| 1. |
- Budayova-Spano, Monika, et al.
(författare)
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Large crystal growth for neutron protein crystallography
- 2020
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Ingår i: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 21-46
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Bokkapitel (refereegranskat)abstract
- The use of neutron protein crystallography (NPX) is expanding rapidly, with most structures determined in the last decade. This growth is stimulated by a number of developments, spanning from the building of new NPX beamlines to the availability of improved software for structure refinement. The main bottleneck preventing structural biologists from adding NPX to the suite of methods commonly used is the large volume of the individual crystals required for a successful experiment. A survey of deposited NPX structures in the Protein Data Bank shows that about two-thirds came from crystals prepared using vapor diffusion, while batch and dialysis-based methods all-together contribute to most of the remaining one-third. This chapter explains the underlying principles of these protein crystallization methods and provides practical examples that may help others to successfully prepare large crystals for NPX.
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| 2. |
- Ishchuk, Olena, 1980, et al.
(författare)
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RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast's intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.
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| 3. |
- Koruza, Katarina, et al.
(författare)
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Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 21:15
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Tidskriftsartikel (refereegranskat)abstract
- Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.
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| 4. |
- Koruza, Katarina, et al.
(författare)
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From initial hit to crystal optimization with microseeding of human carbonic anhydrase IX—A case study for neutron protein crystallography
- 2018
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Ingår i: Crystals. - : MDPI AG. - 2073-4352. ; 8:11
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Tidskriftsartikel (refereegranskat)abstract
- Human carbonic anhydrase IX (CA IX) is a multi-domain membrane protein that is therefore difficult to express or crystalize. To prepare crystals that are suitable for neutron studies, we are using only the catalytic domain of CA IX with six surface mutations, named surface variant (SV). The crystallization of CA IX SV, and also partly deuterated CA IX SV, was enabled by the use of microseed matrix screening (MMS). Only three drops with crystals were obtained after initial sparse matrix screening, and these were used as seeds in subsequent crystallization trials. Application of MMS, commercial screens, and refinement resulted in consistent crystallization and diffraction-quality crystals. The crystallization protocols and strategies that resulted in consistent crystallization are presented. These results demonstrate not only the use of MMS in the growth of large single crystals for neutron studies with defined conditions, but also that MMS enabled re-screening to find new conditions and consistent crystallization success.
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| 5. |
- Koruza, Katarina
(författare)
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Perdeuteration of Biological Macromolecules: A Case Study of Human Carbonic Anhydrases
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Deuterated biomolecules - where hydrogen atoms (H) are exchanged to its isotope deuterium (D) - are essential for biological experiments in neutron scattering, but they are not readily available. In neutron scattering, D has a strong positive coherent scattering length (6.67 fm) in comparison with the negative scattering length of H (-3.74 fm). These different characteristics of neutrons to D and H are used in neutron protein crystallography (NPX) where the use of deuterated proteins maximizes signal-to-noise ratio, reduces background and gives better nuclear scattering length density maps. At the same time, NPX allows at resolutions of ∽2.5 Å or better to unambiguously determine the positions of the exchanged D. This is important because H atoms constitute nearly half of all the atoms in a protein molecule. Accurate structural information about H positions and hydrogen bonds that are involved in most aspects of protein structure and function remains challenging by using X-ray crystallography alone. Therefore, NPX offers a unique and complementary approach to X-rays for locating H atoms. My thesis focuses on production of recombinant proteins for NPX with human carbonic anhydrase IX (hCA IX) as the case study protein. The long-term ambition of my work is to show that joint X-ray crystallography and NPX can provide insight into protein–ligand interactions and can give valuable input into structure-guided drug design and computational chemistry. As an example, for this, an emerging cancer target hCA IX was chosen, that is in need of the development of isoform-specific inhibitors. In paper I, we demonstrate the improvement of binding affinity of the inhibitor acetazolamide to hCA IX when conjugated to a polypeptide. In paper IV, we discuss crystallisation optimisation for large crystal growth, that is often a bottleneck for NPX experiments. Finally, in paper IV we describe jointly refined X-ray and neutron crystal structures of hCA IXmimic (hCA II with mutations that mimic the active site of hCA IX) with two selective inhibitors. This study shows how neutron studies can contribute unique information on inhibitor binding. I was particularly interested in improvement of (per)deuteration in living organisms in order to maximize recombinant protein production and expression of hCA IX under deuterium stress, which are the main discussion points of paper II and III. In paper II, we address some of the technical difficulties of protein deuteration in vivo in bacteria, and study the biophysical effects of deuteration on the protein product. Further on we explore the possibilities improvement of deuteration of proteins by supplementing a minimal growth media with a fully perdeuterated extract from algae, as described in paper III.
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| 6. |
- Koruza, Katarina, et al.
(författare)
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Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative
- 2019
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477. ; 205:2, s. 147-154
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Tidskriftsartikel (refereegranskat)abstract
- Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.
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| 7. |
- Yang, Jie, et al.
(författare)
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Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide
- 2017
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Ingår i: Bioorganic and Medicinal Chemistry. - : Elsevier BV. - 0968-0896 .- 1464-3391. ; 25:20, s. 5838-5848
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Tidskriftsartikel (refereegranskat)abstract
- The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38. nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4. nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3. nM, whereas that of the 4-C10L17-AZM conjugate was 90. pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to μM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.
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