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| 2. |
- Chi, Celestine N., et al.
(författare)
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Interactions outside the Boundaries of the Canonical Binding Groove of a PDZ Domain Influence Ligand Binding
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:44, s. 8971-8979
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Tidskriftsartikel (refereegranskat)abstract
- The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.
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| 3. |
- Gianni, Stefano, et al.
(författare)
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Sequence-specific Long Range networks in PSD-95/Discs Large/ZO-1 (PDZ) Domains Tune Their Binding Selectivity
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:31, s. 27167-27175
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Tidskriftsartikel (refereegranskat)abstract
- Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.
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| 4. |
- Haq, S Raza, et al.
(författare)
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Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains
- 2012
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society. - 0002-7863 .- 1520-5126. ; 134:1, s. 599-605
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Tidskriftsartikel (refereegranskat)abstract
- Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K-d correlate with changes in k(off).
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| 5. |
- Haq, Syed Raza, et al.
(författare)
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The plastic energy landscape of protein folding : a triangular folding mechanism with an equilibrium intermediate for a small protein domain
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:23, s. 18051-18059
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Tidskriftsartikel (refereegranskat)abstract
- Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un) folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on-or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.
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| 6. |
- Khan, M. Ajmal, et al.
(författare)
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Effect of titania concentration on the grain boundary conductivity of calcium-doped ceria electrolyte
- 2014
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Ingår i: Ceramics International. - : Elsevier BV. - 0272-8842 .- 1873-3956. ; 40:7, s. 9775-9781
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Tidskriftsartikel (refereegranskat)abstract
- A solid-state technique was used to synthesize ceria-based (CDC-xT, in which x=0-1 mol%) solid electrolyte ceramics. The effects of doping the ceramic solid electrolyte (CDC) with titanium oxide were studied with regard to densification, crystal structure, morphology, electro-impedance spectroscopy and fuel cell performance. TiO2 doping afforded materials a 95% relative density at 940 degrees C, approximately 200 degrees C lower than the temperature required without titanium oxide. The addition of titanium oxide (TiO2) reduced the CDC sintering temperature and significantly improved the grain boundary conduction. The minimum grain boundary resistivity was obtained at 0.8 mol% TiO2. X-ray diffraction (XRD) results showed that the lattice parameters enhanced with increased titanium oxide concentrations up to 0.8 mol%, revealing the solubility limit for Caria's fluorite structure. The optimum doping level (0.8 mol%) is provided maximum conductivity. Conductivities were measured using EIS (Electrochemical Impedance Spectroscopy) with a two-probe method, and the activation energies were calculated using the Arrhenius plots. The maximum power density (660 mW/cm(2)) was achieved with CDC 0.8T electrolyte at 650 degrees C using LiCuZnNi oxide electrodes.
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| 7. |
- Raza, Rizwan, et al.
(författare)
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Ce-0.8(SmZr)(0.2)O-2-carbonate nanocomposite electrolyte for solid oxide fuel cell
- 2014
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Ingår i: International Journal of Energy Research. - : Hindawi Limited. - 0363-907X .- 1099-114X. ; 38:4, s. 524-529
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Tidskriftsartikel (refereegranskat)abstract
- A nanocomposite Zr/Sm-codoped ceria electrolyte coated with K2CO3/Na2CO3 was synthesized by a coprecipitation method. The electrochemical study of the two-phase nanocomposite electrolytes with carbonate coated on the doped ceria shows high oxygen ion mobility at low temperatures (300-600 degrees C). The interface between the two constituent phases was studied by electrochemical impedance spectroscopy. Ionic conductivities were also measured with electrochemical impedance spectroscopy. The morphology and structure of composite electrolyte were characterized using field-emission scanning electron microscopy and X-ray diffraction. The fuel cell power density is 700 mW cm(-2), and an open-circuit voltage of 1.00 V is achieved at low temperatures (400-550 degrees C). This codoped approach with a second phase provides a good indication regarding overcoming the challenges of solid oxide fuel cell technology.
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