| 1. |
- Andersen, T. C. B., et al.
(författare)
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The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell?specific adapter protein
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:42, s. 15480-15494
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Tidskriftsartikel (refereegranskat)abstract
- T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2?inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239?274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239?256 and aa 257?274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242?268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.
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| 2. |
- Kanter-Smoler, Gunilla, et al.
(författare)
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Novel findings in Swedish patients with MYH-associated polyposis: mutation detection and clinical characterization
- 2006
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Ingår i: Clin Gastroenterol Hepatol. - : Elsevier BV. - 1542-3565. ; 4:4, s. 499-506
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND & AIMS: Biallelic mutations in the base-excision repair gene MYH have recently been associated with recessive inheritance of multiple colorectal adenomas. An investigation and characterization of MYH mutations in Swedish patients were therefore carried out. METHODS: A set of 15 unrelated adenomatous polyposis coli (APC)-mutation negative patients from the Swedish Polyposis Registry was screened for germline mutations in the MYH gene. The patients were clinically characterized and compared with 43 APC-mutation positive probands diagnosed during the same period. RESULTS: Disease-causing biallelic MYH mutations were identified in 6 patients (40%). The mean age at diagnosis was 47.8 years versus 34.1 years in APC-mutation positive patients (P = .015). Colorectal cancer at diagnosis of polyposis was present in 67% (4/6) of the patients, and all were right-sided, compared with only 19% versus 12.5% right-sided cancer in APC-mutation positive patients. Upper gastrointestinal manifestations were diagnosed in 1 of 5 compared with 23 of 27 in APC-mutation positive patients (odds ratio, 23; 95% confidence interval, 2-263; P = .0086). One family exhibited apparent dominant inheritance of colorectal adenomatous polyposis. Two new pathogenic mutations, MYH p.G175E and p.P391L, were identified. The mutations are argued to introduce profound changes in substrate-recognizing domains of the protein. CONCLUSIONS: Biallelic MYH mutations, including 2 novel mutations, were found in a substantial number of the patients with multiple colorectal adenomas who were negative for APC-mutation. The examined MYH-mutation positive patients were found to have higher risks of colorectal cancer at diagnosis, right-sided location of cancers, and a significantly lower incidence of upper gastrointestinal manifestations, compared with APC-mutation positive patients.
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| 3. |
- Bay Nord, Anders, 1976, et al.
(författare)
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Blending Samples to Increase Accuracy and Precision of 1H NMR Urine Metabolomics
- 2024
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Ingår i: ANALYTICAL CHEMISTRY. - 0003-2700 .- 1520-6882. ; 96:32, s. 13078-13085
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Tidskriftsartikel (refereegranskat)abstract
- Urine is an equally attractive biofluid for metabolomics analysis, as it is a challenging matrix analytically. Accurate urine metabolite concentration estimates by Nuclear Magnetic Resonance (NMR) are hampered by pH and ionic strength differences between samples, resulting in large peak shift variability. Here we show that calculating the spectra of original samples from mixtures of samples using linear algebra reduces the shift problems and makes various error estimates possible. Since the use of two-dimensional (2D) NMR to confirm metabolite annotations is effectively impossible to employ on every sample of large sample sets, stabilization of metabolite peak positions increases the confidence in identifying metabolites, avoiding the pitfall of oranges-to-apples comparisons.
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| 4. |
- Bijlmakers, M. J., et al.
(författare)
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A C2HC zinc finger is essential for the RING-E2 interaction of the ubiquitin ligase RNF125
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.
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| 5. |
- Isaksson, Linnéa, et al.
(författare)
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Highly Efficient NMR Assignment of Intrinsically Disordered Proteins: Application to B- and T Cell Receptor Domains
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- We present an integrated approach for efficient characterization of intrinsically disordered proteins. Batch cell-free expression, fast data acquisition, automated analysis, and statistical validation with data resampling have been combined for achieving cost-effective protein expression, and rapid automated backbone assignment. The new methodology is applied for characterization of five cytosolic domains from T- and B-cell receptors in solution.
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| 6. |
- Johansson, Carina B., 1955, et al.
(författare)
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Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.
- 2002
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Ingår i: European journal of biochemistry / FEBS. - 0014-2956. ; 269:18, s. 4505-15
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Tidskriftsartikel (refereegranskat)abstract
- Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.
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| 7. |
- Rohlin, Anna, et al.
(författare)
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A mutation in POLE predisposing to a multi-tumour phenotype
- 2014
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 45:1, s. 77-81
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Tidskriftsartikel (refereegranskat)abstract
- Somatic mutations in the POLE gene encoding the catalytic subunit of DNA polymerase epsilon have been found in sporadic colorectal cancers (CRCs) and are most likely of importance in tumour development and/or progression. Recently, families with dominantly inherited colorectal adenomas and colorectal cancer were shown to have a causative heterozygous germline mutation in the proofreading exonuclease domain of POLE. The highly penetrant mutation was associated with predisposition to CRC only and no extra-colonic tumours were observed. We have identified a mutation in a large family in which the carriers not only developed CRC, they also demonstrate a highly penetrant predisposition to extra-intestinal tumours such as ovarian, endometrial and brain tumours. The mutation, NM_006231.2:c.1089C>A, p.Asn363Lys, also located in the proofreading exonuclease domain is directly involved in DNA binding. Theoretical prediction of the amino acid substitution suggests a profound effect of the substrate binding capability and a more severe impairment of the catalytic activity compared to the previously reported germline mutation. A possible genotype to phenotype correlation for deleterious mutations in POLE might exist that needs to be considered in the follow-up of mutation carriers.
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| 8. |
- Rohlin, Anna, et al.
(författare)
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GREM1 and POLE variants in hereditary colorectal cancer syndromes
- 2016
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Ingår i: Genes, Chromosomes and Cancer. - : Wiley. - 1045-2257 .- 1098-2264. ; 55:1, s. 95-106
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Tidskriftsartikel (refereegranskat)abstract
- Hereditary factors are thought to play a role in at least one third of patients with colorectal cancer (CRC) but only a limited proportion of these have mutations in known high-penetrant genes. In a relatively large part of patients with a few or multiple colorectal polyps the underlying genetic cause of the disease is still unknown. Using exome sequencing in combination with linkage analyses together with detection of copy-number variations (CNV), we have identified a duplication in the regulatory region of the GREM1 gene in a family with an attenuated/atypical polyposis syndrome. In addition, 107 patients with colorectal cancer and/or polyposis were analyzed for mutations in the candidate genes identified. We also performed screening of the exonuclease domain of the POLE gene in a subset of these patients. The duplication of 16 kb in the regulatory region of GREM1 was found to be disease-causing in the family. Functional analyses revealed a higher expression of the GREM1 gene in colorectal tissue in duplication carriers. Screening of the exonuclease domain of POLE in additional CRC patients identified a probable causative novel variant c.1274A>G, p.Lys425Arg. In conclusion a high penetrant duplication in the regulatory region of GREM1, predisposing to CRC, was identified in a family with attenuated/atypical polyposis. A POLE variant was identified in a patient with early onset CRC and a microsatellite stable (MSS) tumor. Mutations leading to increased expression of genes can constitute disease-causing mutations in hereditary CRC syndromes. © 2015 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.
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| 9. |
- Rådjursöga, Millie, 1977, et al.
(författare)
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Metabolic profiles from two different breakfast meals characterized by H-1 NMR-based metabolomics
- 2017
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Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 231, s. 267-274
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Tidskriftsartikel (refereegranskat)abstract
- It is challenging to measure dietary exposure with techniques that are both accurate and applicable to free-living individuals. We performed a cross-over intervention, with 24 healthy individuals, to capture the acute metabolic response of a cereal breakfast (CB) and an egg and ham breakfast (EHB). Fasting and postprandial urine samples were analyzed using H-1 nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis. Metabolic profiles were distinguished in relation to ingestion of either CB or EHB. Phosphocreatine/creatine and citrate were identified at higher concentrations after consumption of EHB. Beverage consumption (i.e., tea or coffee) could clearly be seen in the data. 2-furoylglycine and 5-hydroxymethyl-2-furoic acid - potential biomarkers for coffee consumption were identified at higher concentrations in coffee drinkers. Thus H-1 NMR urine metabolomics is applicable in the characterization of acute metabolic fingerprints from meal consumption and in the identification of metabolites that may serve as potential biomarkers. (C) 2017 Elsevier Ltd. All rights reserved.
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| 10. |
- Rådjursöga, Millie, 1977, et al.
(författare)
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Nutritional metabolomics: Postprandial response of meals relating to vegan, lacto-ovo vegetarian, and omnivore diets
- 2018
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Ingår i: Nutrients. - : MDPI AG. - 2072-6643. ; 10:8
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomics provide an unbiased tool for exploring the modulation of the human metabolome in response to food intake. This study applied metabolomics to capture the postprandial metabolic response to breakfast meals corresponding to vegan (VE), lacto ovo-vegetarian (LOV), and omnivore (OM) diets. In a cross over design 32 healthy volunteers (16 men and 16 females) consumed breakfast meals in a randomized order during three consecutive days. Fasting and 3 h postprandial serum samples were collected and then subjected to metabolite profiling using1 H-nuclear magnetic resonance (NMR) spectroscopy. Changes in concentration of identified and discriminating metabolites, between fasting and postprandial state, were compared across meals. Betaine, choline, and creatine displayed higher concentration in the OM breakfast, while 3-hydroxyisobutyrate, carnitine, proline, and tyrosine showed an increase for the LOV and unidentified free fatty acids displayed a higher concentration after the VE breakfast. Using1 H NMR metabolomics it was possible to detect and distinguish the metabolic response of three different breakfast meals corresponding to vegan, lacto-ovo vegetarian, and omnivore diets in serum. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
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| 11. |
- Rådjursöga, Millie, 1977, et al.
(författare)
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The H-1 NMR serum metabolomics response to a two meal challenge: a cross-over dietary intervention study in healthy human volunteers
- 2019
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Ingår i: Nutrition Journal. - : Springer Science and Business Media LLC. - 1475-2891. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Metabolomics represents a powerful tool for exploring modulation of the human metabolome in response to food intake. However, the choice of multivariate statistical approach is not always evident, especially for complex experimental designs with repeated measurements per individual. Here we have investigated the serum metabolic responses to two breakfast meals: an egg and ham based breakfast and a cereal based breakfast using three different multivariate approaches based on the Projections to Latent Structures framework. Methods: In a cross over design, 24 healthy volunteers ate the egg and ham breakfast and cereal breakfast on four occasions each. Postprandial serum samples were subjected to metabolite profiling using H-1 nuclear magnetic resonance spectroscopy and metabolites were identified using 2D nuclear magnetic resonance spectroscopy. Metabolic profiles were analyzed using Orthogonal Projections to Latent Structures with Discriminant Analysis and Effect Projections and ANOVA-decomposed Projections to Latent Structures. Results: The Orthogonal Projections to Latent Structures with Discriminant Analysis model correctly classified 92 and 90% of the samples from the cereal breakfast and egg and ham breakfast, respectively, but confounded dietary effects with inter-personal variability. Orthogonal Projections to Latent Structures with Effect Projections removed inter-personal variability and performed perfect classification between breakfasts, however at the expense of comparing means of respective breakfasts instead of all samples. ANOVA-decomposed Projections to Latent Structures managed to remove inter-personal variability and predicted 99% of all individual samples correctly. Proline, tyrosine, and N-acetylated amino acids were found in higher concentration after consumption of the cereal breakfast while creatine, methanol, and isoleucine were found in higher concentration after the egg and ham breakfast. Conclusions: Our results demonstrate that the choice of statistical method will influence the results and adequate methods need to be employed to manage sample dependency and repeated measurements in cross-over studies. In addition, H-1 nuclear magnetic resonance serum metabolomics could reproducibly characterize postprandial metabolic profiles and identify discriminatory metabolites largely reflecting dietary composition.
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| 12. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 13. |
- Altincekic, Nadide, et al.
(författare)
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Targeting the Main Protease (Mpro, nsp5) by Growth of Fragment Scaffolds Exploiting Structure-Based Methodologies
- 2024
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 19:2, s. 563-574
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Tidskriftsartikel (refereegranskat)abstract
- The main protease Mpro, nsp5, of SARS-CoV-2 (SCoV2) is one of its most attractive drug targets. Here, we report primary screening data using nuclear magnetic resonance spectroscopy (NMR) of four different libraries and detailed follow-up synthesis on the promising uracil-containing fragment Z604 derived from these libraries. Z604 shows time-dependent binding. Its inhibitory effect is sensitive to reducing conditions. Starting with Z604, we synthesized and characterized 13 compounds designed by fragment growth strategies. Each compound was characterized by NMR and/or activity assays to investigate their interaction with Mpro. These investigations resulted in the four-armed compound 35b that binds directly to Mpro. 35b could be cocrystallized with Mpro revealing its noncovalent binding mode, which fills all four active site subpockets. Herein, we describe the NMR-derived fragment-to-hit pipeline and its application for the development of promising starting points for inhibitors of the main protease of SCoV2.
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| 14. |
- Brunius, Carl, 1974, et al.
(författare)
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Prediction and modeling of pre-analytical sampling errors as a strategy to improve plasma NMR metabolomics data
- 2017
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1460-2059 .- 1367-4811. ; 33:22, s. 3567-3574
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Tidskriftsartikel (refereegranskat)abstract
- Biobanks are important infrastructures for life science research. Optimal sample handling regarding e.g. collection and processing of biological samples is highly complex, with many variables that could alter sample integrity and even more complex when considering multiple study centers or using legacy samples with limited documentation on sample management. Novel means to understand and take into account such variability would enable high-quality research on archived samples. This study investigated whether pre-analytical sample variability could be predicted and reduced by modeling alterations in the plasma metabolome, measured by NMR, as a function of pre-centrifugation conditions (1-36 h pre-centrifugation delay time at 4 A degrees C and 22 A degrees C) in 16 individuals. Pre-centrifugation temperature and delay times were predicted using random forest modeling and performance was validated on independent samples. Alterations in the metabolome were modeled at each temperature using a cluster-based approach, revealing reproducible effects of delay time on energy metabolism intermediates at both temperatures, but more pronounced at 22 A degrees C. Moreover, pre-centrifugation delay at 4 A degrees C resulted in large, specific variability at 3 h, predominantly of lipids. Pre-analytical sample handling error correction resulted in significant improvement of data quality, particularly at 22 A degrees C. This approach offers the possibility to predict pre-centrifugation delay temperature and time in biobanked samples before use in costly downstream applications. Moreover, the results suggest potential to decrease the impact of undesired, delay-induced variability. However, these findings need to be validated in multiple, large sample sets and with analytical techniques covering a wider range of the metabolome, such as LC-MS.
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| 15. |
- Cruz-Gallardo, I., et al.
(författare)
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RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 288:36, s. 25986-25994
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Tidskriftsartikel (refereegranskat)abstract
- T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.
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| 16. |
- Cruz-Gallardo, I., et al.
(författare)
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The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain
- 2014
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Ingår i: Rna Biology. - : Informa UK Limited. - 1547-6286 .- 1555-8584. ; 11:6, s. 766-776
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Tidskriftsartikel (refereegranskat)abstract
- T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C-and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.
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| 17. |
- Diaz-Moreno, I., et al.
(författare)
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NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c(6)
- 2005
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Ingår i: Journal of Biological Chemistry. ; 280:9, s. 7925-7931
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Tidskriftsartikel (refereegranskat)abstract
- A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.
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| 18. |
- Edwin, Aaron, et al.
(författare)
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Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae
- 2015
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 24:12, s. 2076-2080
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Tidskriftsartikel (refereegranskat)abstract
- The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N- terminal domain (residues 23-103) that contains two short alpha-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.
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| 19. |
- Engman, Cecilia, 1974, et al.
(författare)
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Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin.
- 2004
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Ingår i: Protein Science. ; 13:10, s. 2706-2715
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Tidskriftsartikel (refereegranskat)abstract
- The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state.
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| 20. |
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| 21. |
- Gourdon, Pontus Emanuel, 1978, et al.
(författare)
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Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 58:1, s. 103-113
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Tidskriftsartikel (refereegranskat)abstract
- Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0 mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5 mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.
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| 22. |
- Gustavsson, Emil, 1987, et al.
(författare)
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Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching
- 2020
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 118:2, s. 415-421
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Tidskriftsartikel (refereegranskat)abstract
- Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.
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| 23. |
- Isaksson, Linnéa, et al.
(författare)
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Expression screening of membrane proteins with cell-free protein synthesis.
- 2012
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Ingår i: Protein expression and purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 82:1, s. 218-25
-
Tidskriftsartikel (refereegranskat)abstract
- Detailed biophysical studies of integral membrane proteins are often hampered by sample preparation difficulties. Membrane proteins are typically difficult to express in sufficient amounts to enable the use of demanding techniques such as nuclear magnetic resonance and X-ray crystallography for structural biology. Here, we show that an inexpensive batch-based cell-free expression system can be a viable alternative for production of a wide range of different membrane proteins, both of prokaryotic and eukaryotic origin. Out of 38 tested protein constructs, 37 express at levels suitable for structural biology, i.e. enough to produce several milligrams of protein routinely and without excessive costs. This success rate was not anticipated and is even more impressive considering that more than half of the expressed proteins where of mammalian origin. A detergent screen identified Brij-58 as the, in general, most successful choice for co-translational solubilization of the expressed proteins.
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| 24. |
- Isaksson, Linnéa, et al.
(författare)
-
Signaling Mechanism of Phytochromes in Solution.
- 2021
-
Ingår i: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 29:2, s. 151-160
-
Tidskriftsartikel (refereegranskat)abstract
- Phytochrome proteins guide the red/far-red photoresponse of plants, fungi, and bacteria. Crystal structures suggest that the mechanism of signal transduction from the chromophore to the output domains involves refolding of the so-called PHY tongue. It is currently not clear how the two other notable structural features of the phytochrome superfamily, the so-called helical spine and a knot in the peptide chain, are involved in photoconversion. Here, we present solution NMR data of the complete photosensory core module from Deinococcus radiodurans. Photoswitching between the resting and the active states induces changes in amide chemical shifts, residual dipolar couplings, and relaxation dynamics. All observables indicate a photoinduced structural change in the knot region and lower part of the helical spine. This implies that a conformational signal is transduced from the chromophore to the helical spine through the PAS and GAF domains. The discovered pathway underpins functional studies of plant phytochromes and may explain photosensing by phytochromes under biological conditions.
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| 25. |
- Johansson, Tomas, et al.
(författare)
-
X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli.
- 2005
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 352:2, s. 299-312
-
Tidskriftsartikel (refereegranskat)abstract
- The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
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| 26. |
- Karlsson, B Göran, 1962
(författare)
-
Protein Engineering on Azurin. Expression Mutagenesis and Characterisation of Copper Site Mutants
- 1992
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Azurin belongs to a family of small blue copper proteins or cupredoxins which participate in electron transfer processes in plants and bacteria. The type 1 copper site in these proteins is characterised by an intense blue colour, a narrow hyperfine coupling in the EPR signal and, generally, a high reduction potential. A common feature in the cupredoxin structure is the eight stranded b-barrel and a copper binding site situated in a loop between two b-strands. Three of the four copper ligands, two histidines and one cysteine, are conserved in all cupredoxins, whereas the fourth ligand, a methionine, is exchanged in stellacyanin. The gene encoding azurin from Pseudomonas aeruginosa has been expressed in large amounts in Escherichia coli. Cassette mutagenesis was used to exchange the methionine ligand for all other amino acids, including a stop codon. The properties of the mutant proteins were investigated by optical and EPR spectroscopy. The effect of the amino acid exchange on the reduction potential was studied, XAFS was used to analyse the structure of the copper site and the X-ray structure was determined for the Met121Glu mutant protein. It was concluded that the ligand methionine is not needed to create a type 1 copper site. The exchange of the methionine results in mutant proteins with large changes in spectroscopic and redox properties. The introduction of polar residues gives rise to spectroscopic properties similar to properties found in stellacyanin or pseudoazurin. A large increase in reduction potential is observed when hydrophobic residues are introduced, whereas a decrease is observed when the copper ion is solvent exposed. The introduction of a negative group directly coordinating the copper is accompanied by a change in the copper site geometry, a decrease in reduction potential and a drastic change in the spectroscopic properties.
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| 27. |
- Karlsson, B Göran, 1962, et al.
(författare)
-
Solution structure of the calmodulin-like C-terminal domain of Entamoeba-actinin2
- 2016
-
Ingår i: Proteins-Structure Function and Bioinformatics. - : Wiley. - 0887-3585 .- 1097-0134. ; 84:4, s. 461-466
-
Tidskriftsartikel (refereegranskat)abstract
- Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker -actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, -actinin is believed to be required for infection. To better understand the role of -actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker. Proteins 2016; 84:461-466.
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| 28. |
- Karlsson, Fredrik, 1984, et al.
(författare)
-
Gut metagenome in European women with normal, impaired and diabetic glucose control
- 2013
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 498:7452, s. 99-103
-
Tidskriftsartikel (refereegranskat)abstract
- Type 2 diabetes (T2D) is a result of complex gene-environment interactions, and several risk factors have been identified, including age, family history, diet, sedentary lifestyle and obesity. Statistical models that combine known risk factors for T2D can partly identify individuals at high risk of developing the disease. However, these studies have so far indicated that human genetics contributes little to the models, whereas socio-demographic and environmental factors have greater influence(1). Recent evidence suggests the importance of the gut microbiota as an environmental factor, and an altered gut microbiota has been linked to metabolic diseases including obesity(2,3), diabetes(4) and cardiovascular disease(5). Here we use shotgun sequencing to characterize the faecal metagenome of 145 European women with normal, impaired or diabetic glucose control. We observe compositional and functional alterations in the metagenomes of women with T2D, and develop a mathematical model based on metagenomic profiles that identified T2D with high accuracy. We applied this model to women with impaired glucose tolerance, and show that it can identify women who have a diabetes-like metabolism. Furthermore, glucose control and medication were unlikely to have major confounding effects. We also applied our model to a recently described Chinese cohort(4) and show that the discriminant metagenomicmarkers for T2D differ between the European and Chinese cohorts. Therefore, metagenomic predictive tools for T2D should be specific for the age and geographical location of the populations studied.
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| 29. |
- Kristiansen, Per Eugen, et al.
(författare)
-
Nuclear Magnetic Resonance Structure and Mutational Analysis of the Lactococcin A Immunity Protein.
- 2016
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 55:45, s. 6250-6257
-
Tidskriftsartikel (refereegranskat)abstract
- The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins' cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin-man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of LciA in which 6 or 10 of the C-terminal residues were removed yielded a reduced degree of protection, indicating that the unstructured C-terminal tail is important for the functionality of the immunity proteins.
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| 30. |
- Lendel, Christofer, et al.
(författare)
-
Combined Solution- and Magic Angle Spinning NMR Reveals Regions of Distinct Dynamics in Amyloid beta Protofibrils
- 2016
-
Ingår i: Chemistryselect. - : Wiley. - 2365-6549. ; 1:18, s. 5850-5853
-
Tidskriftsartikel (refereegranskat)abstract
- Solid-state magic angle spinning (MAS) NMR has emerged as an important tool for investigations of protein aggregates and amyloid fibrils, which are not accessible for solution NMR experiments. We recently presented a structural model for amyloid beta (A beta) protofibrils based on MAS-NMR data. The absence of resonances for the N-terminus of A beta in this dataset suggested that it might be disordered and more dynamic than the structural core. We here provide evidence for a distinct dynamic regime in the N-terminal part of the peptide and show that the structural characteristics of this region can be elucidated using C-13-detected solution NMR. The results shed more light on the structural properties of pre-fibrillar A beta species and demonstrate the potential of combining MAS and solution NMR experiments for the characterization of structure and dynamics of complex protein assemblies.
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| 31. |
- Malmodin, Daniel, 1974, et al.
(författare)
-
NMR Spectroscopic Analysis to Evaluate the Quality of Insulin: Concentration, Variability, and Excipient Content.
- 2020
-
Ingår i: Journal of diabetes science and technology. - : SAGE Publications. - 1932-2968. ; 14:1, s. 180-184
-
Tidskriftsartikel (refereegranskat)abstract
- Known and consistent bioactivity between samples of insulin is essential to correctly estimate the dose. Insulin concentration is not the same thing as bioactivity, however, and methods to correctly determine both are required. Here we show that one dimensional nuclear magnetic resonance (1D NMR), in contrast to, for example, reverse phase high pressure liquid chromatography, can be used to determine both insulin concentration as well as confirm the structural integrity required for activity. In response to the report by Carter and Heinemann, we decided to investigate insulin intended for public use. Insulin from several manufacturers was investigated. Correct insulin concentrations were found in all tested samples although the general sample variability, which possibly could influence the bioactivity, varied depending on insulin type and manufacturer.
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| 32. |
- Pedersen, Anders, 1976, et al.
(författare)
-
Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis
- 2011
-
Ingår i: Protein Expression and Purification. - : Elsevier Inc. - 1046-5928 .- 1096-0279. ; 77:2, s. 220-223
-
Tidskriftsartikel (refereegranskat)abstract
- The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.
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| 33. |
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| 34. |
- Pedersen, Anders, 1976, et al.
(författare)
-
Rational improvement of cell-free protein synthesis.
- 2011
-
Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 28:3, s. 218-224
-
Tidskriftsartikel (refereegranskat)abstract
- Experimental design principles were applied on cell-free protein synthesis to optimize performance with regard to the expression yield and the incorporation efficiency of amino acid precursors. A versatile screening platform based on batch-mode cell-free expression and central composite design was used. The performance of different extracts (S12 and S30), the concentration dependence of key components and the effect of different additives were investigated. We find that the initial expression yield can be enhanced twofold to threefold in this manner. The improved conditions comprise a modified S12 extract, optimized concentrations of creatine phosphate and key amino acids, as well as introduction of ketoacid additives. Our results show that current cell-free expression technology is far from optimal and that higher yields and increased utilization of the provided precursors are attainable with further optimization.
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| 35. |
- Pedersen, Anders, 1976, et al.
(författare)
-
Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range.
- 2005
-
Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002 .- 0005-2728. ; 1707:2-3, s. 254-8
-
Tidskriftsartikel (refereegranskat)abstract
- A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP(+) or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK(a) values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK(a) values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP(+) and NADPH forms of domain III, but with a marked difference in pK(a) not only attributable to the charge difference between the substrates. Titrating residues found in loop D/alpha5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK(a) values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.
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| 36. |
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| 37. |
- Ross, Alastair, 1976, et al.
(författare)
-
Identification and quantification of even and odd chained 5-n alkylresorcinols, branched chain-alkylresorcinols and methylalkylresorcinols in Quinoa (Chenopodium quinoa)
- 2017
-
Ingår i: Food Chemistry. - : Elsevier BV. - 0308-8146 .- 1873-7072. ; 220, s. 344-351
-
Tidskriftsartikel (refereegranskat)abstract
- Quinoa is a pseudocereal grown in the Andean region of South America that is of increasing interest worldwide as an alternative staple food. We have detected a complex mixture of both odd- and even-alkyl chain alkylresorcinols (AR), branched-chain alkylresorcinols (bcAR) and methylalkylresorcinols (mAR) in ethyl acetate extracts of quinoa. We quantified the content of AR in 17 commercial samples of quinoa, and found that the mean±SD content of AR was 58±16μg/g, bcAR was 182±52μg/g, and mAR was 136±40μg/g. AR from quinoa could also be detected in plasma after eating quinoa, indicating that some of these unique AR could be used as biomarkers of quinoa intake in humans. Further work is required to understand the role of these ARs in the quinoa plant and whether any of the novel ARs may be of particular interest in human nutrition. © 2016 Elsevier Ltd
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| 38. |
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| 39. |
- Saline, Maria, et al.
(författare)
-
Backbone resonance assignment of Staphylococcal Enterotoxin H.
- 2009
-
Ingår i: Biomolecular NMR assignments. - : Springer Science and Business Media LLC. - 1874-270X .- 1874-2718.
-
Tidskriftsartikel (refereegranskat)abstract
- The staphylococcal enterotoxin H (SEH; 217 aa, 25 kD) belongs to a family of superantigens that cause a massive immune response upon simultaneous binding to the T cell receptor (TCR) and the major histocompatibility complex class II. The SEH-TCR interaction is weak and amenable to studies using NMR methodology. Essentially, 2 mg of U{(2)H, (13)C,(15)N}-labeled SEH was used for the complete sequential backbone assignment of SEH at 900 MHz. The protein secondary structure inferred from the chemical shift index (C(alpha) and C(beta)) is in very good agreement with the secondary structure elements of the X-ray structure.
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| 40. |
- Saline, Maria, et al.
(författare)
-
The structure of superantigen complexed with TCR and MHC reveals novel insights into superantigenic T cell activation.
- 2010
-
Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 1:8
-
Tidskriftsartikel (refereegranskat)abstract
- Superantigens (SAgs) are bacterial toxins that interact with immunoreceptors, T cell receptor (TCR) and major histocompatibility complex (MHC) class II, conventionally through the variable β-domain of TCR (TCRVβ). They induce a massive release of cytokines, which can lead to diseases such as food poisoning and toxic shock syndrome. In this study, we report the X-ray structure of the ternary complex between staphylococcal enterotoxin H (SEH) and its human receptors, MHC class II and TCR. The structure demonstrates that SEH predominantly interacts with the variable α-domain of TCR (TCRVα), which is supported by nuclear magnetic resonance (NMR) analyses. Furthermore, there is no contact between MHC and TCR upon complex formation. Structural analyses suggest that the major contact points to TCRVα are conserved among other bacterial SAgs. Consequently, a new dimension of SAg biology emerges, suggesting that in addition to the conventional interactions with the TCRVβ domain, SAgs can also activate T cells through the TCRVα domain.
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| 41. |
- Samuelsson, Linda, 1973, et al.
(författare)
-
Using NMR metabolomics to identify responses of an environmental estrogen in blood plasma of fish
- 2006
-
Ingår i: Aquatic Toxicology (Amsterdam, Netherlands). - : Elsevier BV. - 0166-445X. ; 78:4, s. 341-349
-
Tidskriftsartikel (refereegranskat)abstract
- Nuclear magnetic resonance (NMR) based metabolomics in combination with multivariate data analysis may become valuable tools to study environmental effects of pharmaceuticals and other chemicals in aquatic organisms. To explore the usefulness of this approach in fish, we have used H-1 NMR metabolomics to compare blood plasma and plasma lipid extracts from rainbow trout exposed to the synthetic contraceptive estrogen ethinylestradiol (EE2) with plasma from control fish. The plasma metabolite profile was affected in fish exposed to 10 ng/L but not 0.87 ng/L of EE2, which was in agreement with an induced vitellogenin synthesis in the high dose group only, as measured by ELISA. The main affected metabolites were vitellogenin, alanine, phospholipids and cholesterol. The responses identified by this discovery-driven method could be put in context with previous knowledge of the effects of estrogens on fish. This adds confidence to the approach of using NMR metabolomics to identify environmental effects of pharmaceuticals and other contaminants. (c) 2006 Elsevier B.V. All rights reserved.
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| 42. |
- Sandberg, Anders, 1975, et al.
(författare)
-
Apo-azurin folds via an intermediate that resembles the molten-globule.
- 2004
-
Ingår i: Protein Science. ; 13:10, s. 2628-2638
-
Tidskriftsartikel (refereegranskat)abstract
- The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.
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| 43. |
- Sandberg, Anders, 1975, et al.
(författare)
-
Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen.
- 2003
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42:34, s. 10301-10
-
Tidskriftsartikel (refereegranskat)abstract
- The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins.
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| 44. |
- Schou, M. F., et al.
(författare)
-
Metabolic and functional characterization of effects of developmental temperature in Drosophila melanogaster
- 2017
-
Ingår i: American Journal of Physiology-Regulatory Integrative and Comparative Physiology. - : American Physiological Society. - 0363-6119 .- 1522-1490. ; 312:2
-
Tidskriftsartikel (refereegranskat)abstract
- The ability of ectotherms to respond to changes in their thermal environment through plastic mechanisms is central to their adaptive capability. However, we still lack knowledge on the physiological and functional responses by which ectotherms acclimate to temperatures during development, and in particular, how physiological stress at extreme temperatures may counteract beneficial acclimation responses at benign temperatures. We exposed Drosophila melanogaster to 10 developmental temperatures covering their entire permissible temperature range. We obtained metabolic profiles and reaction norms for several functional traits: egg-to-adult viability, developmental time, and heat and cold tolerance. Females were more heat tolerant than males, whereas no sexual dimorphism was found in cold tolerance. A group of metabolites, mainly free amino acids, had linear reaction norms. Several energy-carrying molecules, as well as some sugars, showed distinct inverted U-shaped norms of reaction across the thermal range, resulting in a positive correlation between metabolite intensities and egg-to-adult viability. At extreme temperatures, low levels of these metabolites were interpreted as a response characteristic of costs of homeostatic perturbations. Our results provide novel insights into a range of metabolites reported to be central for the acclimation response and suggest several new candidate metabolites. Low and high temperatures result in different adaptive physiological responses, but they also have commonalities likely to be a result of the failure to compensate for the physiological stress. We suggest that the regulation of metabolites that are tightly connected to the performance curve is important for the ability of ectotherms to cope with variation in temperature.
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| 45. |
- Stenson, Martin, et al.
(författare)
-
Serum nuclear magnetic resonance-based metabolomics and outcome in diffuse large B-cell lymphoma patients - a pilot study
- 2016
-
Ingår i: Leukemia and Lymphoma. - : Informa UK Limited. - 1042-8194 .- 1029-2403. ; 57:8, s. 1814-1822
-
Tidskriftsartikel (refereegranskat)abstract
- The prognosis for diffuse large B-cell lymphoma (DLBCL) patients with early relapse or refractory disease is dismal. To determine if clinical outcome correlated to diverse serum metabolomic profiles, we used H-1 nuclear magnetic resonance (NMR) spectroscopy and compared two groups of DLBCL patients treated with immunochemotherapy: i) refractory/early relapse (REF/REL; n=27) and ii) long-term progression-free (CURED; n=60). A supervised multivariate analysis showed a separation between the groups. Among discriminating metabolites higher in the REF/REL group were the amino acids lysine and arginine, the degradation product cadaverine and a compound in oxidative stress (2-hydroxybutyrate). In contrast, the amino acids aspartate, valine and ornithine, and a metabolite in the glutathione cycle, pyroglutamate, were higher in CURED patients. Together, our data indicate that NMR-based serum metabolomics can identify a signature for DLBCL patients with high-risk of failing immunochemotherapy, prompting for larger validating studies which could lead to more individualized treatment of this disease.
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| 46. |
- TIGERSTRÖM, ANNA KATARINA, 1976, et al.
(författare)
-
Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin.
- 2004
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:39, s. 12563-74
-
Tidskriftsartikel (refereegranskat)abstract
- Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications. This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures. The atomic displacement parameters describe the molecular movement at higher temperatures. The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry. Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein. Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability. The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution. These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin.
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| 47. |
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| 48. |
- Wallerstein, Johan, 1978, et al.
(författare)
-
Insights into mechanisms of MALT1 allostery from NMR and AlphaFold dynamic analyses
- 2024
-
Ingår i: COMMUNICATIONS BIOLOGY. - : NATURE PORTFOLIO. - 2399-3642. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) is an attractive target for the development of modulatory compounds in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained unexplored. We present here dynamic analyses of the apo MALT1 form along with the E549A mutation. This investigation used NMR 15N relaxation and NOE measurements between side-chain methyl groups. Our findings confirm that MALT1 exists as a monomer in solution, and demonstrate that the domains display semi-independent movements in relation to each other. Our dynamic study, covering multiple time scales, along with the assessment of conformational populations by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, put the side chain of residue W580 in an inward position, shedding light at potential mechanisms underlying the allosteric regulation of this enzyme. NMR relaxation and AlphaFold structural ensemble modelling of MALT1 reveals motions between its PCASP and Ig3 domains. This sheds light into the mechanisms of the protein's allosteric regulation.
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| 49. |
- Wallgren, Marcus, 1978-, et al.
(författare)
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Reconstitution of the Anti-Apoptotic Bcl-2 Protein into Lipid Membranes and Biophysical Evidence for Its Detergent-Driven Association with the Pro-Apoptotic Bax Protein
- 2013
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4
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Tidskriftsartikel (refereegranskat)abstract
- The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A(2) (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.
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| 50. |
- Yang, Jie, et al.
(författare)
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High-affinity recognition of the human C-reactive protein independent of phosphocholine
- 2017
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Ingår i: Organic & Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 15:21, s. 4644-4654
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Tidskriftsartikel (refereegranskat)abstract
- A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 mu M. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the mu M to nM range for which no small molecule ligands are known.
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