| 1. |
- Syed, Zulfeqhar Ali, et al.
(author)
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A potential role for Drosophila mucins in development and physiology.
- 2008
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:8
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Journal article (peer-reviewed)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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| 2. |
- Benrick, Anna, 1979, et al.
(author)
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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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In: Regulatory peptides. - : Elsevier BV. - 0167-0115. ; 146:1-3, s. 189-96
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Journal article (peer-reviewed)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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| 3. |
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| 4. |
- Dogan, Jakob, et al.
(author)
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Thermodynamics of folding and binding in an affibody:affibody complex.
- 2006
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In: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1305-15
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Journal article (peer-reviewed)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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| 5. |
- Guy, Jodie E, et al.
(author)
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New insights into multiple coagulation factor deficiency from the solution structure of human MCFD2.
- 2008
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In: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 381:4, s. 941-55
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Journal article (peer-reviewed)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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| 6. |
- Hammarström, Martin, et al.
(author)
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Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
- 2006
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In: 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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Journal article (peer-reviewed)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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| 7. |
- Hammarström, Martin, 1973-
(author)
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Protein production and purification in structural genomics
- 2006
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Doctoral thesis (other academic/artistic)abstract
- The number of gene products available for structural and functional study is increasing at an unprecedented rate as a result of the successful whole genome sequencing projects. Systematic structure determination of proteins on a genomic scale, called structural genomics, can significantly contribute to the field of protein science and to functional annotation of newly identified genes. This thesis covers different aspects of protein production in Eschericiha coli for structural studies in the context of structural genomics. Protocols have been downscaled and standardized to allow for a rapid assessment of the production characteristics for multiple proteins in parallel under a number of different conditions. Foremost, the ability of different proteins and peptide tags to affect the solubility of the recombinant protein when produced as fusion proteins has been systematically studied. Large differences in the success-rate for production of soluble protein in E. coli were found depending on the fusion partner used, with a more than two-fold increase in the number of proteins produced as soluble when comparing the best and the poorest fusion tags. For different constructs with a histidine tag, commonly used to facilitate protein purification, large differences in yield depending on the design of the expression vector were found. When comparing different fusion proteins produced from identical expression vectors, fusions to the GB1 domain were found to result in the highest yield of purified target protein, on average 25 % higher than any of the other fusions. The suitability for further structural studies was tested at an intermediate scale for proteins that were identified as soluble in the expression screening. For this purpose, protocols for rapid purification and biophysical characterization using nuclear magnetic resonance and circular dichroism spectroscopy were developed and tested on 19 proteins, of which four were structured.
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| 8. |
- Hellgren, Niklas
(author)
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Probing the prospects for structural genomics : expression and characterization of human disease-related proteins in E. coli
- 2005
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Licentiate thesis (other academic/artistic)abstract
- Today a lot of research is directed towards the development of methods to make protein structure determination faster and more generalised. Automated, general methods for protein expression and purification, robotics for crystallisation and automatic methods for data collection will take structural biology into a new era, that of structural genomics. In two previously performed studies applications were developed for potential use in a structural genomics context. In the first, a fast method to screen for soluble proteins expressed in E. coli using seven N-terminal fusion proteins as solubility enhancing tags was developed. The method was tested on 32 small human proteins suitable for structural study with NMR (Hammarström et al, 2002). In the second, a biophysical screen for fast identification of proteins suitable for structure determination was developed (Woestenenk et al, 2003). This thesis is an extension of the two previous studies. We have tested the solubility enhancing effect of four N-terminal fusion proteins on 47 cancer and developmental disease related human target proteins, of sizes ranging from 6.6 to 126.5 kDa. We have indications that the solubility enhancing effect is dependent on target protein size. Small proteins of size less than 20 kDa are more likely to be generally affected by the solubility enhancing effect of the fusion partners. All targets that were overexpressed in the present screen and ten previously uncharacterized targets, related to cancer, from an earlier project were selected for biophysical characterization trials. We were able to purify 15 targets for NMR, CD spectroscopy or both methods. A majority of the targets were estimated to be unstructured or partially unstructured. Only one target was considered directly suitable for structure determination using NMR. The solubility screen and biophysical results were compared to DisEMBL™ and GlobPlot™ disorder predictions of all targets. We found a close match between biophysical data and prediction results. We concluded that, even though we obtain an increased solubility using fusion proteins, many of our targets are likely to be unstructured or partially unstructured and are thus not suitable for structure determination.
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| 9. |
- Hoyer, Wolfgang, 1975, et al.
(author)
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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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In: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 378:2, s. 398-411
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Journal article (peer-reviewed)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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| 10. |
- Hoyer, Wolfgang, 1975, et al.
(author)
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Stabilization of a beta-hairpin in monomeric Alzheimer's amyloid-beta peptide inhibits amyloid formation.
- 2008
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In: 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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Journal article (peer-reviewed)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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