| 1. |
- Järver, Peter, et al.
(författare)
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Chemical synthesis and evaluation of a backbone-cyclized minimized 2-helix Z-domain
- 2011
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 17:6, s. 463-469
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Tidskriftsartikel (refereegranskat)abstract
- The Z-molecule is a small, engineered IgG-binding affinity protein derived from the immunoglobulin-binding domain B of Staphylococcus aureus protein A. The Z-domain consists of 58 amino acids forming a well-defined antiparallel three-helix structure. Two of the three helices are involved in ligand binding, whereas the third helix provides structural support to the three-helix bundle. The small size and the stable three-helix structure are two attractive properties comprised in the Z-domain, but a further reduction in size of the protein is valuable for several reasons. Reduction in size facilitates synthetic production of any protein-based molecule, which is beneficial from an economical viewpoint. In addition, a smaller protein is easier to manipulate through chemical modifications. By omitting the third stabilizing helix from the Z-domain and joining the N- and C-termini by a native peptide bond, the affinity protein obtains the advantageous properties of a smaller scaffold and in addition becomes resistant to exoproteases. We here demonstrate the synthesis and evaluation of a novel cyclic two-helix Z-domain. The molecule has retained affinity for its target protein, is resistant to heat treatment, and lacks both N- and C-termini. Copyright (C) 2011 European Peptide Society and John Wiley & Sons, Ltd.
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| 2. |
- Mikaelsson, Cecilia
(författare)
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Structural and biophysical studies of pneumococcal capsular surface proteins
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Streptococcus pneumoniae (pneumococcus) is a major human pathogen and a leading cause of morbidity and mortality worldwide, especially in children and the elderly. The casualties due to respiratory infections are estimated to be over 4 million per year, where pneumococcus is the predominant species. Moreover, the increasing number of antibiotic-resistant strains and the suboptimal clinical efficacy of available vaccines confines control of this pathogen. In view of this situation, substantial attention has focused on novel virulence-related pneumococcal proteins as potential targets for future drug targets. One such target is the pneumococcal serine-rich repeat protein (PsrP), an important virulence factor present in a majority of the strains capable of causing invasive pneumococcal disease (IPD). The functional binding region (BR) of this protein binds to keratin-10 (KRT10) and also promotes biofilm formation through self-oligomerization. The crystal structure of the KRT10-binding region of PsrP (BR187-385) reveals an extended β-sheet on one side of a compressed two-sided β-barrel presents a basic groove that could accommodate the acidic helical rod domain of KRT10. Well-ordered loop regions distort the other side of the barrel and form a papercliplike sub-structure for more specific interaction with KRT10. In vitro alanine substitution of residues localized within this paperclip structure efficiently disrupted BR187-385/KRT10 complex formation. Within the work of this thesis we also found that BR187-385, which lacks the putative oligomerization region, forms stable oligomers in vitro. Small angle X-ray scattering and circular dichroism experiments revealed a non-globular and possibly disordered structure of the N-terminal region. A comparative analysis of the long (BR120-395) and short (BR187-385) domain constructs even suggested an inhibitory role for the N-terminal BR122-166 domain in oligomerization. Indeed, we could show that the N-terminal region is released by cleavage through the human furin protease that specifically recognizes a sequence localized between the globular BR187-385 domain and the disordered N-terminal part. The crystal structure of the dimer of the KRT10-binding domain of PsrP reveals a domain swap mechanism for dimerization, this process, although energetically costly, is probable when PsrP is involved in biofilm formation. The minor ancillary pilus protein RrgC is believed to anchor the pneumococcal pilus to the cell wall, and until recently very little was known about its structure. In this work we have evaluated the structure of the protein in solution with SAXS, and shown that the protein is a multidomain protein with flexible linkers and adopts extended conformations in solution. Through this work we have proposed a more specific interaction between PsrP and KRT10 than previously reported, as well as shown that oligomerization is possible despite the removal of the N-terminal region. These findings are very important for a deeper understanding of the details in PsrP’s role in pneumococcal invasion. The structural studies of RrgC provide a platform for future studies. The results presented within this thesis will hopefully aid in the future development of novel drugs and vaccines.
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| 3. |
- Schulte, Tim, et al.
(författare)
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The BR domain of PsrP interacts with extracellular DNA to promote bacterial aggregation; structural insights into pneumococcal biofilm formation
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The major human pathogen Streptococcus pneumoniae is a leading cause of disease and death worldwide. Pneumococcal biofilm formation within the nasopharynx leads to long-term colonization and persistence within the host. We have previously demonstrated that the capsular surface-associated pneumococcal serine rich repeat protein (PsrP), key factor for biofilm formation, binds to keratin-10 (KRT10) through its microbial surface component recognizing adhesive matrix molecule (MSCRAMM)-related globular binding region domain (BR187-385). Here, we show that BR187-385 also binds to DNA, as demonstrated by electrophoretic mobility shift assays and size exclusion chromatography. Further, heterologous expression of BR187-378 or the longer BR120-378 construct on the surface of a Gram-positive model host bacterium resulted in the formation of cellular aggregates that was significantly enhanced in the presence of DNA. Crystal structure analyses revealed the formation of BR187-385 homo-dimers via an intermolecular beta-sheet, resulting in a positively charged concave surface, shaped to accommodate the acidic helical DNA structure. Furthermore, small angle X-ray scattering and circular dichroism studies indicate that the aggregate-enhancing N-terminal region of BR120-166 adopts an extended, non-globular structure. Altogether, our results suggest that PsrP adheres to extracellular DNA in the biofilm matrix and thus promotes pneumococcal biofilm formation.
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