| 11. |
- Hammarström, Per, 1972-, et al.
(author)
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Is the unfolded state the Rosetta Stone of the protein folding problem?
- 2000
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 276:2, s. 393-398
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Journal article (peer-reviewed)abstract
- Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in hydrophobic regions clustered together in the protein core. These stable structures can work as seeds in the folding process. In addition, local preferences for secondary structure in the form of turns for ▀-sheet initiation and helical turns for a-helix formation can guide the folding reaction. In this respect the unfolded states, studied at increasing structural resolution, can be the Rosetta Stone of the protein folding problem. (C) 2000 Academic Press.
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| 12. |
- Hammarström, Per, et al.
(author)
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Protein compactness measured by fluorescence resonance energy transfer - Human carbonic anhydrase II Is considerably expanded by the interaction of GroEL
- 2001
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:24, s. 21765-21775
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Journal article (peer-reviewed)abstract
- Nine single-cysteine mutants were labeled with 5-(2-iodoacetylaminoethylamino)naphthalene-1-sulfonic acid, an efficient acceptor of Trp fluorescence in fluorescence resonance energy transfer. The ratio between the fluorescence intensity of the 5-(2-acetylaminoethylamino)naphthalene-1-sulfonic acid (AEDANS) moiety excited at 295 nm (Trp absorption) and 350 nn (direct AEDANS absorption) was used to estimate the average distances between the seven Trp residues in human carbonic anhydrase II (HCA II) and the AEDANS label, Guanidine HCl denaturation of the HCA II variants was also performed to obtain a curve that reflected the compactness of the protein at various stages of the unfolding, which could serve as a scale of the expansion of the protein. This approach was developed in this study and was used to estimate the compactness of HCA II during heat denaturation and interaction with GroEL, It was shown that thermally induced unfolding of HCA II proceeded only to the molten globule state. Reaching this state was sufficient to allow HCA II to bind to GroEL, and the volume of the molten globule intermediate increased similar to2.2-fold compared with that of the native state. GroEL-bound HCA II expands to a volume three to four times that of the native state (to similar to 117,000 Angstrom (3)), which correlates well with a stretched and loosened-up HCA II molecule in an enlarged GroEL cavity, Recently, we found that HCA II binding causes such an inflation of the GroEL molecule, and this probably represents the mechanism by which GroEL actively stretches its protein substrates apart (Hammarstrom, P., Persson, M., Owenius, R., Lindgren, M., and Carlsson, U. (2000) J. Biol. Chem. 275, 22832-22838), thereby facilitating rearrangement of misfolded structure.
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| 13. |
- Hammarström, Per, 1972-, et al.
(author)
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Protein substrate binding induces conformational changes in the chaperonin GroEL : A suggested mechanism for unfoldase activity
- 2000
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:30, s. 22832-22838
-
Journal article (peer-reviewed)abstract
- Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxy1-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.
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| 14. |
- Hammarström, Per, et al.
(author)
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Sequence-dependent denaturation energetics : A major determinant in amyloid disease diversity
- 2002
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 99:SUPPL. 4, s. 16427-16432
-
Conference paper (other academic/artistic)abstract
- Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease, however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance-presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.
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| 15. |
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| 16. |
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| 17. |
- Hammarström, Ulf, et al.
(author)
-
Validering och utvärdering av AUT/TRANSYT
- 2000
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Reports (pop. science, debate, etc.)abstract
- AUT är ett system för optimal styrning av samordnadetrafiksignaler. Systemet har utvecklats föratt kunna installeras i befintliga signalsystem utankrav på omfattande investeringar. AUT innehållerde modeller för effektberäkning som krävs förutvärdering. Sådana effektdata redovisas normaltav AUT tidplan för tidplan och dag för dag.En meningsfull utvärdering förutsätter validering.Validering har genomförts både genom att urvideoupptagningar genomföra detaljerade analyser pålänknivå och genom att använda en mätbil.Ur videon utvärderades: länkflöden; matande delflöden;mättnadsflöden; stopp och fördröjning.Mätbilen användes för registrering av körförlopp ochbränsleförbrukning. Efter justering för påvisadesystematiska avvikelser avseende stopp och fördröjninghar AUT bedömts, att inom försöksområdet,kunna ge representativa eller åtminstone inteöverskattande beskrivningar av: stopp;fördröjning; olika avgaser och trafikantkostnader.Systemet ger initialt en betydande reduktion av bådeavgasutsläpp och övriga trafikantkostnader.Initialt har i försöksområdets korsningaravgasutsläppen reducerats med minst 10%. Den störstareduktionen gäller kväveoxider, vilka under ett andradriftår skulle reduceras med ca 16%.Kostnadsreduktionen under andra året skulle bli drygten miljon kr/korsning och år, vilket kanjämföras med en investering av maximalt 100 000 kr perkorsning. Genom att AUT kan arbeta medbefintlig signalutrustning kan befintliga påvisatgynnsamma signalfunktioner för trafiksäkerhetbibehållas.
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| 18. |
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| 19. |
- Huber, M., et al.
(author)
-
Phase memory relaxation times of spin labels in human carbonic anhydrase II : Pulsed EPR to determine spin label location
- 2001
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In: Biophysical Chemistry. - 0301-4622 .- 1873-4200. ; 94:3, s. 245-256
-
Journal article (peer-reviewed)abstract
- Phase memory relaxation times (TM or T2) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters TM and x. TM values of seven positions are between 1.6 ╡s for the most buried residue (L79C) and 4.7 ╡s for a residue at the protein surface (W245C). In deuteriated buffer, longer TM are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose TM as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism. ⌐ 2001 Elsevier Science B.V. All rights reserved.
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| 20. |
- Knudsen, J.F., et al.
(author)
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The cyclooxygenase-2 inhibitor celecoxib is a potent inhibitor of human carbonic anhydrase II
- 2004
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In: Inflammation. - : Springer Science and Business Media LLC. - 0360-3997 .- 1573-2576. ; 28:5, s. 285-290
-
Journal article (peer-reviewed)abstract
- Cyclooxygenase-2 (COX-2) is up-regulated in stromal and inflammatory cells. The inducible COX-2 isoform is expressed during inflammation, in some cancers, and in brain tissue after global and focal ischemia. Tissue acidosis is a dominant factor in inflammation, and contributes to pain and hyperalgesia. Recently, compelling epidemiological and clinical evidence has documented the COX-independent effects of some COX-2 inhibitors (i.e., celecoxib, valdecoxib, and rofecoxib), among these effects are carbonic anhydrase (CA) inhibition. Carbonic anhydrases are zinc metalloenzymes expressed in various cell types, including those of the kidney, where they act as general acid-base catalysts. The kidneys are also known to express the highest concentration of COX-2 messenger ribonucleic acid. Celecoxib, like the prototypic CA inhibitor acetazolamide, is structurally characterized by an unsubstituted sulfonamide moiety. In the present study, we report that celecoxib exhibits the characteristics of a potent CA inhibitor, showing inhibitory human carbonic anhydrase II (hCAII) activity in the nanomolar range. Valdecoxib was relatively less potent. Rofecoxib, which lacks the unsubstituted sulfonamide moiety characteristic of CA inhibitors, showed no significant hCAII inhibitory activity. The current study corroborates our earlier report of structure-activity relationships as predictors of such metabolic events as hyperchloremia, acidosis, and changes in calcium and phosphate disposition, and clinical manifestations associated with CA inhibition reported with celecoxib. These data showing inhibition of hCAII by the unsubstituted sulfonamides celecoxib and valdecoxib, but not by rofecoxib, may have important implications for the elucidation of the mechanisms of action as well as the side effects associated with COX-2 inhibitors. © 2004 Springer Science+Business Media, Inc.
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