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Broo, Kerstin
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Publications (8 of 8) Show all publications
Viljanen, J., Larsson, J., Larsson (Kaiser), A. & Broo, K. S. (2007). A Multipurpose Receptor Composed of Promiscuous Proteins. Analyte Detection through Pattern Recognition. Bioconjugate Chemistry, 18(6), 1935-1945
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, p. 1935-1945Article 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
Hederos, S., Tegler, L., Carlsson, J., Persson, B., Viljanen, J. & Kerstin S., B. (2006). A Promiscuous Glutathione Transferase Transformed into a Selective Thiolester Hydrolase. Organic and biomolecular chemistry, 4(1), 90-97
Open this publication in new window or tab >>A Promiscuous Glutathione Transferase Transformed into a Selective Thiolester Hydrolase
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2006 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 4, no 1, p. 90-97Article in journal (Refereed) Published
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13365 (URN)10.1039/b510115h (DOI)
Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2017-10-27
Viljanen, J., Tegler, L., Larsson, J. & Broo, K. S. (2006). Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant. Bioconjugate Chemistry, 17(2), 429-437
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, p. 429-437Article 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.

Keywords
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
Vahlberg, C., Petoral, R. J., Lindell, C., Broo, K. & Uvdal, K. (2006). α2A-adrenergic receptor derived peptide adsorbates: a g-protein interaction study. Langmuir, 22(17), 7260-7264
Open this publication in new window or tab >>α2A-adrenergic receptor derived peptide adsorbates: a g-protein interaction study
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2006 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 22, no 17, p. 7260-7264Article in journal (Refereed) Published
Abstract [en]

The affinity of α2A-adrenergic receptor (α2A-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-50157 (URN)10.1021/la052801r (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-12Bibliographically approved
Hederos (Håkansson), S., Karlsson, B., Tegler, L. & Kerstin S., B. (2005). Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants. Bioconjugate chemistry, 16(4), 1009-1018
Open this publication in new window or tab >>Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants
2005 (English)In: Bioconjugate chemistry, ISSN 1043-1802, Vol. 16, no 4, p. 1009-1018Article in journal (Refereed) Published
Abstract [en]

Previously, we discovered that human glutathione transferase (hGST) A1-1 could be site-specifically acylated on a tyrosine residue (Y9) to form ester products using thiolesters of glutathione (GS-thiolesters) as acylating reagents. Out of a total of 20 GS-thiolester reagents tested, 15 (75%) are accepted by hGST A1-1 and thus this is a very versatile reaction. The present investigation was aimed at obtaining a more stable product, an amide bond, between the acyl group and the protein, in order to further increase the value of the reaction. Three lysine mutants (Y9K, A216K, and Y9F/A216K) were therefore prepared and screened against a panel of 18 GS-thiolesters. The Y9K mutant did not react with any of the reagents. The double mutant Y9F/A216K reacted with only one reagent, but in contrast, the A216K mutant could be acylated at the introduced lysine 216 with eight (44%) of the GS-thiolesters. The reaction can take place in the presence of glutathione and even in a crude cell lysate for five (28%) of the reagents. Through the screening process we obtained some basic rules relating to reagent requirements. We have thus produced a mutant (A216K) that can be rapidly and site-specifically modified at a lysine residue to form a stable amide linkage with a range of acyl groups. One of the successful reagents is a fluorophore that potentially can be used in downstream protein purification and protein fusion applications.

Keywords
human GST A1-1, site-specific covalent modification, tyrosine 9, alanine 216, lysine mutant, pre-programmed, ligand-directed protein modification, intramolecular acyl-transfer reaction
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-13367 (URN)10.1021/bc050111t (DOI)
Available from: 2005-09-23 Created: 2005-09-23 Last updated: 2017-10-27
Viljanen, J., Tegler, L. & Broo, K. (2004). Combinatorial Chemical Reengineering of the Alpha Class Glutathione Transferases. Bioconjugate Chemistry, 15(4), 718-727
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, p. 718-727Article 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
Petoral Jr., R. M., Herland, A., Broo, K. & Uvdal, K. (2003). G-protein interactions with receptor-derived peptides chemisorbed on gold. Langmuir, 19(24), 10304-10309
Open this publication in new window or tab >>G-protein interactions with receptor-derived peptides chemisorbed on gold
2003 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 19, no 24, p. 10304-10309Article in journal (Refereed) Published
Abstract [en]

Interactions between the functional bovine brain G-protein and receptor-derived peptidea chemically adsorbed on gold surfaces are studied. The peptides are designed to mimic the third ic-loop (aa 361-373) of the Alpha 2a-adrenergic receptor (α 2AR). These segments are linked to a surface using the thiol-gold chemistry, and the protein interaction studies are conducted to investigate the key function of recognition. The chemical composition and binding strength of the peptide monolayers onto a gold surface are characterized using X-ray photoelectron spectroscopy and infrared (IR) spectroscopy. Strong molecular binding of the adsorbates to the gold surface is attained, and the presence of amide-related IR vibrations verified the composition of the peptides. Bovine brain G-protein adsorption studies on these molecular monolayers are performed using the surface plasmon resonance technique. The arginine-rich peptide, which is a direct mimicry of the receptor, has a higher affinity for G-protein than the lysine-rich and alanine-rich derived peptides, showing that arginine residue has special importance for the G-protein interaction with the receptor.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46427 (URN)10.1021/la035046v (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13
Håkansson Hederos, S., Viljanen, J. & Broo, K. (2003). Programmed Delivery of Novel Functional Groups to the Alpha Class Glutathione Transferases. Biochemistry, 42(34), 10260-10268
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, p. 10260-10268Article 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
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