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- Hallin, Erik Ingmar, et al.
(författare)
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Functional and structural characterization of domain truncated violaxanthin de-epoxidase
- 2016
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317. ; 157:4, s. 414-421
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic organisms need protection against excessive light. By using non-photochemical quenching, where the excess light is converted into heat, the organism can survive at higher light intensities. This process is partly initiated by the formation of zeaxanthin, which is achieved by the de-epoxidation of violaxanthin and antheraxanthin to zeaxanthin. This reaction is catalyzed by violaxanthin de-epoxidase (VDE). VDE consists of three domains of which the central lipocalin-like domain has been the most characterized. By truncating the domains surrounding the lipocalin-like domain, we show that VDE activity is possible without the C-terminal domain but not without the N-terminal domain. The N-terminal domain shows no VDE activity by itself but when separately expressed domains are mixed, VDE activity is possible. This shows that these domains can be folded separately and could therefore be studied separately. An increase of the hydrodynamic radius of wild-type VDE was observed when pH was lowered toward the pH required for activity, consistent with a pH-dependent oligomerization. The C-terminally truncated VDE did not show such an oligomerization, was relatively more active at higher pH but did not alter the KM for ascorbate. Circular dichroism measurements revealed the presence of α-helical structure in both the N- and C-terminal domains. By measuring the initial formation of the product, VDE was found to convert a large number of violaxanthin molecules to antheraxanthin before producing any zeaxanthin, favoring a model where violaxanthin is bound non-symmetrically in VDE.
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- Hallin, Erik Ingmar, et al.
(författare)
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Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation
- 2016
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Ingår i: Photosynthesis Research. - : Springer Science and Business Media LLC. - 0166-8595 .- 1573-5079. ; 129:1, s. 29-41
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.
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- Hallin, Erik, et al.
(författare)
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Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.
- 2015
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Ingår i: Photosynthesis Research. - : Springer Science and Business Media LLC. - 0166-8595 .- 1573-5079. ; 124:2, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9 %. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9-C27, C14-C21, C33-C50, C37-C46, C65-C72 and C118-C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.
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- Uhlén, Mathias, et al.
(författare)
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A human protein atlas for normal and cancer tissues based on antibody proteomics
- 2005
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 4:12, s. 1920-1932
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, similar to 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.
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- Bayram, Firas, et al.
(författare)
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A Drift Handling Approach for Self-Adaptive ML Software in Scalable Industrial Processes
- 2022
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Ingår i: Proceedings of the 37th IEEE/ACM International Conference on Automated Software Engineering. - New York, NY, USA : Association for Computing Machinery (ACM). - 9781450394758 ; , s. 1-5
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Konferensbidrag (refereegranskat)abstract
- Most industrial processes in real-world manufacturing applications are characterized by the scalability property, which requires an automated strategy to self-adapt machine learning (ML) software systems to the new conditions. In this paper, we investigate an Electroslag Remelting (ESR) use case process from the Uddeholms AB steel company. The use case involves predicting the minimum pressure value for a vacuum pumping event. Taking into account the long time required to collect new records and efficiently integrate the new machines with the built ML software system. Additionally, to accommodate the changes and satisfy the non-functional requirement of the software system, namely adaptability, we propose an automated and adaptive approach based on a drift handling technique called importance weighting. The aim is to address the problem of adding a new furnace to production and enable the adaptability attribute of the ML software. The overall results demonstrate the improvements in ML software performance achieved by implementing the proposed approach over the classical non-adaptive approach.
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- Bayram, Firas, et al.
(författare)
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DQSOps : Data Quality Scoring Operations Framework for Data-Driven Applications
- 2023
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Ingår i: EASE '23: Proceedings of the 27<sup>th</sup> International Conference on Evaluation and Assessment in Software Engineering. - : Association for Computing Machinery (ACM). - 9798400700446 ; , s. 32-41
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Konferensbidrag (refereegranskat)abstract
- Data quality assessment has become a prominent component in the successful execution of complex data-driven artificial intelligence (AI) software systems. In practice, real-world applications generate huge volumes of data at speeds. These data streams require analysis and preprocessing before being permanently stored or used in a learning task. Therefore, significant attention has been paid to the systematic management and construction of high-quality datasets. Nevertheless, managing voluminous and high-velocity data streams is usually performed manually (i.e. offline), making it an impractical strategy in production environments. To address this challenge, DataOps has emerged to achieve life-cycle automation of data processes using DevOps principles. However, determining the data quality based on a fitness scale constitutes a complex task within the framework of DataOps. This paper presents a novel Data Quality Scoring Operations (DQSOps) framework that yields a quality score for production data in DataOps workflows. The framework incorporates two scoring approaches, an ML prediction-based approach that predicts the data quality score and a standard-based approach that periodically produces the ground-truth scores based on assessing several data quality dimensions. We deploy the DQSOps framework in a real-world industrial use case. The results show that DQSOps achieves significant computational speedup rates compared to the conventional approach of data quality scoring while maintaining high prediction performance.
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