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A Novel Route for Construction of Multipurpose Receptors through Chemical Modification of Glutathione Transferases
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry . Linköping University, The Institute of Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes how the human Alpha class glutathione transferase (GST) A1-1 can be reprogrammed either to function as a multipurpose biosensor for detection of small molecule analytes, or as a handle providing for more efficient protein purification.

A novel, user-friendly, and efficient method for site-specific introduction of functional groups into the active site of hGST A1-1 is the platform for these achievements. The designed thioester reagents are glutathione-based and they are able to label one single nucleophile (Y9) and leave the other 50 nucleophiles (in hGST A1-1) intact. The modification reaction was tested with five classes of GSTs (Alpha, Mu, Pi, Theta and Omega) and was found to be specific for the Alpha class isoenzymes. The reaction was further refined to target a single lysine residue, K216 in the hGST A1-1 mutant A216K, providing a stable amide bond between the protein and the labeling group. To further improve the labeling process, biotinylated reagents that could deliver the acyl group to Y9 (wt hGST A1-1) or K216 in the lysine mutant, while attached to streptavidin-coated agarose beads, were designed and synthesized.

A focused library of eleven A216K/M208X mutants was made via random mutagenesis to provide an array of proteins with altered micro-environments in the hydrophobic binding site, where M208 is situated. Through the invented route for site-specific labeling, a fluorescent probe (coumarin) was introduced on K216 in all double mutants, with the purpose of developing a protein-based biosensor, akin to the olfactory system. The array of coumarin-labeled proteins responded differently to the addition of different analytes, and the responses were analyzed through pattern recognition of the fluorescence signals. The labeled proteins could also be site-specifically immobilized on a PEG-based biosensor chip via the single C112 on the surface of the protein, enabling development of surface-based biosensing systems.

Also, a refined system for efficient detection and purification of GST-fusion proteins is presented. Through a screening process involving A216K and all produced A216K/M208X mutants, two candidates (A216K and A216K/M208F) were singled out as scaffolds for the next generation of fusion proteins. In addition to the features present in commercially available GST fusion constructs, the new mutants can be site-specifically labeled with a fluorophore in bacterial lysates providing quick and sensitive monitoring of expression and purification. Furthermore, the proteins could be labeled with a unique aldehyde moiety providing for a novel protein purification scheme.

Place, publisher, year, edition, pages
Institutionen för teknik och naturvetenskap , 2008. , 79 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1184
Keyword [en]
Human GST A1-1, site-specific covalent modification, tyrosine 9, lysine 216, methionine 208, multipurpose receptor, pattern recognition, protein purification
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:liu:diva-11612ISBN: 978-91-7393-893-8 (print)OAI: oai:DiVA.org:liu-11612DiVA: diva2:18024
Public defence
2008-05-23, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-05-18
List of papers
1. Programmed Delivery of Novel Functional Groups to the Alpha Class Glutathione Transferases
Open this publication in new window or tab >>Programmed Delivery of Novel Functional Groups to the Alpha Class Glutathione Transferases
2003 (English)In: Biochemistry, ISSN 0006-2960, Vol. 42, no 34, 10260-10268 p.Article in journal (Refereed) Published
Abstract [en]

Here we describe a new route to site- and class-specific protein modification that will allow us to create novel functional proteins with artificial chemical groups. Glutathione transferases from the alpha but not the mu, pi, omega, or theta classes can be rapidly and site-specifically acylated with thioesters of glutathione (GS-thioesters) that are similar to compounds that have been demonstrated to occur in vivo. The human isoforms A1-1, A2-2, A3-3, and A4-4 from the alpha class all react with the reagent at a conserved tyrosine residue (Y9) that is crucial in catalysis of detoxication reactions. The yield of modified protein is virtually quantitative in less than 30 min under optimized conditions. The acylated product is stable for more than 24 h at pH 7 and 25 °C. The modification is reversible in the presence of excess glutathione, but the labeled protein can be protected by adding S-methylglutathione. The stability of the ester with respect to added glutathione depends on the acyl moiety. The reaction can also take place in Escherichia coli lysates doped with alpha class glutathione transferases. A control substance that lacks the peptidyl backbone required for binding to the glutathione transferases acylates surface-exposed lysines. There is some acyl group specificity since one out of the three different GS-thioesters that we tried was not able to acylate Y9.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13363 (URN)10.1021/bi0343525 (DOI)
Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2017-10-27
2. Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases
Open this publication in new window or tab >>Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases
2004 (English)In: Bioconjugate Chemistry, ISSN 1043-1802, Vol. 15, no 4, 718-727 p.Article in journal (Refereed) Published
Abstract [en]

Previously, we discovered that human glutathione transferases (hGSTs) from the alpha class can be rapidly and quantitatively modified on a single tyrosine residue (Y9) using thioesters of glutathione (GS-thioesters) as acylating reagents. The current work was aimed at exploring the potential of this site-directed acylation using a combinatorial approach, and for this purpose a panel of 17 GS-thioesters were synthesized in parallel and used in screening experiments with the isoforms hGSTs A1-1, A2-2, A3-3, and A4-4. Through analytical HPLC and MALDI-MS experiments, we found that between 70 and 80% of the reagents are accepted and this is thus a very versatile reaction. The range of ligands that can be used to covalently reprogram these proteins is now expanded to include functionalities such as fluorescent groups, a photochemical probe, and an aldehyde as a handle for further chemical derivatization. This site-specific modification reaction thus allows us to create novel functional proteins with a great variety of artificial chemical groups in order to, for example, specifically tag GSTs in biological samples or create novel enzymatic function using appropriate GS-thioesters.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13196 (URN)10.1021/bc034192+ (DOI)
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-06-08
3. Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant
Open this publication in new window or tab >>Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant
2006 (English)In: Bioconjugate Chemistry, ISSN 1043-1802, Vol. 17, no 2, 429-437 p.Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the α-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.

Keyword
human GST A1-1, site-specific covalent modification, tyrosine 9, alanine 216, lysine 216, pre-programmed, solid support delivery, biotin, streptavidin
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13197 (URN)10.1021/bc0502762 (DOI)
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-06-08
4. A Multipurpose Receptor Composed of Promiscuous Proteins. Analyte Detection through Pattern Recognition
Open this publication in new window or tab >>A Multipurpose Receptor Composed of Promiscuous Proteins. Analyte Detection through Pattern Recognition
2007 (English)In: Bioconjugate Chemistry, ISSN 1043-1802, Vol. 18, no 6, 1935-1945 p.Article in journal (Refereed) Published
Abstract [en]

A multipurpose receptor akin to the “electronic nose” was composed of coumarin-labeled mutants of human glutathione transferase A1. We have previously constructed a kit for site-specific modification of a lysine residue (A216K) using a thiol ester of glutathione (GSC-Coubio) as a modifying reagent. In the present investigation, we scrambled the hydrophobic binding site (H-site) of the protein scaffold through mutations at position M208 via random mutagenesis and isolated a representative library of 11 A216K/M208X mutants. All of the double mutants could be site-specifically labeled to form the K216Cou conjugates. The labeled proteins responded to the addition of different analytes with signature changes in their fluorescence spectra resulting in a matrix of 96 data points per analyte. Ligands as diverse as n-valeric acid, fumaric acid monoethyl ester, lithocholic acid, 1-chloro-2,4-dinitrobenzene (CDNB), glutathione (GSH), S-methyl-GSH, S-hexyl-GSH, and GS-DNB all gave rise to signals that potentially can be interpreted through pattern recognition. The measured Kd values range from low micromolar to low millimolar. The cysteine residue C112 was used to anchor the coumarin-labeled protein to a PEG-based hydrogel chip in order to develop surface-based biosensing systems. We have thus initiated the development of a multipurpose, artificial receptor composed of an array of promiscuous proteins where detection of the analyte occurs through pattern recognition of fluorescence signals. In this system, many relatively poor binders each contribute to detailed readout in a truly egalitarian fashion.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13198 (URN)10.1021/bc700247x (DOI)
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2015-10-13
5. Orthogonal Protein Purification - Expanding the Repertoire of GST Fusion Systems
Open this publication in new window or tab >>Orthogonal Protein Purification - Expanding the Repertoire of GST Fusion Systems
2008 (English)In: Protein Expression & Purification, ISSN 1046-5928, Vol. 57, no 1, 17-26 p.Article in journal (Refereed) Published
Abstract [en]

We have previously developed a labeling scheme that can be used to site-specifically link human glutathione transferases (hGSTs) from the alpha class to chemical entities such as fluorophores and aldehydes. The reagents are in-house synthesized derivatives of glutathione (GS-derivatives). We have focused on a lysine mutant of hGST A1:A216K. In this study, we wanted to utilize these findings and improve on protein purification schemes that are using GSTs as fusion partners. We have used random mutagenesis to scramble the hydrophobic binding site of A216K through mutations at position M208 and isolated a library of 11 A216K/M208X mutants. All mutants were easily expressed and purified and retained all or parts of the catalytic properties of the parent GST. The mutants were stable over several days at room temperature. The A216K/M208X mutants could be site-specifically labeled using our designed fluorescent reagents. Furthermore, reaction with an aldehyde-containing reagent termed GS-Al results in site-specific introduction of an orthogonal handle for subsequent conjugation with aldehyde-reactive probes. Labeling with coumarin results in a fluorescent protein-conjugate that can bind glutathione (GSH) derivatives for subsequent affinity purification. The Kd for S-hexyl-GSH of coumarin-labeled A216K was measured to be 2.5 μM. The candidate proteins A216K and A216K/M208F could be purified in high yield in a one-step procedure through affinity chromatography (Glutathione Sepharose™ 4B). The proteins can readily be perceived as improved GST fusion partners.

Keyword
Human GST A1-1 mutants; Site-specific covalent modification; Lysine 216; Methionine 208; Protein purification; Fusion partner
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13199 (URN)10.1016/j.pep.2007.09.011 (DOI)
Available from: 2008-04-28 Created: 2008-04-28 Last updated: 2009-05-18

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