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- Duroux, Meg, et al.
(författare)
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Light-induced immobilization of biomolecules as an attractive alternative to micro-droplet dispensing-based arraying technologies
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:19, s. 1113-1113
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Tidskriftsartikel (refereegranskat)abstract
- The present work shows how UV light-induced molecular immobilisation (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using LIMI, arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 m in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies.
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| 3. |
- Neves-Petersen, Maria Teresa, et al.
(författare)
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Flash Photolysis of Cutinase: Identification and Decay Kinetics of Transient Intermediates Formed upon UV Excitation of Aromatic Residues
- 2009
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 97:1, s. 211-226
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Tidskriftsartikel (refereegranskat)abstract
- Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after similar to 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR center dot-), with concomitant faster decay kinetics and near disappearance of the Trp(center dot) radical peak at 330 nm, indicating possible additional formation of TyrO(center dot) formed upon reaction of Trp(center dot) with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR center dot-. Additional mechanisms that may lead to the near disappearance of Trp(center dot) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.
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| 4. |
- Nilsson, Anna, et al.
(författare)
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Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems
- 2003
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - 1570-9639. ; 1646:1-2, s. 57-66
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Tidskriftsartikel (refereegranskat)abstract
- Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C12EOn). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)4 and cutinase-TGGSGG-(WP)4, showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C12EOn detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C12EOn detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.
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