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- Digre, Andreas, et al.
(författare)
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The Human Protein Atlas - Spatial localization of the human proteome in health and disease
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:1, s. 218-233
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Tidskriftsartikel (refereegranskat)abstract
- For a complete understanding of a system's processes and each protein's role in health and disease, it is essential to study protein expression with a spatial resolution, as the exact location of proteins at tissue, cellular, or subcellular levels is tightly linked to protein function. The Human Protein Atlas (HPA) project is a large-scale initiative aiming at mapping the entire human proteome using antibody-based proteomics and integration of various other omics technologies. The publicly available knowledge resource www.proteinatlas.org is one of the world's most visited biological databases and has been extensively updated during the last few years. The current version is divided into six main sections, each focusing on particular aspects of the human proteome: (a) the Tissue Atlas showing the distribution of proteins across all major tissues and organs in the human body; (b) the Cell Atlas showing the subcellular localization of proteins in single cells; (c) the Pathology Atlas showing the impact of protein levels on survival of patients with cancer; (d) the Blood Atlas showing the expression profiles of blood cells and actively secreted proteins; (e) the Brain Atlas showing the distribution of proteins in human, mouse, and pig brain; and (f) the Metabolic Atlas showing the involvement of proteins in human metabolism. The HPA constitutes an important resource for further understanding of human biology, and the publicly available datasets hold much promise for integration with other emerging efforts focusing on single cell analyses, both at transcriptomic and proteomic level.
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- Elfageih, Rageia, et al.
(författare)
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Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli
- 2020
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 29:10, s. 2028-2037
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Tidskriftsartikel (refereegranskat)abstract
- Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of similar to 160 angstrom, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
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- Karlsson, Elin, et al.
(författare)
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High affinity between CREBBP/p300 and NCOA evolved in vertebrates
- 2020
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 29:7, s. 1687-1691
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between the transcriptional coactivators CREBBP/p300 and NCOA is governed by two intrinsically disordered domains called NCBD and CID, respectively. The CID domain emerged within the NCOA protein in deuterostome animals (including vertebrates) after their split from the protostomes (molluscs, worms, and arthropods). However, it has not been clear at which point a high affinity interaction evolved within the deuterostome clade and whether all present-day deuterostome animals have a high affinity NCBD:CID interaction. We have here expressed and measured affinity for NCBD and CID domains from animal species representing different evolutionary branches of the deuterostome tree. While all vertebrate species have high-affinity NCBD:CID interactions we found that the interaction in the echinoderm purple sea urchin is of similar affinity as that of the proposed ancestral domains. Our findings demonstrate that the high-affinity NCBD:CID interaction likely evolved in the vertebrate branch and question whether the interaction between CREBBP/p300 and NCOA is essential in nonvertebrate deuterostomes. The data provide an example of evolution of transcriptional regulation through protein-domain based inventions.
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- Kelpšas, Vinardas, et al.
(författare)
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Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:12, s. 2457-2473
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Tidskriftsartikel (refereegranskat)abstract
- Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
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- Møller, Marie Sofie, et al.
(författare)
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An ultra-high affinity protein–protein interface displaying sequence-robustness
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:6, s. 1144-1156
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Tidskriftsartikel (refereegranskat)abstract
- Protein–protein interactions are crucial in biology and play roles in for example, the immune system, signaling pathways, and enzyme regulation. Ultra-high affinity interactions (Kd <0.1 nM) occur in these systems, however, structures and energetics behind stability of ultra-high affinity protein–protein complexes are not well understood. Regulation of the starch debranching barley limit dextrinase (LD) and its endogenous cereal type inhibitor (LDI) exemplifies an ultra-high affinity complex (Kd of 42 pM). In this study the LD–LDI complex is investigated to unveil how robust the ultra-high affinity is to LDI sequence variation at the protein–protein interface and whether alternative sequences can retain the ultra-high binding affinity. The interface of LD–LDI was engineered using computational protein redesign aiming at identifying LDI variants predicted to retain ultra-high binding affinity. These variants present a very diverse set of mutations going beyond conservative and alanine substitutions typically used to probe interfaces. Surface plasmon resonance analysis of the LDI variants revealed that high affinity of LD–LDI requires interactions of several residues at the rim of the protein interface, unlike the classical hotspot arrangement where key residues are found at the center of the interface. Notably, substitution of interface residues in LDI, including amino acids with functional groups different from the wild-type, could occur without loss of affinity. This demonstrates that ultra-high binding affinity can be conferred without hotspot residues, thus making complexes more robust to mutational drift in evolution. The present mutational analysis also demonstrates how energetic coupling can emerge between residues at large distances at the interface.
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- Norn, Christoffer, et al.
(författare)
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A thermodynamic model of protein structure evolution explains empirical amino acid substitution matrices
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:10, s. 2057-2068
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Tidskriftsartikel (refereegranskat)abstract
- Proteins evolve under a myriad of biophysical selection pressures that collectively control the patterns of amino acid substitutions. These evolutionary pressures are sufficiently consistent over time and across protein families to produce substitution patterns, summarized in global amino acid substitution matrices such as BLOSUM, JTT, WAG, and LG, which can be used to successfully detect homologs, infer phylogenies, and reconstruct ancestral sequences. Although the factors that govern the variation of amino acid substitution rates have received much attention, the influence of thermodynamic stability constraints remains unresolved. Here we develop a simple model to calculate amino acid substitution matrices from evolutionary dynamics controlled by a fitness function that reports on the thermodynamic effects of amino acid mutations in protein structures. This hybrid biophysical and evolutionary model accounts for nucleotide transition/transversion rate bias, multi-nucleotide codon changes, the number of codons per amino acid, and thermodynamic protein stability. We find that our theoretical model accurately recapitulates the complex yet universal pattern observed in common global amino acid substitution matrices used in phylogenetics. These results suggest that selection for thermodynamically stable proteins, coupled with nucleotide mutation bias filtered by the structure of the genetic code, is the primary driver behind the global amino acid substitution patterns observed in proteins throughout the tree of life.
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