| 1. |
- Andersson, Per Ola, et al.
(författare)
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A Novel ATR-FTIR Approach for Characterisation and Identification of Ex Situ Immobilised Species
- 2007
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Ingår i: ChemPhysChem. - : Wiley. - 1439-4235 .- 1439-7641. ; 8:5, s. 712-722
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a novel method to analyse ex situ prepared protein chips by attenuated total reflection Fourier IR spectroscopy (ATR-FTIR), which circumvents tedious functionalisation steps of internal reflection elements (IREs), and simultaneously allows for complementary measurements by other analytical techniques. This concept is proven by utilising immobilised metal affinity capture (IMACTM) chips containing about 10 m thick films of copolymers coated with nitrilotriacetic acid (NTA) groups, which originally was manufactured for surface enhanced laser desorption ionisation (SELDI) spectrometry. Three immobilisation steps were analysed by ATR-FTIR spectroscopy: 1) NTA complexation with nickel(II) ions 2) binding of two histidine (His)-tagged synthetic peptides of 25 (25-His6) and 48 (48-His6) amino acids to the NTA-groups and 3) attachment of a ligand, mesyl amide, to the surface-bound 48-His6. Despite interference from H2O, both amide I and II were well resolved. Utilising peptide adsorption in the thick copolymer matrix yields a high saturation peptide concentration of 100 mg mL-1 and a dissociation constant of 116±11 M, as determined by a detailed analysis of the Langmuir adsorption isotherm. The mesyl amide ligand was directly seen in the raw ATR-FTIR spectrum with specific peaks in the fingerprint region at 1172 and 1350 cm-1. Several aspects of the fine structure of the amide I band of the peptide were analysed: influences from secondary structure, amino side chains and competing contamination product. We believe that this approach has great potential as a stand-alone or complementary analytical tool for determination of the chemical composition of functionalised surfaces. We emphasise further that with this approach no chemical treatment of IREs is needed; the chips can be regenerated and reused, and applied in other experimental set-ups.
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| 2. |
- Hederos (Håkansson), Sofia, et al.
(författare)
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Ligand-Directed Labeling of a Single Lysine Residue in hGST A1-1 Mutants
- 2005
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 16:4, s. 1009-1018
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Hederos, Sofia, et al.
(författare)
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A promiscuous glutathione transferase transformed into a selective thiolester hydrolase
- 2006
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Ingår i: Organic & Biomolecular Chemistry. - 1477-0520. ; 4:1, s. 90-97
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Tidskriftsartikel (refereegranskat)abstract
- Human glutathione transferase A1-1 (hGST A1-1) can be reengineered by rational design into a catalyst for thiolester hydrolysis with a catalytic proficiency of 1.4 × 107 M–1. The thiolester hydrolase, A216H that was obtained by the introduction of a single histidine residue at position 216 catalyzed the hydrolysis of a substrate termed GSB, a thiolester of glutathione and benzoic acid. Here we investigate the substrate requirements of this designed enzyme by screening a thiolester library. We found that only two thiolesters out of 18 were substrates for A216H. The A216H-catalyzed hydrolysis of GS-2 (thiolester of glutathione and naphthalenecarboxylic acid) exhibits a kcat of 0.0032 min–1 and a KM of 41 µM. The previously reported catalysis of GSB has a kcat of 0.00078 min–1 and KM of 5 µM. The kcat for A216H-catalyzed hydrolysis of GS-2 is thus 4.1 times higher than for GSB. The catalytic proficiency (kcat/KM)/kuncat for GS-2 is 3 × 106 M–1. The promiscuous feature of the wt protein towards a range of different substrates has not been conserved in A216H but we have obtained a selective enzyme with high demands on the substrate.
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| 4. |
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| 5. |
- Lignell, Martin, 1967-, et al.
(författare)
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Hydrated and Dehydrated Tertiary Interactions - Opening and Closing - of a Four-helix Bundle Peptide
- 2009
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Ingår i: Biophysical Journal. - : Elsevier. - 0006-3495 .- 1542-0086. ; 97:2, s. 572-580
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Tidskriftsartikel (refereegranskat)abstract
- The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide has been studied with steady state and time-resolved fluorescence spectroscopy and with circular dichroism. At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations we can use the lifetime analysis to monitor the change of hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9 °C to 23 °C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30 °C the structural dynamics involve transient openings of the four-helix bundle structure as evidenced by the emergence of a water-quenched component and less negative CD. Above 60 °C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions.
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| 6. |
- T. Tegler, Lotta, et al.
(författare)
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A highly selective polypeptide binder for human Acetylcholine esterase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A highly selective high-affinity polypeptide conjugate binder for human Acetylcholine Esterase (hAChE) has been obtained by coupling a derivative of acridine, a known medium-affinity inhibitor of hAChE, to each member of a 16-membered set of 42-residue polypeptide scaffolds. The best candidate, 4-C10L17-Ac, was identified to have a KD of 10 nM or less in an assay where each polypeptide conjugate was titrated with hAChE in 50 mM phosphate buffer at pH 7.0 and 298K. It was found in a sandwich ELISA to have high selectivity for hAChE in cerebrospinal fluid. Targeting the active site of hAChE by a polypeptide conjugate binder presents a special problem as it is buried deep inside the protein in a cavity that is approximately 20 Å deep. In order to permit simultaneous and cooperative binding of the acridine and the polypeptide to the active site and the AChE surface a fourteen atom spacer was needed. The 9-aminoacridine group was linked to the side chain of a lysine residue in each polypeptide via a spacer prepared from two aminohexanoic acid fragments. The results reinforce the impression that polypeptide conjugates are excellent alternatives to currently used protein binder technologies in diagnostic and therapeutic applications and that the conjugation of enzyme inhibitors to polypeptide scaffolds is a strategy of general applicability in the design of high-affinity binders for enzymes.
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| 7. |
- T. Tegler, Lotta, et al.
(författare)
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Efficient protein binders for the C-reactive protein from a designed chemically modified peptide library
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A polypeptide conjugate synthesized by coupling a small organic molecule to the side chain of an amino acid residue in a designed 42-residue polypeptide binds the C-reactive protein (CRP) essentially irreversibly. The specificity in human serum is equal to that of an avian antibody although the size is only 1/30 and the structure unordered. The polypeptide conjugate binds CRP several orders of magnitude more tightly than the small molecule due to the fact that one amino acid has been modified to include a more strongly interacting side chain. The polypeptide was selected from a 16-membered set of sequences with no prior relationship to the target protein and designed to fold into a helix-loop-helix motif. The results suggest that synthetic amino acid alphabets with more strongly interacting side chains can be used to form polypeptides with improved binding properties in comparison to those engineered by biological methods.
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| 8. |
- T. Tegler, Lotta, et al.
(författare)
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Synthetic binder molecules that discriminate between isoforms of human Carbonic Anhydrase in blood.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- From a 16-membered set of 42 residue polypeptide conjugates designed to bind human Carbonic Anhydrases (HCA), two binder candidates 4-C37L34-B och 3-C15L8-B were shown to bind the isoform HCAII with high affinity in a fluorescence based screening assay. To determine their specificity for Carbonic Anhydrases the binders were immobilized on polystyrene beads and submerged in lysed blood, washed three times, cleaved from the beads, analyzed by SDS-PAGE and shown to specifically extract Carbonic Anhydrases from the complex biological mixture. Two Carbonic Anhydrase isoforms with 60% homology exist in human blood with HCAI being present in 5-7 fold excess over HCAII and the ability of the binder molecules to discriminate between HCAI and HCAII was investigated. Due to the high degree of homology HCAI and HCAII could not be separated by electrophoresis and the instead affinities were determined by SPR biosensor analysis. The polypeptide conjugate 4-C37L34-B bound HCAII with a KD of 12 nM whereas it was 90 nM for the binding of HCAI, a ratio of 7.5. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two Carbonic Anhydrases were X and Y. This demonstration of selectivity between two very similar proteins is conspicuous in view of the fact that the molecular weight of each one of the binder molecules is little more than 5000, the fold is unordered and the polypeptide sequences are designed from scratch and have no prior relationship to Carbonic Anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
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| 9. |
- Tegler, Lotta T., et al.
(författare)
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Polypeptide Conjugate Binders that Discriminate between Two Isoforms of Human Carbonic Anhydrase in Human Blood
- 2011
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 12:4, s. 559-566
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Tidskriftsartikel (refereegranskat)abstract
- Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60% homology exist in human blood with HCAI being present in five-to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K-D of 17 nm and HCAI with a K-D of 470 nm, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nm, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
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| 10. |
- Tegler, Lotta T., et al.
(författare)
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Powerful protein binders from designed polypeptides and small organic molecules : a general concept for protein recognition
- 2011
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 50:8, s. 1823-1827
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Tidskriftsartikel (refereegranskat)abstract
- High-affinity binders for the C-reactive protein (CRP), with dissociation constants in the pM to nM range and selectivities in human serum comparable to those of antibodies, were obtained by conjugation of 16 designed polypeptides to phosphocholine, a small molecule that binds CRP with a KDvalue of 5I . The polypeptides were not designed specifically to recognize CRP and bind by an adapted fit mechanism.
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