| 1. |
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| 2. |
- Syed, Zulfeqhar Ali, et al.
(författare)
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A potential role for Drosophila mucins in development and physiology.
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:8
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Tidskriftsartikel (refereegranskat)abstract
- Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300-23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis.
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| 3. |
- van den Berg, Susanne, et al.
(författare)
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Improved solubility of TEV protease by directed evolution.
- 2006
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Ingår i: Journal of biotechnology. - Amsterdam : Elsevier BV. - 0168-1656 .- 1873-4863. ; 121:3, s. 291-8
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Tidskriftsartikel (refereegranskat)abstract
- The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.
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| 4. |
- Benrick, Anna, 1979, et al.
(författare)
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A non-conservative polymorphism in the IL-6 signal transducer (IL6ST)/gp130 is associated with myocardial infarction in a hypertensive population.
- 2008
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Ingår i: Regulatory peptides. - : Elsevier BV. - 0167-0115. ; 146:1-3, s. 189-96
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Tidskriftsartikel (refereegranskat)abstract
- Inflammation is a key component in the development of atherosclerosis, and myocardial infarction (MI); therefore we investigated the association between an interleukin-6 signal transducer (IL6ST)/gp130 polymorphism, gp130 function and risk of MI. Structural modeling suggested that a non-conservative single nucleotide polymorphism in the gp130, Gly148Arg, can change the stability and functional properties of the molecule. In vitro studies were done with BAF/3 cells lacking endogenous gp130. Cells stably transfected with the gp130 148Arg variant proliferated less and showed slightly lower STAT-3 phosphorylation in response to gp130 stimulation as compared to cells transfected with gp130 148Gly. In a prospectively followed hypertensive cohort we identified 167 patients who suffered a MI during the study and compared them to matched controls (mean age 57 years, 73% males, n=482). Carriers of the 148Arg variant (f(Arg)=0.12) of the gp130 receptor had decreased odds ratio for MI in univariate analysis (0.56, 95% CI 0.34-0.91, p=0.02). In conclusion, a genetically determined structural variant of the IL-6 receptor subunit gp130 is, independently of other known risk factors, associated with decreased risk of MI. The variant is also associated with decreased IL-6 responsiveness and could lead to a configuration change in the gp130 receptor.
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| 5. |
- Dincbas-Renqvist, Vildan, et al.
(författare)
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Thermodynamics of folding, stabilization, and binding in an engineered protein--protein complex.
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 126:36, s. 11220-30
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.
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| 6. |
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| 7. |
- Dogan, Jakob, et al.
(författare)
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Thermodynamics of folding and binding in an affibody:affibody complex.
- 2006
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1305-15
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Tidskriftsartikel (refereegranskat)abstract
- Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.
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| 8. |
- Guy, Jodie E, et al.
(författare)
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New insights into multiple coagulation factor deficiency from the solution structure of human MCFD2.
- 2008
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 381:4, s. 941-55
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Tidskriftsartikel (refereegranskat)abstract
- Human MCFD2 (multiple coagulation factor deficiency 2) is a 16-kDa protein known to participate in transport of the glycosylated human coagulation factors V and VIII along the secretory pathway. Mutations in MCFD2 or in its binding partner, the membrane-bound transporter ERGIC (endoplasmic reticulum-Golgi intermediate compartment)-53, cause a mild form of inherited hemophilia known as combined deficiency of factors V and VIII (F5F8D). While ERGIC-53 is known to be a lectin-type mannose binding protein, the role of MCFD2 in the secretory pathway is comparatively unclear. MCFD2 has been shown to bind both ERGIC-53 and the blood coagulation factors, but little is known about the binding sites or the true function of the protein. In order to facilitate understanding of the function of MCFD2 and the mechanism by which mutations in the protein cause F5F8D, we have determined the structure of human MCFD2 in solution by NMR. Our results show the folding of MCFD2 to be dependent on availability of calcium ions. The protein, which is disordered in the apo state, folds upon binding of Ca(2+) to the two EF-hand motifs of its C-terminus, while retaining some localized disorder in the N-terminus. NMR studies on two disease-causing mutant variants of MCFD2 show both to be predominantly disordered, even in the presence of calcium ions. These results provide an explanation for the previously observed calcium dependence of the MCFD2-ERGIC-53 interaction and, furthermore, clarify the means by which mutations in this protein result in inefficient secretion of blood coagulation factors V and VIII.
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| 9. |
- Hammarström, Martin, et al.
(författare)
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Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
- 2006
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Ingår i: Journal of structural and functional genomics. - : Springer Science and Business Media LLC. - 1345-711X .- 1570-0267. ; 7:1, s. 1-14
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.
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| 10. |
- Hoyer, Wolfgang, 1975, et al.
(författare)
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Interaction of Alzheimer's A beta peptide with an engineered binding protein--thermodynamics and kinetics of coupled folding-binding.
- 2008
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 378:2, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- The oligomerization and aggregation of the amyloid-beta (A beta) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of A beta-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying A beta interactions is currently very limited. We have earlier determined the structure of A beta (1-40) in complex with the affibody protein Z(A beta 3), a selected binding protein based on a three-helix bundle scaffold (Z domain). Z(A beta 3) is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z(A beta 3) binds to the central and C-terminal part of A beta (residues 17-36), which adopts a beta-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z(A beta 3):A beta (1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z(A beta 3) is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z(A beta 3):A beta (1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both A beta and Z(A beta 3) fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and A beta is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of A beta. Options for improvement of Z(A beta) binding proteins are discussed.
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| 11. |
- Hoyer, Wolfgang, 1975, et al.
(författare)
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Stabilization of a beta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation.
- 2008
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:13, s. 5099-104
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Tidskriftsartikel (refereegranskat)abstract
- According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-beta (Abeta) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Abeta assemblies is accompanied by a conformational change toward a high content of beta-structure. Here, we report the solution structure of Abeta(1-40) in complex with the phage-display selected affibody protein Z(Abeta3), a binding protein of nanomolar affinity. Bound Abeta(1-40) features a beta-hairpin comprising residues 17-36, providing the first high-resolution structure of Abeta in beta conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Abeta. Z(Abeta3) stabilizes the beta-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Abeta hairpin within a large hydrophobic tunnel-like cavity. Consequently, Z(Abeta3) acts as a stoichiometric inhibitor of Abeta fibrillation. The selected Abeta conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.
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| 12. |
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| 13. |
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| 14. |
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| 15. |
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| 16. |
- Johansson, Denny, 1980, et al.
(författare)
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Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.
- 2009
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 131:27, s. 9475-7
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Tidskriftsartikel (refereegranskat)abstract
- Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --> O or N --> S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.
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| 17. |
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| 18. |
- 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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| 19. |
- Lendel, Christofer, et al.
(författare)
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Structural basis for molecular recognition in an affibody:affibody complex.
- 2006
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1293-304
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules constitute a class of engineered binding proteins based on the 58-residue three-helix bundle Z domain derived from staphylococcal protein A (SPA). Affibody proteins are selected as binders to target proteins by phage display of combinatorial libraries in which typically 13 side-chains on the surface of helices 1 and 2 in the Z domain have been randomized. The Z(Taq):anti-Z(Taq) affibody-affibody complex, consisting of Z(Taq), originally selected as a binder to Taq DNA polymerase, and anti-Z(Taq), selected as binder to Z(Taq), is formed with a dissociation constant K(d) approximately 100 nM. We have determined high-precision solution structures of free Z(Taq) and anti-Z(Taq), and the Z(Taq):anti-Z(Taq) complex under identical experimental conditions (25 degrees C in 50 mM NaCl with 20 mM potassium phosphate buffer at pH 6.4). The complex is formed with helices 1 and 2 of anti-Z(Taq) in perpendicular contact with helices 1 and 2 of Z(Taq). The interaction surface is large ( approximately 1670 A(2)) and unusually non-polar (70 %) compared to other protein-protein complexes. It involves all varied residues on anti-Z(Taq), most corresponding (Taq DNA polymerase binding) side-chains on Z(Taq), and several additional side-chain and backbone contacts. Other notable features include a substantial rearrangement (induced fit) of aromatic side-chains in Z(Taq) upon binding, a close contact between glycine residues in the two subunits that might involve aliphatic glycine Halpha to backbone carbonyl hydrogen bonds, and four hydrogen bonds made by the two guanidinium N(eta)H(2) groups of an arginine side-chain. Comparisons of the present structure with other data for affibody proteins and the Z domain suggest that intrinsic binding properties of the originating SPA surface might be inherited by the affibody binders. A thermodynamic characterization of Z(Taq) and anti-Z(Taq) is presented in an accompanying paper.
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| 20. |
- Lindborg, M., et al.
(författare)
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High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule
- 2013
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 26:10, s. 635-644
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K-D, of 20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)(2)htt dimer binds with an off-rate of k(off) 5 10(6) s(1) and an estimated K-D 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
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| 21. |
- Macao, Bertil, 1969, et al.
(författare)
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Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin.
- 2006
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Ingår i: Nature structural & molecular biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 13:1, s. 71-6
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Tidskriftsartikel (refereegranskat)abstract
- The single cell layer of the lungs and the gastrointestinal tract is protected by the mucus formed by large glycoproteins called mucins. Transmembrane mucins typically contain 110-residue SEA domains located next to the membrane. These domains undergo post-translational cleavage between glycine and serine in a characteristic GSVVV sequence, but the two peptides remain tightly associated. We show that the SEA domain of the human MUC1 transmembrane mucin undergoes a novel type of autoproteolysis, which is catalyzed by conformational stress and the conserved serine hydroxyl. We propose that self-cleaving SEA domains have evolved to dissociate as a result of mechanical rather than chemical stress at the apical cell membrane and that this protects epithelial cells from rupture. We further suggest that the cell can register mechanical shear at the mucosal surface if the dissociation is signaled via loss of a SEA-binding protein.
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| 22. |
- Macao, Bertil, 1969, et al.
(författare)
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Recombinant amyloid beta-peptide production by coexpression with an affibody ligand.
- 2008
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Ingår i: BMC biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Oligomeric and fibrillar aggregates of the amyloid beta-peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Abeta assemblies is essential for the elucidation of the mechanisms of Abeta neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Abeta. The method is based on the coexpression of the affibody protein ZAbeta3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAbeta3 binds to the amyloidogenic central and C-terminal part of Abeta with nanomolar affinity and consequently inhibits aggregation. RESULTS: Coexpression of ZAbeta3 affords the overexpression of both major Abeta isoforms, Abeta(1-40) and Abeta(1-42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Abeta. ZAbeta3 coexpression moreover permits the recombinant production of Abeta(1-42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Abeta(1-42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies. CONCLUSION: The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Abeta peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Abeta(1-42) is reported.
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| 23. |
- Rahman, Mahafuzur, et al.
(författare)
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Binding of Human Proteins to Amyloid-beta Protofibrils
- 2015
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 10:3, s. 766-774
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Tidskriftsartikel (refereegranskat)abstract
- The progressive neurodegeneration in Alzheimers disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-beta (A beta) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric A beta may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of A beta 42 (A beta(42CC)). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system, or in hemostasis. Binding of representative proteins from all of these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of A beta enhances protein binding, as lower amounts of proteins bind monomeric A beta. Proteins that bind to A beta protofibrils might contribute to biological effects in which these aggregates are involved. Our results therefore suggest that an improved understanding of the mechanisms by which A beta causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which A beta-binding proteins are present.
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| 24. |
- Sandberg, Anders, 1975, et al.
(författare)
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SEA domain autoproteolysis accelerated by conformational strain: energetic aspects.
- 2008
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 377:4, s. 1117-29
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Tidskriftsartikel (refereegranskat)abstract
- A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G(-1)S+1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N-->O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t((1/2))) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by approximately 10 kcal mol(-1) compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to approximately 11 and approximately 15 kcal mol(-1), respectively, but approximately 7 kcal mol(-1) of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of approximately 7 kcal mol(-1) was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G(-1)A+1VVV motif) and the wild-type protein. The results imply that cleavage by N-->O acyl shift alone would proceed with a t((1/2)) of approximately 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.
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| 25. |
- Wahlberg, Elisabet, et al.
(författare)
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Conformational stabilization of an engineered binding protein.
- 2006
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 128:23, s. 7651-60
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Tidskriftsartikel (refereegranskat)abstract
- We analyzed the thermodynamic basis for improvement of a binding protein by disulfide engineering. The Z(SPA)(-)(1) affibody binds to its Z domain binding partner with a dissociation constant K(d) = 1.6 microM, and previous analyses suggested that the moderate affinity is due to the conformational heterogeneity of free Z(SPA)(-)(1) rather than to a suboptimal binding interface. Studies of five stabilized Z(SPA)(-)(1) double cystein mutants show that it is possible to improve the affinity by an order of magnitude to K(d) = 130 nM, which is close to the range (20 to 70 nM) observed with natural Z domain binders, without altering the protein-protein interface obtained by phage display. Analysis of the binding thermodynamics reveals a balance between conformational entropy and desolvation entropy: the expected and favorable reduction of conformational entropy in the best-binding Z(SPA)(-)(1) mutant is completely compensated by an unfavorable loss of desolvation entropy. This is consistent with a restriction of possible conformations in the disulfide-containing mutant and a reduction of average water-exposed nonpolar surface area in the free state, resulting in a smaller conformational entropy penalty, but also a smaller change in surface area, for binding of mutant compared to wild-type Z(SPA)(-)(1). Instead, higher Z domain binding affinity in a group of eight Z(SPA)(-)(1) variants correlates with more favorable binding enthalpy and enthalpy-entropy compensation. These results suggest that protein-protein binding affinity can be improved by stabilizing conformations in which enthalpic effects can be fully explored.
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| 26. |
- Woestenenk, Esmeralda A., et al.
(författare)
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His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors.
- 2004
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Ingår i: Journal of structural and functional genomics. - 1345-711X .- 1570-0267. ; 5:3, s. 217-29
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Tidskriftsartikel (refereegranskat)abstract
- We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.
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