| 1. |
- Almstedt, Karin, 1980-, et al.
(författare)
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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
-
Ingår i: Journal of Molecular Biology. - Oxford : Elsevier. - 0022-2836 .- 1089-8638. ; 342:2, s. 619-633
-
Tidskriftsartikel (refereegranskat)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. |
- Zhang, Wei
(författare)
-
Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles : A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.
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| 3. |
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| 4. |
- Ahl, Ing-Marie, 1974-
(författare)
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Protein Engineering of Extracellular Superoxide Dismutase : Characterization of Binding to Heparin and Cellular Surfaces
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accumulating evidence indicates that oxygen free radicals are involved in many diseases and pathological conditions, such as aging, inflammation, reperfusion damage of ischemic tissue and various cardiovascular diseases. Extracellular superoxide dismutase (ECSOD) thus plays a major role in the maintenance of cells by providing protection against these toxic substances in the extracellular space. Various animal studies have shown that ECSOD has the ability to protect against many of these disorders, and interest has therefore evolved in the potential therapeutic use of the enzyme.However, despite strenuous efforts, large-scale production of the enzyme has not been achieved. To overcome this problem, a mimic of the enzyme, PseudoECSOD, has previouslybeen constructed. This chimera is easy to produce in large amounts and has all the structural, enzymatic and heparin-binding characteristics of ECSOD, making it a potential substitute for ECSOD in therapeutic situations. However, the copper content of PseudoECSOD has been shown to be rather low, and since the copper ion is very important for the catalytic function of the enzyme, a production system that utilizes a copper chaperone for proper insertion of copper into the active site of the enzyme was constructed. The results show that the copper content of PseudoECSOD produced by this system is close to 100 %.In order to use PseudoECSOD therapeutically, further investigations of its binding capability and protective properties are needed. Therefore, the binding of ECSOD and PseudoECSOD to heparin was investigated using isothermal titration calorimetry. The results show that although some purely ionic interactions are important for the binding between ECSOD and heparin, there is also a substantial contribution from non-ionic interactions. The investigation also showed that the C-terminal domain is the only part of ECSOD that contributes to productive binding, and that the binding of PseudoECSOD and ECSOD to heparin is similar.In addition, analysis of mutant proteins strongly indicated that the amino acids R210, K211 and R214 are important for optimal binding of ECSOD to heparin, accounting for about 30 % of the total binding energy. The structural placement of these amino acids in an α-helix also confirms the hypothesis postulated by Margalit et al., that a common structural motif for heparin-binding proteins may be two positively charged amino acids at a distance of approximately 20 Å in the 3D-structure, facing opposite directions of a α-helix. The importance of these residues was also confirmed by analysis of a phage display library of the C-terminal domain of ECSOD.The binding of PseudoECSOD to heparan sulfate on cell surfaces of two different cell types, HepG2 and endothelial cells, was also investigated. The results clearly show that PseudoECSOD binds to these cells in a very similar manner to ECSOD. To investigate the protective properties of PseudoECSOD against ischemia-reperfusion injuries, an isolated rabbit heart model was used. The results indicate that the enzyme has a protective effect. However, more experiments using the rabbit heart and other animal models are needed to identify the optimal dose for protective purposes. The protective properties of PseudoECSOD in human tissue should also be thoroughly investigated.In summary, the findings in these studies, together with earlier results showing the close resemblance of PseudoECSOD to ECSOD in structural, enzymatic and heparin-binding properties, further support the proposition that PseudoECSOD may be a good substitute for ECSOD to use in therapeutic interventions.
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| 5. |
- Ahl, Ing-Marie, et al.
(författare)
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Thermodynamic Characterization of the Interaction between the C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin by Isothermal Titration Calorimetry
- 2009
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Ingår i: BIOCHEMISTRY. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:41, s. 9932-9940
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular superoxide dismutase (ECSOD) interacts with heparin through its C-terminal domain. In this study we used isothermal titration calorimetry (ITC) to get detailed thermodynamic information about the interaction. We have shown that the interaction between ECSOD and intestinal mucosal heparin (M-w 6000-30000 Da) is exothermic and driven by enthalpy at physiological salt concentration. However, the contribution from entropy is favorable for binding or small isolated heparin fragments. By studying different size-defined heparin fragments, we also concluded that it hexasaccharide moiety is sufficient for strong binding to ECSOD. The binding involves proton transfer from the buffer to the ECSOD-heparin complex, and the results indicate that the number of ionic interactions made between ECSOD and heparin upon binding varies from three to five for heparin and an octasaccharide fragment, respectively. Surprisingly and despite the many charges found oil both the protein and the polysaccharide, our results indicate that the nonionic contribution to the binding is large. From the temperature dependence we have calculated the constant pressure heat capacity change (Delta C-p) of the interaction to -644 J K-1 mol(-1) and -306 J K-1 mol(-1) for heparin and all octasaccharide, respectively
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| 6. |
- Ahlner, Alexandra, 1984-
(författare)
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Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins.To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals.To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism.
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| 7. |
- Ahlner, Alexandra, et al.
(författare)
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PINT: a software for integration of peak volumes and extraction of relaxation rates
- 2013
-
Ingår i: Journal of Biomolecular NMR. - : Springer Verlag (Germany). - 0925-2738 .- 1573-5001. ; 56:3, s. 191-202
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Tidskriftsartikel (refereegranskat)abstract
- We present the software Peak INTegration (PINT), designed to perform integration of peaks in NMR spectra. The program is very simple to run, yet powerful enough to handle complicated spectra. Peaks are integrated by fitting predefined line shapes to experimental data and the fitting can be customized to deal with, for instance, heavily overlapped peaks. The results can be inspected visually, which facilitates systematic optimization of the line shape fitting. Finally, integrated peak volumes can be used to extract parameters such as relaxation rates and information about low populated states. The utility of PINT is demonstrated by applications to the 59 residue SH3 domain of the yeast protein Abp1p and the 289 residue kinase domain of murine EphB2.
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| 8. |
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| 9. |
- Andersson, Theresa, et al.
(författare)
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The binding of human Carbonic Anhydrase II by functionalized folded polypeptide receptors
- 2005
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Ingår i: Chemistry and Biology. - : Elsevier BV. - 1074-5521 .- 1879-1301. ; 12:11, s. 1245-1252
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Tidskriftsartikel (refereegranskat)abstract
- Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 Å deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.
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| 10. |
- Andrasko, Jan, et al.
(författare)
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Analysis of Explosives by GC-UV
- 2017
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Ingår i: Journal of Forensic Sciences. - : WILEY. - 0022-1198 .- 1556-4029. ; 62:4, s. 1022-1027
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Tidskriftsartikel (refereegranskat)abstract
- A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178-330 nm. The gas-phase UV spectra of 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.
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| 11. |
- Aronsson, Göran, et al.
(författare)
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Remarkably slow folding of a small protein.
- 1997
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 411:2-3, s. 359-364
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Tidskriftsartikel (refereegranskat)abstract
- Equilibrium denaturation of the 72 amino acid alpha/beta-protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two-state model in which only the native and unfolded states are populated. A cis-trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow- and a fast folding population. With a rate constant of 1.2 s(-1) for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.
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| 12. |
- Babu Moparthi, Satish, et al.
(författare)
-
Differential conformational modulations of MreB folding upon interactions with GroEL/ES and TRiC chaperonin components
- 2016
-
Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 6:28386
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 13. |
- Babu Moparthi, Satish, et al.
(författare)
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Transient conformational remodeling of folding proteins by GroES - Individually and in concert with GroEL
- 2014
-
Ingår i: Journal of chemical biology. - : Springer Berlin/Heidelberg. - 1864-6158 .- 1864-6166. ; 7:1, s. 1-15
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 14. |
- Basaiawmoit, R V, et al.
(författare)
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SAXS Models of TGFBIp Reveal a Trimeric Structure and Show That the Overall Shape Is Not Affected by the Arg124His Mutation
- 2011
-
Ingår i: JOURNAL OF MOLECULAR BIOLOGY. - : Elsevier Science B.V., Amsterdam. - 0022-2836. ; 408:3, s. 503-513
-
Tidskriftsartikel (refereegranskat)abstract
- Human transforming growth factor beta induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple "beads-on-a-string" arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.
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| 15. |
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| 16. |
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| 17. |
- Berg, Therese, et al.
(författare)
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Regular Inference for State Machines Using Domains with Equality Tests
- 2008
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Ingår i: Fundamental Approaches to Software Engineering. - Berlin : Springer-Verlag. - 9783540787426 ; , s. 317-331
-
Konferensbidrag (refereegranskat)abstract
- Existing algorithms for regular inference (aka automata learning) allows to infer a finite state machine by observing the output that the machine produces in response to a selected sequence of input strings. We generalize regular inference techniques to infer a class of state machines with an infinite state space. We consider Mealy machines extended with state variables that can assume values from a potentially unbounded domain. These values can be passed as parameters in input and output symbols, and can be used in tests for equality between state variables and/or message parameters. This is to our knowledge the first extension of regular inference to infinite-state systems. We intend to use these techniques to generate models of communication protocols from observations of their input-output behavior. Such protocols often have parameters that represent node adresses, connection identifiers, etc. that have a large domain, and on which test for equality is the only meaningful operation. Our extension consists of two phases. In the first phase we apply an existing inference technique for finite-state Mealy machines to generate a model for the case that the values are taken from a small data domain. In the second phase we transform this finite-state Mealy machine into an infinite-state Mealy machine by folding it into a compact symbolic form.
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| 18. |
- Berg, Therese, et al.
(författare)
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Regular Inference for State Machines with Parameters.
- 2006
-
Ingår i: Fundamental Approaches to Software Engineering, 9th International Conference, FASE 2006. - 3540330933 ; , s. 107-121
-
Konferensbidrag (refereegranskat)abstract
- We present experiences from a case study where a model-based approach to black-box testing is applied to verify that a Wireless Application Protocol (WAP) gateway conforms to its specification.The WAP gateway is developed by Ericsson and used in mobile telephone networks to connect mobile phones with the Internet. We focus on testing the software implementing the session (WSP) and transaction (WTP) layers of the WAP protocol. These layers, and their surrounding environment, are described as a network of timed automata. To model the many sequence numbers (from a large domain) used in the protocol, we introduce an abstraction technique. We believe the suggested abstractiontechnique will prove useful to model and analyse other similar protocols with sequence numbers, in particular in the context of model-based testing.A complete test bed is presented, which includes generation and execution of test cases. It takes as input a model and a coverage criterion expressed as an observer, and returns a verdict for each test case. The test bed includes existingtools from Ericsson for test-case execution. To generate test suites, we use our own tool CoVer --- a new test-case generation tool based on the real-time model-checker Uppaal.
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| 19. |
- Berg, Therese, et al.
(författare)
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Regular Inference for State Machines with Parameters
- 2006
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Ingår i: Fundamental approaches to software engineering. - Berlin : Springer. - 3540330933 ; , s. 107-121
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Techniques for inferring a regular language, in the form of a finite automaton, from a sufficiently large sample of accepted and nonaccepted input words, have been employed to construct models of software and hardware systems, for use, e.g., in test case generation. We intend to adapt these techniques to construct state machine models of entities of communication protocols. The alphabet of such state machines can be very large, since a symbol typically consists of a protocol data unit type with a number of parameters, each of which can assume many values. In typical algorithms for regular inference, the number of needed input words grows with the size of the alphabet and the size of the minimal DFA accepting the language. We therefore modify such an algorithm (Angluin's algorithm) so that its complexity grows not with the size of the alphabet, but only with the size of a certain symbolic representation of the DFA. The main new idea is to infer, for each state, a partitioning of input symbols into equivalence classes, under the hypothesis that all input symbols in an equivalence class have the same effect on the state machine. Whenever such a hypothesis is disproved, equivalence classes are refined. We show that our modification retains the good properties of Angluin's original algorithm, but that its complexity grows with the size of our symbolic DFA representation rather than with the size of the alphabet. We have implemented the algorithm; experiments on synthesized examples are consistent with these complexity results.
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| 20. |
- Berglund, Anders, et al.
(författare)
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The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data
- 2005
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Ingår i: Journal of magnetic resonance. - San Diego : Academic Press. - 1090-7807 .- 1096-0856. ; 172:1, s. 24-30
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Tidskriftsartikel (refereegranskat)abstract
- A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NNIR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NNIR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis.
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| 21. |
- Bivall Persson, Petter, 1979-, et al.
(författare)
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Designing and Evaluating a Haptic System for Biomolecular Education
- 2007
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Ingår i: IEEE Virtual Reality Conference, 2007. VR '07.. - Piscataway, NJ, USA : IEEE. - 1424409063 ; , s. 171-178
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Konferensbidrag (refereegranskat)abstract
- In this paper we present an in situ evaluation of a haptic system, with a representative test population, we aim to determine what, if any, benefit haptics can have in a biomolecular education context. We have developed a haptic application for conveying concepts of molecular interactions, specifically in protein-ligand docking. Utilizing a semi-immersive environment with stereo graphics, users are able to manipulate the ligand and feel its interactions in the docking process. The evaluation used cognitive knowledge tests and interviews focused on learning gains. Compared with using time efficiency as the single quality measure this gives a better indication of a system's applicability in an educational environment. Surveys were used to gather opinions and suggestions for improvements. Students do gain from using the application in the learning process but the learning appears to be independent of the addition of haptic feedback. However the addition of force feedback did decrease time requirements and improved the students understanding of the docking process in terms of the forces involved, as is apparent from the students' descriptions of the experience. The students also indicated a number of features which could be improved in future development.
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| 22. |
- Bivall Persson, Petter, 1979-, et al.
(författare)
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Evaluating the Effectiveness of Haptic Visualization in Biomolecular Education - Feeling Molecular Specificity in a Docking Task
- 2006
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Ingår i: 12th IOSTE Symposium. - : Universiti Science Malaysia. - 9832700396 ; , s. 745-752
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Konferensbidrag (refereegranskat)abstract
- Within the molecular life sciences extensive use is made of visual representations, ranging from sketches to advanced computer graphics, often used to convey abstract knowledge that is difficult for the student to grasp. This work evaluates a new visual and haptic (tactile/kinetic) tool for protein docking in an in situ learning situation by combining qualitative and quantitative methods, performing tests and interviews with students; all aiming at a proper inclusion of visualization tools into biomolecular education. Preliminary results indicate time gains, strong positive affective responses and learning gains from the tasks, however the influence of haptics needs further investigation.
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| 23. |
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| 24. |
- Brorsson, Ann-Christin, et al.
(författare)
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GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern
- 2006
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 357:5, s. 1634-1646
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Tidskriftsartikel (refereegranskat)abstract
- We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (ΔGHX) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions. © 2006 Elsevier Ltd. All rights reserved.
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| 25. |
- Brorsson, Ann-Christin, 1969-
(författare)
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The Folding Energy Landscape of MerP
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4.The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size.The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O.In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl.Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.
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| 26. |
- Brorsson, Ann-Christin, et al.
(författare)
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The “Two-State folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange
- 2004
-
Ingår i: Journal of Molecular Biology. - London : Academic Press. - 0022-2836 .- 1089-8638. ; 340:2, s. 333-344
-
Tidskriftsartikel (refereegranskat)abstract
- We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55 degrees C. The temperature dependence of the hydrogen exchange has allowed us to determine DeltaG, DeltaH and DeltaC(p) values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP.
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| 27. |
- Byström, Roberth, 1971-
(författare)
-
SOD1´s Law : An Investigation of ALS Provoking Properties in SOD1
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.
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| 28. |
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| 29. |
- Chilkova, O., et al.
(författare)
-
The quaternary structure of DNA polymerase e from Saccharomyces cerevisiae
- 2003
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278:16, s. 14082-14086
-
Tidskriftsartikel (refereegranskat)abstract
- DNA polymerase e (Pol e) trom Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol e have been cumbersome due to protease sensitivity and the limited amounts of Pol e in cells. We have developed a protocol for overexpression and purification of Pol e from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol e was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 Å), a molecular mass for Pol e of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol e. Thus, both DNA polymerase d and Pol e are purified as monomeric complexes, in agreement with accumulating evidence that Pol d and Pol e are located on opposite strands of the eukaryotic replication fork.
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| 30. |
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| 31. |
- Chirica, Laura C., et al.
(författare)
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Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori
- 2002
-
Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - 1570-9639 .- 1878-1454. ; 1601:2, s. 192-199
-
Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (α and β), catalyzing the reversible hydration of CO2. Presumably, the high CO2 requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO2 concentration in the investigated range (0.1–10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the α-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the β-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the β-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The α-enzyme was found attached to the surface of the bacterium.
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| 32. |
- Gaulton, Kyle J, et al.
(författare)
-
Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.
- 2015
-
Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1546-1718 .- 1061-4036. ; 47:12, s. 1415-1415
-
Tidskriftsartikel (refereegranskat)abstract
- We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.
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| 33. |
- Hammarström, Per, 1972-, et al.
(författare)
-
Protein denaturation and the denatured state
- 2005
-
Ingår i: Encyclopedia of Life Sciences. - : Wiley-Blackwell.
-
Bokkapitel (populärvet., debatt m.m.)abstract
- Protein denaturation experiments are routinely used to determine protein stability and to elucidate structural and dynamic effects of mutations, cofactors and ligands. Denatured states of proteins have gained wide interest in recent years owing to their fundamental importance in a wide variety of phenomena such as deciphering the protein folding problem and the molecular understanding of many diseases.
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| 34. |
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| 35. |
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| 36. |
- Helmfors, Linda, 1983-
(författare)
-
Understanding the dual nature of lysozyme: part villain – part hero : A Drosophila melanogaster model of lysozyme amyloidosis
- 2014
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid proteins are a distinct class of proteins that can misfold into β-sheet rich structures that later mature to form the characteristic species known as amyloid fibrils, and accumulate in tissues in the human body. The misfolding event is often caused by mutations (or outer factors such as changes in pH) that destabilize the native protein structure. The mature amyloid fibrils were initially believed to be associated with diseases connected to protein misfolding such as Alzheimer’s disease (AD), Parkinson’s disease, transthyretin amyloidosis and lysozyme amyloidosis. However, now it is known that many different factors are involved in these diseases such as failure in protein clearance, lysosomal dysfunction and formation of intermediate misfolded protein species, which possess cytotoxic properties, preceding the formation of mature fibrils.In this thesis the amyloidogenic protein lysozyme has been examined in vivo by using Drosophila melanogaster (fruit fly) as a model organism. The effects of over-expressing human lysozyme and amyloidogenic variants in Drosophila have been investigated both in the absence and presence of the serum amyloid P component (SAP), a protein known to interact with amyloid species. In addition, the role of lysozyme in AD has been investigated by co-expressing human lysozyme and amyloid β in Drosophila.The lysozyme protein is an enzyme naturally found in bodily fluids such as tears, breast milk and saliva. It is engaged in the body’s defense and acts by hydrolyzing the cell wall of invading bacteria. Certain disease-associated point mutations in the gene encoding lysozyme destabilize the protein and cause it to misfold which results in systemic amyloidosis. To investigate the in vivo misfolding behavior of lysozyme we developed and established a Drosophila model of lysozyme amyloidosis. SAP is commonly found attached to amyloid deposits in the body; however, the role of SAP in amyloid diseases is unknown. To investigate the effect of SAP in lysozyme misfolding, these two proteins were co-expressed in Drosophila.The amyloid β peptide is involved in AD, building up the plaques found in AD patient brains. These plaques trigger neuroinflammation and since lysozyme is upregulated during various inflammation conditions, a possible role of lysozyme in AD was investigated by overexpressing lysozyme in a Drosophila model of AD. Interaction between lysozyme and the amyloid β protein was also studied by biophysical measurements.During my work with this thesis, the dual nature of lysozyme emerged; on the one hand a villain, twisted by mutations, causing the lysozyme amyloidosis disease. On the other hand a hero, delaying the toxicity and maybe the neurological damage caused by the amyloid β peptide.
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| 37. |
- Hennig, Janosch, et al.
(författare)
-
Local Destabilization of the Metal-Binding Region in Human Copper-Zinc Superoxide Dismutase by Remote Mutations Is a Possible Determinant for Progression of ALS
- 2015
-
Ingår i: Biochemistry. - : American Chemical Society. - 0006-2960 .- 1520-4995. ; 54:2, s. 323-333
-
Tidskriftsartikel (refereegranskat)abstract
- More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 degrees C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 degrees C, where (15)N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer beta-strands I and VI of the beta-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 degrees C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local beta-strands.
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| 38. |
- Huang, Shenghua, et al.
(författare)
-
Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.
- 1998
-
Ingår i: J Mol Biol. - 0022-2836. ; 283:1, s. 301-10
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.
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| 39. |
- Huang, Shenghua, et al.
(författare)
-
Organization of an efficient carbonic anhydrase : implications for the mechanism based on structure-function studies of a T199P/C206S mutant.
- 2002
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:24, s. 7628-35
-
Tidskriftsartikel (refereegranskat)abstract
- Substitution of Pro for Thr199 in the active site of human carbonic anhydrase II (HCA II)(1) reduces its catalytic efficiency about 3000-fold. X-ray crystallographic structures of the T199P/C206S variant have been determined in complex with the substrate bicarbonate and with the inhibitors thiocyanate and beta-mercaptoethanol. The latter molecule is normally not an inhibitor of wild-type HCA II. All three ligands display novel binding interactions to the T199P/C206S mutant. The beta-mercaptoethanol molecule binds in the active site area with its sulfur atom tetrahedrally coordinated to the zinc ion. Thiocyanate binds tetrahedrally coordinated to the zinc ion in T199P/C206S, in contrast to its pentacoordinated binding to the zinc ion in wild-type HCA II. Bicarbonate binds to the mutant with two of its oxygens at the positions of the zinc water (Wat263) and Wat318 in wild-type HCA II. The environment of this area is more hydrophilic than the normal bicarbonate-binding site of HCA II situated in the hydrophobic part of the cavity normally occupied by the so-called deep water (Wat338). The observation of a new binding site for bicarbonate has implications for understanding the mechanism by which the main-chain amino group of Thr199 acquired an important role for orientation of the substrate during the evolution of the enzyme.
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| 40. |
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| 41. |
- Höst, Gunnar, et al.
(författare)
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Combined Enzyme and Substrate Design : Grafting of a cooperative two-histidine catalytic motif into a protein targeted at the scissile bond in a designed ester substrate
- 2007
-
Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:13, s. 1570-1576
-
Tidskriftsartikel (refereegranskat)abstract
- A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum kcat/KM value of approximately 14 000 M-1 s-1, with a kcat value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.
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| 42. |
- Höst, Gunnar, et al.
(författare)
-
Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign
- 2008
-
Ingår i: Biochimica et Biophysica Acta. - : Institutionen för fysik, kemi och biologi. - 0006-3002 .- 1878-2434. ; 1784:5, s. 811-815
-
Tidskriftsartikel (refereegranskat)abstract
- Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occuring esterases. The design was based on a previously developed strategy,[1] in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with kcat/KM = 625 (± 38) M-1s-1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with kcat/KM = 101700 (± 4800) M-1s-1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable KM values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV),[1] but for V121A/V143A/T200A no KM could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, kcat/KM is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.
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| 43. |
- Höst, Gunnar, 1976-
(författare)
-
Engineering carbonic anhydrase for highly selective ester hydrolysis
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna.Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat.I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen.Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede.
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| 44. |
- Höst, Gunnar, et al.
(författare)
-
Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
- 2006
-
Ingår i: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics. - : Elsevier BV. - 1570-9639. ; 1764:10, s. 1601-1606
-
Tidskriftsartikel (refereegranskat)abstract
- The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.
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| 45. |
- Johansson, Mikaela
(författare)
-
Metaproteogenomics-guided enzyme discovery : Targeted identification of novel proteases in microbial communities
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Industrial biotechnology is a large and growing industry as it is part of establishing a “greener” and more sustainable bioeconomy-based society. Using enzymes as biocatalysts is a viable alternative to chemicals and energy intense industrial processes and is en route to a more sustainable industry. Enzymes have been used in different areas for ages and are today used in many industrial processes such as biofuels production, food industry, tanning, chemical synthesis, pharmaceuticals etc. Enzymes are today a billion-dollar industry in itself and the demand for novel catalysts for various present and future processes of renewable resources are high and perfectly in line with converting to a more sustainable society.Most enzymes used in industry today have been identified from isolated and pure cultured microorganisms with identified desirable traits and enzymatic capacities. However, it is known that less than 1% of all microorganisms can be can be obtained in pure cultures. Thus, if we were to rely solely on pure culturing, this would leave the 99% of the microorganisms that constitutes the “microbial dark matter” uninvestigated for their potential in coding for and producing valuable novel enzymes. Therefore, to investigate these “unculturable” microorganisms for novel and valuable enzymes, pure-culture independent methods are needed.During the last two decades there has been a fast and extensive development in techniques and methods applicable for this purpose. Especially important has been the advancements made in mass spectrometry for protein identification and next generation sequencing of DNA. With these technical developments new research fields of proteomics and genomics have been developed, by which the complete protein complement of cells (the proteome) and all genes (the genome) of organisms can be investigated. When these techniques are applied to microbial communities these fields of research are known as meta-proteomics and meta-genomics.However, when applied to complex microbial communities, difficulties different from those encountered in their original usage for analysis of single multicellular organisms or cell linages arises, and when used independently both methods have their own limitations and bottlenecks. In addition, both metaproteomics and metagenomics are largely non-targeting techniques. Thus, if the purpose is still to - somewhat contradictory – use these non-targeting methods for targeted identification of novel enzymes with certain desired activities and properties from within microbial communities, special measures need to be taken.The work presented in this thesis describes the development of a method that combinesmetaproteomics and metagenomics (i.e. metaproteogenomics) for the targeted discovery of novel enzymes with desired activities, and their correct coding genes, from within microbial communities. Thus, what is described is a method that can be used to circumvent the pure-culturing problem so that a much larger fraction of the microbial dark matter can be specifically investigated for the identification of novel valuable enzymes.
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| 46. |
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| 47. |
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| 48. |
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| 49. |
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| 50. |
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