uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Selenomethionine labeling of recombinant proteins
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2007 (English)In: Pichia Protocols / [ed] James M. Cregg, Humana Press, 2007, Vol. 389, p. 165-174Chapter in book (Refereed)
Description
Abstract [en]

Selenomethionine incorporation is a standard method for determining the phases in protein crystallography by single- or multiwavelength anomalous dispersion. Recombinant expression of selenomethionine-containing protein in non-auxotrophic Pichia pastoris strains yield an incorporation of about 50%. The expression of a mutated variant of Penicillium minioluteum dextranase in P. pastoris is used to illustrate the method utilized to obtain selenomethionyl-substituted protein and to show the phasing power of the acquired anomalous signal. The dextranase structure was solved using the anomalous signal achieved from 50% selenomethionine incorporation.

Place, publisher, year, edition, pages
Humana Press, 2007. Vol. 389, p. 165-174
Series
Methods in Molecular Biology, ISSN 1064-3745 ; 389
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94013DOI: 10.1007/978-1-59745-456-8_12PubMedID: 17951642ISBN: 978-1-58829-429-6 (print)ISBN: 978-1-59745-456-8 (print)OAI: oai:DiVA.org:uu-94013DiVA, id: diva2:167695
Available from: 2006-02-17 Created: 2006-02-17 Last updated: 2016-05-12Bibliographically approved
In thesis
1. Structural Studies of Three Glycosidases
Open this publication in new window or tab >>Structural Studies of Three Glycosidases
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work.

Dextranase catalyzes the hydrolysis of α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49.

Endo-β-1,4-D-mannanase catalyzes the depolymerization of β-1,4-mannan polymers. The structure of endo-1,4-β-mannanase Man5A from blue mussel Mytilus edulis has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5.

Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium K-edge with selenomethionyl enzymes expressed in the yeast Pichia pastoris.

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the β-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from T. fusca in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted 2SO conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed 4C1 chair conformation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. p. 47
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 145
Keyword
Cell and molecular biology, glycosidase, dextranase, mannanase, endoglucanase, SeMet, MAD, crystal structure, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6339 (URN)91-554-6461-0 (ISBN)
Public defence
2006-03-10, B22, Biomedical centre, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2006-02-17 Created: 2006-02-17Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Authority records BETA

Larsson, AnnaJones, Alwyn

Search in DiVA

By author/editor
Larsson, AnnaJones, Alwyn
By organisation
Department of Cell and Molecular Biology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
isbn
urn-nbn

Altmetric score

doi
pubmed
isbn
urn-nbn
Total: 478 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf