| 1. |
- Meiby, Elinor, et al.
(författare)
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Fragment Screening by Weak Affinity Chromatography : Comparison with Established Techniques for Screening against HSP90
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:14, s. 6756-6766
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Tidskriftsartikel (refereegranskat)abstract
- The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.
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| 2. |
- Mrosek, Michael, et al.
(författare)
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Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin.
- 2007
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 21:7, s. 1383-1392
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Tidskriftsartikel (refereegranskat)abstract
- Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C-terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (Ig(A168)-Ig(A169)-FnIII(A170), hereby A168-A170) that recruits the E3 ubiquitin-ligase MuRF-1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168-A170, characterized MuRF-1 variants by circular dichroism (CD) and SEC-MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull-down assays. This has led to the identification of the molecular determinants of the binding. A168-A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF-1, a C-terminal helical domain is sufficient to bind A168-A170 with high affinity. This helical region predictably docks into the surface groove of A168-A170. Furthermore, pull-down assays demonstrate that the loop protrusion in A168-A170 is a key mediator of MuRF-1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF-1 access.
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| 3. |
- Mrosek, Michael, et al.
(författare)
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Structural analysis of B-Box 2 from MuRF1 : identification of a novel self-association pattern in a RING-like fold.
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:40, s. 10722-10730
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Tidskriftsartikel (refereegranskat)abstract
- The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.
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