| 1. |
- Altschuh, Danièle, et al.
(författare)
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Deciphering complex protein interaction kinetics using Interaction Map
- 2012
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 428:1, s. 74-79
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Tidskriftsartikel (refereegranskat)abstract
- Cellular receptor systems are expected to present complex ligand interaction patterns that cannot beevaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluatedheterogeneous interactions using an alternative method to regression analysis, called Interaction Map(IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic,scalar kinetic association rate constant ka and dissociation rate constant kd with a two-dimensionaldistribution of ka and kd, it is possible to display heterogeneous data as a map where each peakcorresponds to one of the components that contribute to the cumulative binding curve. Here we challengethe Interaction Map approach by artificially generating heterogeneous data from two known interactions,on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM toaccurately decompose these man-made heterogeneous binding curves composed of two different interactions.We conclude that the Interaction Map approach is well suited for the analysis of complex bindingdata and forecast that it has a potential to resolve previously uninterpretable data, in particular thosegenerated in cell-based assays.
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| 2. |
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| 3. |
- Choulier, Laurence, et al.
(författare)
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Delineation of a linear epitope by multiple peptide synthesis and phage display
- 2001
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Ingår i: JOURNAL OF IMMUNOLOGICAL METHODS. - : ELSEVIER SCIENCE BV. - 0022-1759. ; 249:1-2, s. 253-264
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Tidskriftsartikel (refereegranskat)abstract
- Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composit
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| 4. |
- Christopeit, Tony, 1982-
(författare)
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Protein Interaction Studies with Low Molecular Weight Ligands : Applications for Drug Discovery, Basic Research and Diagnostic Tool Design
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays. For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions. The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings.The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins. The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes.The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.
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| 5. |
- Enander, Karin, 1972-, et al.
(författare)
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A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte
- 2008
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 19:9, s. 1864-1870
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Tidskriftsartikel (refereegranskat)abstract
- We present the design, synthesis, and functional evaluation of peptide-based fluorescent constructs for wavelength-ratiometric biosensing of a protein analyte. The concept was shown using the high-affinity model interaction between the 18 amino acid peptide pTMVP and a recombinant antibody fragment, Fab57P. pTMVP was functionalized in two different positions with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive fluorophore with a two-band emission. The equilibrium dissociation constant of the interaction between pTMVP and Fab57P was largely preserved upon labeling. The biosensor ability of the labeled peptide constructs was evaluated in terms of the relative intensity change of the emission bands from the normal (N*) and tautomer (T*) excited-state species of the fluorophore (IN*/IT*) upon binding of Fab57P. When the peptide was labeled in the C terminus, the IN*/I T* ratio changed by 40% upon analyte binding, while labeling close to the residues most important for binding resulted in a construct that completely lacked ratiometric biosensor ability. Integrated biosensor elements for reagentless detection, where peptides and ratiometric fluorophores are combined to ensure robustness in both recognition and signaling, are expected to become an important contribution to the design of future protein quantification assays in immobilized formats. © 2008 American Chemical Society.
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