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Carlsson, Uno
Publications (10 of 54) Show all publications
Babu Moparthi, S., Carlsson, U., Vincentelli, R., Jonsson, B.-H., Hammarström, P. & Wenger, J. (2016). Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components. Scientific Reports, 6(28386)
Open this publication in new window or tab >>Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, no 28386Article in journal (Refereed) Published
Abstract [en]

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2016
National Category
Biophysics
Identifiers
urn:nbn:se:liu:diva-130280 (URN)10.1038/srep28386 (DOI)000378228700001 ()27328749 (PubMedID)
Note

Funding Agencies|European Commission [FP7-ICT-2011-7, ERC StG 278242]; Goran Gustafsson Foundation; Swedish Alzheimer Foundation

Available from: 2016-08-01 Created: 2016-07-28 Last updated: 2018-04-25
Babu Moparthi, S., Sjölander, D., Villebeck, L., Jonsson, B.-H., Hammarström, P. & Carlsson, U. (2014). Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL. Journal of chemical biology, 7(1), 1-15
Open this publication in new window or tab >>Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL
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2014 (English)In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 7, no 1, p. 1-15Article, review/survey (Refereed) Published
Abstract [en]

The commonly accepted dogma of the bacterial GroE chaperonin system entails protein folding mediated by cycles of several ATP-dependent sequential steps where GroEL interacts with the folding client protein. In contrast, we herein report GroES-mediated dynamic remodeling (expansion and compression) of two different protein substrates during folding: the endogenous substrate MreB and carbonic anhydrase (HCAII), a well-characterized protein folding model. GroES was also found to influence GroEL binding induced unfolding and compression of the client protein underlining the synergistic activity of both chaperonins, even in the absence of ATP. This previously unidentified activity by GroES should have important implications for understanding the chaperonin mechanism and cellular stress response. Our findings necessitate a revision of the GroEL/ES mechanism.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2014
Keywords
Carbonic anhydrase; Chaperone; FRET; Molten globule; MreB; Protein folding
National Category
Chemical Sciences Biological Sciences
Identifiers
urn:nbn:se:liu:diva-110534 (URN)10.1007/s12154-013-0106-5 (DOI)24386013 (PubMedID)2-s2.0-84891782616 (Scopus ID)
Available from: 2014-09-14 Created: 2014-09-12 Last updated: 2018-04-25
Karabencheva-Christova, T. G., Carlsson, U., Balali-Mood, K., Black, G. W. & Christov, C. Z. (2013). Conformational Effects on the Circular Dichroism of Human Carbonic Anhydrase II: A Multilevel Computational Study. PLoS ONE, 8(2)
Open this publication in new window or tab >>Conformational Effects on the Circular Dichroism of Human Carbonic Anhydrase II: A Multilevel Computational Study
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2Article in journal (Refereed) Published
Abstract [en]

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-92709 (URN)10.1371/journal.pone.0056874 (DOI)000316849500038 ()
Note

Funding Agencies|UK National Service for Computational Chemistry Software||UK National Supercomputer Service Hector||Marie Curie Fellowships (FP7 of EU)||

Available from: 2013-05-16 Created: 2013-05-16 Last updated: 2017-12-06
Carlsson, K., Persson, E., Østergaard, H., Lindgren, M., Carlsson, U. & Svensson, M. (2011). Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching. Biochemical and Biophysical Research Communications - BBRC, 413(4), 545-549
Open this publication in new window or tab >>Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, p. 545-549Article in journal (Refereed) Published
Abstract [en]

The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent  fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.

Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

Place, publisher, year, edition, pages
Elsevier, 2011
Keywords
Factor VIIa, Fluorescence quenching, Fluorescence resonance energy transfer, Tissue factor, Fluorescein, Rhodamine, Conformational dynamics
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-63686 (URN)10.1016/j.bbrc.2011.08.135 (DOI)000295912800010 ()
Note
Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation||Available from: 2010-12-30 Created: 2010-12-30 Last updated: 2017-12-11Bibliographically approved
Moparthi, S. B., Fristedt, R., Mishra, R., Almstedt, K., Karlsson, M., Hammarström, P. & Carlsson, U. (2010). Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates. Biochemistry, 49(6), 1137-1145
Open this publication in new window or tab >>Chaperone activity of Cyp18 through hydrophobic condensation that enables rescue of transient misfolded molten globule intermediates
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2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 6, p. 1137-1145Article in journal (Refereed) Published
Abstract [en]

The single-domain cyclophilin 18 (Cyp18) has long been known to function as a peptidyl-prolyl cis/trans isomerase (PPI) and was proposed by us to also function as a chaperone [Freskgård, P.-O., Bergenhem, N., Jonsson, B.-H., Svensson, M., and Carlsson, U. (1992) Science 258, 466−468]. Later several multidomain PPIs were demonstrated to work as both a peptidyl-prolyl cis/trans isomerase and a chaperone. However, the chaperone ability of Cyp18 has been debated. In this work, we add additional results that show that Cyp18 can both accelerate the rate of refolding and increase the yield of native protein during the folding reaction, i.e., function as both a folding catalyst and a chaperone. Refolding experiments were performed using severely destabilized mutants of human carbonic anhydrase II under conditions where the unfolding reaction is significant and a larger fraction of a more destabilized variant populates molten globule-like intermediates during refolding. A correlation of native state protein stability of the substrate protein versus Cyp18 chaperone activity was demonstrated. The induced correction of misfolded conformations by Cyp18 likely functions through rescue from misfolding of transient molten globule intermediates. ANS binding data suggest that the interaction by Cyp18 leads to an early stage condensation of accessible hydrophobic portions of the misfolding-prone protein substrate during folding. The opposite effect was observed for GroEL known as an unfoldase at early stages of refolding. The chaperone effect of Cyp18 was also demonstrated for citrate synthase, suggesting a general chaperone effect of this PPI.

Keywords
Chaperone, carbonic anhydrase, citrate synthase, peptidyl‐prolyl cis/trans isomerase, proline isomerase, cyclophilin
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51602 (URN)10.1021/bi901997q (DOI)20070121 (PubMedID)
Available from: 2009-11-09 Created: 2009-11-09 Last updated: 2018-04-25Bibliographically approved
Owenius, R., Jarl, A., Jonsson, B.-H., Carlsson, U. & Hammarström, P. (2010). GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study. Journal of chemical biology, 3(3), 127-39
Open this publication in new window or tab >>GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study
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2010 (English)In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 3, no 3, p. 127-39Article in journal (Refereed) Published
Abstract [en]

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-100534 (URN)10.1007/s12154-010-0038-2 (DOI)21479077 (PubMedID)
Note

At the time of the licentiate theses defence this article was submitted.

Available from: 2013-11-08 Created: 2013-11-08 Last updated: 2018-04-25
Moparthi, S. B., Hammarström, P. & Carlsson, U. (2009). A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding. Protein Science, 18(2), 475-479
Open this publication in new window or tab >>A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding
2009 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 18, no 2, p. 475-479Article in journal (Refereed) Published
Abstract [en]

The protein folding process is often in vitro rate-limited by slow cis-trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active-site located Arg 55 is not required for catalysis of the rate-limiting prolyl cis-trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis-trans proline isomerization in HCA II occurs in the active-site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding-assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.

Keywords
cis-trans proline isomerization, cyclophilin 18, prolyl isomerases, human carbonic anhydrase II
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-17882 (URN)10.1002/pro.28 (DOI)
Available from: 2009-04-25 Created: 2009-04-24 Last updated: 2018-04-25Bibliographically approved
Almstedt, K., Rafstedt, T., Supuran, C. T., Carlsson, U. & Hammarström, P. (2009). Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease. Biochemistry, 48(23), 5358-5364
Open this publication in new window or tab >>Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 23, p. 5358-5364Article in journal (Refereed) Published
Abstract [en]

Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-19548 (URN)10.1021/bi900128e (DOI)
Note
On the day of the defence date tha status of this articel was In Manuscript.Available from: 2009-06-29 Created: 2009-06-26 Last updated: 2018-04-25Bibliographically approved
Mishra, R., Olofsson, L., Karlsson, M., Carlsson, U., Nicholls, I. A. & Hammarström, P. (2008). A conformationally isoformic thermophilic protein with high kinetic unfolding barriers. Cellular and Molecular Life Sciences (CMLS), 65(5), 827-839
Open this publication in new window or tab >>A conformationally isoformic thermophilic protein with high kinetic unfolding barriers
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2008 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, no 5, p. 827-839Article in journal (Refereed) Published
Abstract [en]

The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH. © 2008 Birkhaueser.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-43773 (URN)10.1007/s00018-008-7517-4 (DOI)74765 (Local ID)74765 (Archive number)74765 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2018-04-25
Almstedt, K., Mårtensson, L.-G., Carlsson, U. & Hammarström, P. (2008). Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.. Biochemistry, 47(5), 1288-1298
Open this publication in new window or tab >>Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 5, p. 1288-1298Article in journal (Refereed) Published
Abstract [en]

Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order:  wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion:  (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.

Place, publisher, year, edition, pages
Washington: ACS, 2008
Keywords
mutant mapping, carbonic anhydrase, misfolding, loss-of-function, molten globule
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-21056 (URN)10.1021/bi701720p (DOI)
Available from: 2009-09-28 Created: 2009-09-28 Last updated: 2018-04-25Bibliographically approved
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