annotation of these loci by utilizing gene expression, methylation and neuropathological data identified 199 genes putatively implicated in neurodevelopment, synaptic signaling, axonal transport, apoptosis, inflammation/infection and susceptibility to neurological disorders. This set of genes is significantly enriched for Drosophila orthologs associated with neurodevelopmental phenotypes, suggesting evolutionarily conserved mechanisms. Our findings uncover novel biology and potential drug targets underlying brain development and disease.
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| 4. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 5. |
- Chen, Eefei, et al.
(författare)
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Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:49, s. 9836-9845
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Tidskriftsartikel (refereegranskat)abstract
- Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.
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| 6. |
- Collett-Solberg, Paulo F., et al.
(författare)
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Diagnosis, Genetics, and Therapy of Short Stature in Children : A Growth Hormone Research Society International Perspective
- 2019
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Ingår i: Hormone Research in Paediatrics. - : S. Karger. - 1663-2818 .- 1663-2826. ; 92:1, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- The Growth Hormone Research Society (GRS) convened a Workshop in March 2019 to evaluate the diagnosis and therapy of short stature in children. Forty-six international experts participated at the invitation of GRS including clinicians, basic scientists, and representatives from regulatory agencies and the pharmaceutical industry. Following plenary presentations addressing the current diagnosis and therapy of short stature in children, breakout groups discussed questions produced in advance by the planning committee and reconvened to share the group reports. A writing team assembled one document that was subsequently discussed and revised by participants. Participants from regulatory agencies and pharmaceutical companies were not part of the writing process. Short stature is the most common reason for referral to the pediatric endocrinologist. History, physical examination, and auxology remain the most important methods for understanding the reasons for the short stature. While some long-standing topics of controversy continue to generate debate, including in whom, and how, to perform and interpret growth hormone stimulation tests, new research areas are changing the clinical landscape, such as the genetics of short stature, selection of patients for genetic testing, and interpretation of genetic tests in the clinical setting. What dose of growth hormone to start, how to adjust the dose, and how to identify and manage a suboptimal response are still topics to debate. Additional areas that are expected to transform the growth field include the development of long-acting growth hormone preparations and other new therapeutics and diagnostics that may increase adult height or aid in the diagnosis of growth hormone deficiency.
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| 7. |
- Sodergren, Erica, et al.
(författare)
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The genome of the sea urchin Strongylocentrotus purpuratus.
- 2006
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 314:5801, s. 941-52
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Tidskriftsartikel (refereegranskat)abstract
- We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.
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| 8. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Effects of macromolecular crowding agents on protein folding in vitro and in silico
- 2013
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Ingår i: Biophysical Reviews. - : Springer. - 1867-2450 .- 1867-2469. ; 5:2, s. 137-145
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Forskningsöversikt (refereegranskat)abstract
- Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects-present when polypeptides normally fold in vivo-modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton's early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i. e., less misfolding and/or kinetic heterogeneity) than in buffer.
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| 9. |
- Christiansen, Alexander, 1982-, et al.
(författare)
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Factors defining effects of Macromolecular crowding on protein stability : an in vitro/in silico case study using cytochrome c
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:31, s. 6519-6530
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Tidskriftsartikel (refereegranskat)abstract
- Previous experiments with two single-domain proteins showed that macromolecular crowding can stabilize dramatically toward heat perturbation and modulate native-state structure and shape. To assess the generality of this, we here tested the effects of the synthetic crowding agents on cytochrome c, a small single-domain protein. Using far-UV circular dichroism (CD), we discovered that there is no effect on cytochrome c's secondary structure upon addition of Ficoll or dextran (0-400 mg/mL, pH 7). Thermal experiments revealed stabilizing effects (5-10 degrees C) of Ficoll 70 and dextran 70; this effect was enhanced by the presence of low levels of guanidine hydrochloride (GuHCl) that destabilize the protein. When using a smaller dextran, dextran 40, the thermal effects were larger (10-20 degrees C). In silico analysis, using structure-based (Go-like) interactions for cytochrome c, is in excellent agreement with the in vitro thermodynamic data and also agrees with scaled particle theory. Simulations of a range of crowder size and shape demonstrated that the smaller the crowder the larger the favorable effect on cytochrome c's folded-state stability. Together with previous data, we conclude that protein size, stability, conformational malleability, and folding routes, as well as crowder size and shape, are key factors that modulate the net effect of macromolecular crowding on proteins.
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| 10. |
- Homouz, Dirar, et al.
(författare)
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Crowded, cell-like environment induces shape changes in aspherical protein
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - Washington, DC : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:33, s. 11754-11759
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Tidskriftsartikel (refereegranskat)abstract
- How the crowded environment inside cells affects the structures of proteins with aspherical shapes is a vital question because many proteins and protein–protein complexes in vivo adopt anisotropic shapes. Here we address this question by combining computational and experimental studies of a football-shaped protein (i.e., Borrelia burgdorferi VlsE) in crowded, cell-like conditions. The results show that macromolecular crowding affects protein-folding dynamics as well as overall protein shape. In crowded milieus, distinct conformational changes in VlsE are accompanied by secondary structure alterations that lead to exposure of a hidden antigenic region. Our work demonstrates the malleability of “native” proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo.
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| 11. |
- Homouz, Dirar M., et al.
(författare)
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Role of Zero-Order Loop in Protein Unfolding Case Study with Apoazurin
- 2020
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:3, Supplement 1, s. 197A-197A
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Konferensbidrag (refereegranskat)abstract
- Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) is a two-state folder that has a single disulfide bond between residues 3 and 26. This bond covalently connects the N-termini of beta-strands 1 and 3; thereby it creates a zero-order loop. The loop restricts the conformational space for the apoazurin polypeptide. In order to understand the role played by the zero-order loop, we used molecular dynamics (MD) simulations to compare two variants of apoazurin; one variant called “loop” which contained the disulfide and another called “open” in which the disulfide bond between residues 3 and 26 was removed. MD simulations were performed to probe the unfolding pathway and stability of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the folded structure apoazurin is somewhat more stable due to the presence of the disulfide bond. However, the disulfide bond plays a prominent role in the apoazurin unfolding mechanism: we find that it changes both the folding-transition state and the unfolded-state ensemble of conformations.
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| 12. |
- Homouz, Dirar, et al.
(författare)
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Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin
- 2009
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Ingår i: Biophysical Journal. - : Elsevier Inc. - 0006-3495. ; 96:2, s. 671-80
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Tidskriftsartikel (refereegranskat)abstract
- Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (c) as well as of crowding agent geometry (sphere or spherocylinder) at high c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin's time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.
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| 13. |
- Samiotakis, Antonios, et al.
(författare)
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Folding, stability and shape of proteins in crowded environments : experimental and computational approaches
- 2009
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Ingår i: International journal of molecular sciences. - : MDPI AG. - 1422-0067. ; 10:2, s. 572-88
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Tidskriftsartikel (refereegranskat)abstract
- How the crowded environment inside cells affects folding, stability and structures of proteins is a vital question, since most proteins are made and function inside cells. Here we describe how crowded conditions can be created in vitro and in silico and how we have used this to probe effects on protein properties. We have found that folded forms of proteins become more compact in the presence of macromolecular crowding agents; if the protein is aspherical, the shape also changes (extent dictated by native-state stability and chemical conditions). It was also discovered that the shape of the macromolecular crowding agent modulates the folding mechanism of a protein; in addition, the extent of asphericity of the protein itself is an important factor in defining its folding speed.
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| 14. |
- Stagg, Loren, et al.
(författare)
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Residue-specific analysis of frustration in the folding landscape of repeat beta/alpha protein apoflavodoxin
- 2010
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Ingår i: Journal of molecular biology. - : Elsevier Ltd. - 1089-8638 .- 0022-2836. ; 396:1, s. 75-89
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Tidskriftsartikel (refereegranskat)abstract
- Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding.
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| 15. |
- Wang, Qian, et al.
(författare)
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Comparison of chemical and thermal protein denaturation by combination of computational and experimental approaches. II
- 2011
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Ingår i: Journal of Chemical Physics. - Lancaster, Pa. : American Institute of Physics (AIP). - 0021-9606 .- 1089-7690. ; 135:17, s. 175102-
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Tidskriftsartikel (refereegranskat)abstract
- Chemical and thermal denaturation methods have been widely used to investigate folding processes of proteins in vitro. However, a molecular understanding of the relationship between these two perturbation methods is lacking. Here, we combined computational and experimental approaches to investigate denaturing effects on three structurally different proteins. We derived a linear relationship between thermal denaturation at temperature T(b) and chemical denaturation at another temperature T(u) using the stability change of a protein (Delta G). For this, we related the dependence of Delta G on temperature, in the Gibbs-Helmholtz equation, to that of Delta G on urea concentration in the linear extrapolation method, assuming that there is a temperature pair from the urea (T(u)) and the aqueous (T(b)) ensembles that produces the same protein structures. We tested this relationship on apoazurin, cytochrome c, and apoflavodoxin using coarse-grained molecular simulations. We found a linear correlation between the temperature for a particular structural ensemble in the absence of urea, T(b), and the temperature of the same structural ensemble at a specific urea concentration, T(u). The in silico results agreed with in vitro far-UV circular dichroism data on apoazurin and cytochrome c. We conclude that chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions; however, deviations were found at high concentrations of denaturant. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3656692]
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| 16. |
- Zegarra, Fabio C., et al.
(författare)
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Crowding-Induced Elongated Conformation of Urea-Unfolded Apoazurin: Investigating the Role of Crowder Shape in Silico
- 2019
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 123:17, s. 3607-3617
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Tidskriftsartikel (refereegranskat)abstract
- American Chemical Society. Here, we show by solution nuclear magnetic resonance measurements that the urea-unfolded protein apoazurin becomes elongated when the synthetic crowding agent dextran 20 is present, in contrast to the prediction from the macromolecular crowding effect based on the argument of volume exclusion. To explore the complex interactions beyond volume exclusion, we employed coarse-grained molecular dynamics simulations to explore the conformational ensemble of apoazurin in a box of monodisperse crowders under strong chemically denaturing conditions. The elongated conformation of unfolded apoazurin appears to result from the interplay of the effective attraction between the protein and crowders and the shape of the crowders. With a volume-conserving crowder model, we show that the crowder shape provides an anisotropic direction of the depletion force, in which a bundle of surrounding rodlike crowders stabilize an elongated conformation of unfolded apoazurin in the presence of effective attraction between the protein and crowders.
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| 17. |
- Zegarra, Fabio C., et al.
(författare)
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The Zero-Order Loop in Apoazurin Modulates Folding Mechanism In Silico
- 2021
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 125:14, s. 3501-3509
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas aeruginosa apoazurin (apo, without the copper cofactor) has a single disulfide bond between residues 3 and 26 and unfolds in a two-state reaction in vitro. The disulfide bond covalently connects the N-termini of β-strands 1 and 3; thereby, it creates a zero-order loop or a "cinch" that restricts conformational space. Covalent loops and threaded topologies are emerging as important structural elements in folded proteins and may be important for function. In order to understand the role of a zero-order loop in the folding process of a protein, here we used coarse-grained molecular dynamics (CGMD) simulations in silico to compare two variants of apoazurin: one named "loop" which contained the disulfide, and another named "open" in which the disulfide bond between residues 3 and 26 was removed. CGMD simulations were performed to probe the stability and unfolding pathway of the two apoazurin variants at different urea concentrations and temperatures. Our results show that the covalent loop plays a prominent role in the unfolding mechanism of apoazurin; its removal alters both the folding-transition state and the unfolded-state ensemble of conformations. We propose that modulation of azurin's folding landscape by the disulfide bridge may be related to both copper capturing and redox sensing.
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