| 1. |
- Almstedt, Karin, 1980-, et al.
(author)
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Unfolding a folding disease: folding, misfolding and aggregation of the marble brain syndrome-associated mutant H107Y of human carbonic anhydrase II
- 2004
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In: Journal of Molecular Biology. - Oxford : Elsevier. - 0022-2836 .- 1089-8638. ; 342:2, s. 619-633
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Journal article (peer-reviewed)abstract
- Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to mutations. The disease marble brain syndrome (MBS), known also as carbonic anhydrase II deficiency syndrome (CADS), can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. One mutation associated with MBS entails the His107Tyr substitution. Here, we demonstrate that this mutation is a remarkably destabilizing folding mutation. The loss-of-function is clearly a folding defect, since the mutant shows 64% of CO2 hydration activity compared to that of the wild-type at low temperature where the mutant is folded. On the contrary, its stability towards thermal and guanidine hydrochloride (GuHCl) denaturation is highly compromised. Using activity assays, CD, fluorescence, NMR, cross-linking, aggregation measurements and molecular modeling, we have mapped the properties of this remarkable mutant. Loss of enzymatic activity had a midpoint temperature of denaturation (Tm) of 16 °C for the mutant compared to 55 °C for the wild-type protein. GuHCl-denaturation (at 4 °C) showed that the native state of the mutant was destabilized by 9.2 kcal/mol. The mutant unfolds through at least two equilibrium intermediates; one novel intermediate that we have termed the molten globule light state and, after further denaturation, the classical molten globule state is populated. Under physiological conditions (neutral pH; 37 °C), the His107Tyr mutant will populate the molten globule light state, likely due to novel interactions between Tyr107 and the surroundings of the critical residue Ser29 that destabilize the native conformation. This intermediate binds the hydrophobic dye 8-anilino-1-naphthalene sulfonic acid (ANS) but not as strong as the molten globule state, and near-UV CD reveals the presence of significant tertiary structure. Notably, this intermediate is not as prone to aggregation as the classical molten globule. As a proof of concept for an intervention strategy with small molecules, we showed that binding of the CA inhibitor acetazolamide increases the stability of the native state of the mutant by 2.9 kcal/mol in accordance with its strong affinity. Acetazolamide shifts the Tm to 34 °C that protects from misfolding and will enable a substantial fraction of the enzyme pool to survive physiological conditions.
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| 2. |
- Hammarström, Martin, et al.
(author)
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Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
- 2002
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In: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 11:2, s. 313-321
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Journal article (peer-reviewed)abstract
- A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
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| 3. |
- Malmström, Jonas, et al.
(author)
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The antioxidant profile of 2,3-dihydrobenzo[b]furan-5-ol and its 1-thio, 1-seleno, and 1-telluro analogues
- 2001
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 123:15, s. 3434-3440
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Journal article (peer-reviewed)abstract
- A novel synthesis of 2,3-dihydrobenzo[b]thiophene-S-of based on intramolecular homolytic substitution on sulfur was reported. The ''antioxidant profile" of the series of 2,3-dihydrobenzo[b] furan-5-ol (2a) its l-thio (2b), I-seleno (2c) and 1-telluro (2d) analogues was determined by studies of redox properties, the capacity to inhibit stimulated lipid peroxidation, the reactivity toward tert-butoxyl radicals, the ability to catalyze decomposition of hydrogen peroxide in the presence of glutathione, and the inhibiting effect on stimulated peroxidation in liver microsomes. The one-electron reduction potentials of the aroxyl radicals corresponding to compounds 2a-2d, E degrees (ArO./ArO-) were 0.49, 0.49, 0.49, and 0.52 V vs NHE, respectively, as determined by pulse radiolysis. With increasing chalcogen substitution the compounds become slightly more acidic (pK(a) 10.6, 10.0, 9.9, and 9.5, respectively, for compounds 2a-2d). By using Hess' law, the homolytic O-H bond dissociation enthalpies of compounds 2a-2d (340, 337, 336, and 337 kJ mol(-1), respectively) were calculated. The reduction potentials for the proton coupled oxidation of compounds 2a-2d (ArOH --> ArO. + H+) as determined by cyclic voltammetry in acetonitrile were 1.35 (irreversible), 1.35 (quasireversible) 1.13 (reversible), and 0.74 (reversible) V vs NHE, respectively. As judged by the inhibited rates of peroxidation, R-inh, in a water/chlorobenzene two-phase lipid peroxidation system containing N-acetylcysteine as a thiol-reducing agent in the aqueous phase, the antioxidant capacity increases (2d > 2c = 2b > 2a) as one traverses the group of chalcogens. Whereas the times of inhibition, T-inh were slightly reduced for the oxygen (2a) and sulfur (2b) derivatives in the absence of the thiol-reducing agent, they were drastically reduced for the selenium (2c) and tellurium (2d) derivatives. This seems to indicate that the organochalcogen compounds are continuously regenerated at the lipid aqueous interphase and that regeneration is much more efficient for the selenium and tellurium compounds. The absolute rate constants for the oxidation of compounds 2a-2b by the tert-butoxyl radical in acetonitrile/di-tert-butyl peroxide (10/1) were the same-2 x 10(8) M-1 s(-1). Whereas the oxygen, sulfur, and selenium derivatives 2a-2c were essentially void of any glutathione peroxidase-like activity, the organotellurium compound 2d accelerated the initial reduction of hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide in the presence of glutathione 100, 333, and 213 times, respectively, as compared to the spontaneous reaction. Compounds 2a-2d were assessed for their capacity to inhibit lipid peroxidation in liver microsomes stimulated by Fe(II)/ADP/ascorbate. Whereas the oxygen, sulfur, and selenium compounds showed weak inhibiting activity (IC50 values of similar to 250, 25, and 13 muM, respectively), the organotellurium compound 2d was a potent inhibitor with an IC50 value of 0.13 muM.
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| 5. |
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| 6. |
- Sjödin, Martin, 1974-
(author)
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Regulation of Proton Coupled Electron Transfer from Amino Acids in Artificial Model Systems: A Mechanistic Study
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Amino acid radicals are key redox intermediates in several natural enzymes including Cytochrome c peroxidase, DNA photolyase, ribonucletide reductase, cytochrome c oxidase and photosystem II. Electron transfer from amino acids is often coupled to deprotonation and this thesis concerns the coupling of electron transfer from tyrosine and tryptophan to trisbipyridineruthenium(III) with deprotonation in model complexes. Specifically the mechanisms for these proton coupled electron transfer reactions have been studied and the controlling parameters have been identified, the possible mechanisms being stepwise electron transfer followed by deprotonation and deprotonation followed by electron transfer or concerted electron transfer/deprotonation.Proton coupled electron transfer reactions have been studied using nano-second flash photolysis in water solution and the effect of pH, temperature, reaction driving force, deuteration and nature of the amino acid has been determined. I have shown that the rate constant for the concerted reaction depends intrinsically on the mixing entropy of the released proton and that the pH-dependence can be used as an experimental tool for mechanistic discrimination. Moreover I have shown that the concerted reaction inherently has a high reorganisation energy due to the coupling of the electron motion with deprotonation. Hydrogen bonding to the transferring proton however significantly reduces this reorganisation energy. The concerted reaction also has a relatively high driving force counteracting the high reorganisation energy in the competition between the concerted reaction and the stepwise electron transfer first reaction. The relative importance of the high reorganisation energy and the high driving force for the concerted reaction determines the mechanistic outcome of the reaction, the stepwise reaction being favoured by high over-all driving forces and the concerted reaction by high pH.By comparing my results from model complexes with tyrosineZ oxidation in photosystem II, I give strong evidence for a concerted electron transfer/deprotonation mechanism.
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| 7. |
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| 8. |
- Woestenenk, Esmeralda A., et al.
(author)
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His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors.
- 2004
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In: Journal of structural and functional genomics. - 1345-711X .- 1570-0267. ; 5:3, s. 217-29
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Journal article (peer-reviewed)abstract
- We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.
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| 9. |
- Woestenenk, Esmeeralda A., et al.
(author)
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Screening methods to determine biophysical properties of proteins in structural genomics
- 2003
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In: Analytical biochemistry. - 0003-2697. ; 318:1, s. 71-79
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Journal article (peer-reviewed)abstract
- We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarström et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.
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