| 1. |
- Edwin, Aaron, et al.
(författare)
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Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae
- 2015
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 24:12, s. 2076-2080
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Tidskriftsartikel (refereegranskat)abstract
- The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N- terminal domain (residues 23-103) that contains two short alpha-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.
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| 2. |
- Lendel, Christofer, et al.
(författare)
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Biophysical characterization of Z(SPA-1)--a phage-display selected binder to protein A.
- 2004
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Ingår i: Protein science : a publication of the Protein Society. - : Wiley. - 0961-8368 .- 1469-896X. ; 13:8, s. 2078-88
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Tidskriftsartikel (refereegranskat)abstract
- Affibodies are a novel class of binding proteins selected from phagemid libraries of the Z domain from staphylococcal protein A. The Z(SPA-1) affibody was selected as a binder to protein A, and it binds the parental Z domain with micromolar affinity. In earlier work we determined the structure of the Z:Z(SPA-1) complex and noted that Z(SPA-1) in the free state exhibits several properties characteristic of a molten globule. Here we present a more detailed biophysical investigation of Z(SPA-1) and four Z(SPA-1) mutants with the objective to understand these properties. The characterization includes thermal and chemical denaturation profiles, ANS binding assays, size exclusion chromatography, isothermal titration calorimetry, and an investigation of structure and dynamics by NMR. The NMR characterization of Z(SPA-1) was facilitated by the finding that trimethylamine N-oxide (TMAO) stabilizes the molten globule conformation in favor of the fully unfolded state. All data taken together lead us to conclude the following: (1) The topology of the molten globule conformation of free Z(SPA-1) is similar to that of the fully folded structure in the Z-bound state; (2) the extensive mutations in helices 1 and 2 destabilize these without affecting the intrinsic stability of helix 3; (3) stabilization and reduced aggregation can be achieved by replacing mutated residues in Z(SPA-1) with the corresponding wild-type Z residues. This stabilization is better correlated to changes in helix propensity than to an expected increase in polar versus nonpolar surface area of the fully folded state.
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| 3. |
- Sjöhamn, Jennie, 1986, et al.
(författare)
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Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.
- 2016
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Ingår i: Protein science : a publication of the Protein Society. - : Wiley. - 1469-896X. ; 25:12, s. 2196-2208
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Tidskriftsartikel (refereegranskat)abstract
- Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.
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