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Mutational effects on protein structure and function
Linköping University, Department of Physics, Chemistry and Biology, Bioinformatics . Linköping University, The Institute of Technology.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1).

When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations.

For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients.

The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1.

The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements.

In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes.

A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro.

ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated.

To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.

 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 80 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1271
Keyword [en]
Mutation, prediction, phenotypes, homology model, virtual screening, molecular dynamics, amyloid, cancer, membrane protein
National Category
Bioinformatics and Systems Biology
Identifiers
URN: urn:nbn:se:liu:diva-50491ISBN: 978-91-7393-539-9 OAI: oai:DiVA.org:liu-50491DiVA: diva2:271401
Public defence
2009-11-06, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:00 (English)
Opponent
Supervisors
Available from: 2009-10-19 Created: 2009-10-12 Last updated: 2009-10-19Bibliographically approved
List of papers
1. Molecular Model of Human CYP21 Based onMammalian CYP2C5: Structural Features Correlatewith Clinical Severity of Mutations CausingCongenital Adrenal Hyperplasia
Open this publication in new window or tab >>Molecular Model of Human CYP21 Based onMammalian CYP2C5: Structural Features Correlatewith Clinical Severity of Mutations CausingCongenital Adrenal Hyperplasia
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2006 (English)In: Molecular Endocrinology, ISSN 0888-8809, E-ISSN 1944-9917, Vol. 20, no 11, 2946-2964 p.Article in journal (Refereed) Published
Abstract [en]

Enhanced understanding of structure-function relationshipsof human 21-hydroxylase, CYP21, is requiredto better understand the molecular causesof congenital adrenal hyperplasia. To this end, astructural model of human CYP21 was calculatedbased on the crystal structure of rabbit CYP2C5.All but two known allelic variants of missense type,a total of 60 disease-causing mutations and sixnormal variants, were analyzed using this model. Astructural explanation for the corresponding phenotypewas found for all but two mutants for whichavailable clinical data are also discrepant with invitro enzyme activity. Calculations of protein stabilityof modeled mutants were found to correlateinversely with the corresponding clinical severity.Putative structurally important residues were identifiedto be involved in heme and substrate binding,redox partner interaction, and enzyme catalysisusing docking calculations and analysis of structurallydetermined homologous cytochrome P450s(CYPs). Functional and structural consequences ofseven novel mutations, V139E, C147R, R233G,T295N, L308F, R366C, and M473I, detected inScandinavian patients with suspected congenitaladrenal hyperplasia of different severity, were predictedusing molecular modeling. Structural featuresdeduced from the models are in good correlationwith clinical severity of CYP21 mutants,which shows the applicability of a modeling approachin assessment of new CYP21 mutations.

Place, publisher, year, edition, pages
Stanford: The endocrin society, 2006
Keyword
Mutations, prediction, CAH, CYP21, homology model
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:liu:diva-21305 (URN)10.1210/me.2006-0172 (DOI)
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2017-12-13Bibliographically approved
2. Investigation and prediction of the severity of p53 mutants using parameters from structural calculations
Open this publication in new window or tab >>Investigation and prediction of the severity of p53 mutants using parameters from structural calculations
2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 15, 4142-4155 p.Article in journal (Refereed) Published
Abstract [en]

A method has been developed to predict the effects of mutations in the p53 cancer suppressor gene. The new method uses novel parameters combined with previously established parameters. The most important parameter is the stability measure of the mutated structure calculated using molecular modelling. For each mutant, a severity score is reported, which can be used for classification into deleterious and nondeleterious. Both structural features and sequence properties are taken into account. The method has a prediction accuracy of 77% on all mutants and 88% on breast cancer mutations affecting WAF1 promoter binding. When compared with earlier methods, using the same dataset, our method clearly performs better. As a result of the severity score calculated for every mutant, valuable knowledge can be gained regarding p53, a protein that is believed to be involved in over 50% of all human cancers.

Keyword
Cancer; molecular modelling; mutations; p53; structural prediction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20141 (URN)10.1111/j.1742-4658.2009.07124.x (DOI)
Available from: 2009-08-31 Created: 2009-08-31 Last updated: 2017-12-13Bibliographically approved
3. A structural model of human steroid 11-betahydroxylase,CYP11B1, used to predict consequences of mutations
Open this publication in new window or tab >>A structural model of human steroid 11-betahydroxylase,CYP11B1, used to predict consequences of mutations
2009 (English)Article in journal (Other academic) Submitted
Abstract [en]

A prediction method has been developed to estimate the severity of amino acid residue exchanges in human steroid 11-beta-hydroxylase, CYP11B1, due to mutations in the corresponding gene. The prediction is based both on structural and on sequence dependent parameters. The method uses two approaches; one with general molecular property weights and one with a consensus voting strategy based upon distribution of molecular properties, which does not require any training. Both methods are tested on known mutations in CYP11B1 and result in 85% prediction accuracy. The consensus voting method is then further evaluated on 9 proteins with an average of 81% prediction accuracy. A server utilizing the results from the consensus voting on CYP11B1 is provided where the user can extract information about new mutants. A similar server is also provided for mutants in human steroid 21-hydroxylase (CYP21).

Keyword
CYP11B1, steroid 11-beta-hydroxylase, molecular modeling, structural prediction, mutations
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51118 (URN)
Available from: 2009-10-19 Created: 2009-10-19 Last updated: 2009-10-19Bibliographically approved
4. Disruption of the GDNF Binding Site in NCAM DissociatesLigand Binding and Homophilic Cell Adhesion
Open this publication in new window or tab >>Disruption of the GDNF Binding Site in NCAM DissociatesLigand Binding and Homophilic Cell Adhesion
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2007 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 17, 12734-12740 p.Article in journal (Refereed) Published
Abstract [en]

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent towhich this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM tomediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor α1, shedding light on the molecular architecture of a multicomponent GDNF receptor.

Place, publisher, year, edition, pages
Bethesda, MD: American Society for Biochemistry and Molecular Biology, 2007
Keyword
homology model, protein complex, interaction interface, mutagenesis
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:liu:diva-21306 (URN)10.1074/jbc.M701588200 (DOI)
Available from: 2009-09-30 Created: 2009-09-30 Last updated: 2017-12-13Bibliographically approved
5. Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-directed Mutagenesis
Open this publication in new window or tab >>Functionally Important Amino Acids in the Arabidopsis Thylakoid Phosphate Transporter: Homology Modeling and Site-directed Mutagenesis
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2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 30, 6430-6439 p.Article in journal (Other academic) Published
Abstract [en]

The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian SLC17 family, has recently been localized to the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acids involved in substrate binding/translocation by ANTR1 and in the Na+-dependence of its activity. A threedimensional structural model of ANTR1 was constructed using the crystal structure of glycerol-3-phosphate/phosphate antiporter (GlpT) from E.coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely Arg-120, Ser-124 and Arg-201 inside the putative translocation pathway, Arg-228 and Asp-382 exposed at the cytosolic surface of the protein. The activities of the wild type and mutant proteins have been analyzed using expression in E. coli and radioactive transport assays, and compared with bacterial cells carrying an empty plasmid. Based on Pi- and Na+-dependent kinetics, we propose that Arg-120, Arg-201 and Arg-228 are involved in binding and translocation of the substrate, Ser-124 functions as a periplasmic gate for Na+ ions, and finally Asp-382 participates in the turnover of the transporter via ionic interaction with either Arg-228 or Na+ ions. We also propose that the corresponding residues may have a similar function in other plant and mammalian SLC17 homologous transporters.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-51119 (URN)10.1021/bi100239j (DOI)
Note
On the day of the defence day the status of this article was ManuscriptAvailable from: 2009-10-19 Created: 2009-10-19 Last updated: 2017-12-12Bibliographically approved
6. A folding study on IAPP (Islet Amyloid Polypeptide) using molecular dynamics simulations
Open this publication in new window or tab >>A folding study on IAPP (Islet Amyloid Polypeptide) using molecular dynamics simulations
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Amyloidosis is the largest group among the protein misfolding diseases, and includes well known diseases such as Alzheimer’s disease and type 2 diabetes. In the latter, islet amyloid is present in the pancreas in almost all individuals. Today, more than 25 different proteins have been isolated from amyloid deposits in human. Even though these proteins differ in size, charge and sequence they all have the capacity to assemble in to fibrillar structures with inseparable morphological appearance. Therefore, it can be assumed that the fibril process is based upon principles that are general for all proteins and knowledge derived from one protein can be used for other amyloid proteins. In this paper, we study the process of amyloid formation in parts of islet amyloid polypeptide (residues 18-29 and 11-37) by analyzing mutations using three different in silico methods. Finally, we use the methods to predict the amyloidogenic properties of the native IAPP and 16 variants thereof and compare the result with in vitro measurements. Using a consensus prediction of the three methods we managed to correctly classify all but two peptides. We have also given further evidence to the importance of S28P for inhibiting amyloid fibre formation, found evidence for antiparallel stacking, and identified important regions for beta sheet stability.

Keyword
IAPP, molecular modeling, amyloid, prediction, molecular dynamics, Monte Carlo
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51120 (URN)
Available from: 2009-10-19 Created: 2009-10-19 Last updated: 2010-01-14Bibliographically approved
7. Virtual screening for ligands to human alcohol dehydrogenase 3
Open this publication in new window or tab >>Virtual screening for ligands to human alcohol dehydrogenase 3
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alcohol dehydrogenase 3 (ADH3) has been suggested a role in nitric oxide homeostasis due to its function as a S-nitrosoglutathione (GSNO) reductase. This has requested a modulator of the ADH3 activity for control of GSNO levels. Today virtual screenings are frequently used in drug discovery to dock and rank a large number of compounds. With molecular dockings of more than 40,000 compounds into the active site pocket of human ADH3 we ranked compounds with a novel method. Six top ranked compounds that were not known to interact with ADH3 were tested in vitro, where two showed substrate activity (9-decen-1-ol and dodecyltetraglycol), two showed inhibition capacity (deoxycholic acid and doxorubicin) and two did not have any detectable effect. For the substrates, site specific interactions and calculated binding scoring energies were determined with an extended docking simulation including flexible side chains of amino acids residues. The binding scoring energies correlated well with the logarithm of the substrates kcat over Km values. Furthermore, with these computational and experimental data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs and in addition deoxycholic acids.

Keyword
Alcohol dehydrogenase, Enzyme kinetics, Molecular docking, Virtual screening
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-51121 (URN)
Available from: 2009-10-19 Created: 2009-10-19 Last updated: 2010-01-14Bibliographically approved

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