Proteins are the most important functional units of living matter: all cellular processes are governed by specific proteins possessing highly specialized functional roles. Understanding the intimate mechanisms of cell functions necessitates a detailed knowledge of the 3D-structure of proteins.
The project aims to elucidate the three-dimensional structure of several scientifically significant proteins using X-ray diffraction. Understanding protein structures is essential as proteins govern cellular processes with specialized functional roles. The proteins targeted in this study include phosphoketolase from Lactococcus lactis, the recently identified KIND domain of protein tyrosine phosphatase PTP-BL, and PTP-EYA.
1. Phosphoketolase (PK): A key enzyme in the pentose phosphate pathway, representing the largest group of thiamin diphosphate-dependent enzymes. Despite its importance, many aspects of PK remain unknown. The project aims to uncover the stereospecificity of the PK-catalyzed reaction, the conformation of key intermediates, and the overall reaction mechanism.
2. Protein Tyrosine Phosphatases (PTP): This large family of proteins is involved in critical cellular signaling pathways. The EYA proteins, part of this family, lack sequence homology with other PTPs. Determining their 3D structure will highlight the active site nucleophile and the role of the essential Mg2+ ion.
3. KIND Domain of PTP-BL: Understanding the physiological role of this catalytically inactive C-terminal domain of protein kinases will be enhanced by elucidating its 3D crystal structure.
Achieving the project objectives requires interdisciplinary collaboration among biochemistry, molecular biology, crystallography, and X-ray diffraction specialists. The outcomes are expected to advance our understanding of these proteins' functions and mechanisms significantly.
Head of Department
Prof. Dr. Stefan Szedlacsek is the Head of the Enzymology Department at the Institute of Biochemistry of the Romanian Academy. He holds a PhD degree in Biotechnology from Polytechnical University of Bucharest as well as a MSc in Organic Synthesis (Polytechnical University, Bucharest) and MSc in Mathematics (University of Bucharest). As a visiting scientist, he performed research in the field of cholesterol metabolism at the University of Illinois at Urbana-Champaign (USA), where he succeeded to evidence a new pathway in the metabolism of oxysterols. He is an “Alexander von Humboldt” fellow and worked in Germany, in the Institute of Biochem More...
Conceptual approach of the project: the conceptual approach of this project, which focuses on the determination of the three-dimensional structure of key proteins using X-ray diffraction, is both ambitious and methodologically sound. The comprehensive framework of the project integrates several critical stages of structural biology, from gene cloning and protein expression to advanced purification, crystallisation and structural modelling. Here are some key aspects of the conceptual approach:
Integration of multi-disciplinary techniques: one of the most commendable aspects of the project is the seamless integration of different disciplines and techniques within molecular biology and structural biochemistry. By using cloning, protein expression, advanced purification methods and crystallisation, the project ensures a thorough investigation of each protein of interest. This holistic approach is critical to obtaining high quality crystals and reliable structural data.
Focus on advanced cleaning processes: of particular note is the project's focus on the development of advanced purification processes. Given that obtaining pure and homogeneous protein samples is a prerequisite for successful crystallisation and subsequent X-ray diffraction analysis, the multi-step purification protocols implemented - such as affinity chromatography, ion exchange and gel filtration - demonstrate a strategic focus on optimising protein quality. This meticulous attention to purification reflects a deep understanding of the complexities involved in protein crystallography.
Systematic crystallisation strategy: the project takes a systematic approach to protein crystallisation, using different crystallisation screens and optimising conditions to achieve high quality crystals. The use of different kits and methodical variation of parameters such as pH and precipitant concentration underlines the thoroughness and adaptability of the project. This strategic rigour is essential to overcome the inherent challenges of protein crystallisation, particularly for difficult-to-crystallise proteins.
Use of seleno-methionine labeling: the incorporation of selenomethionine labeling to facilitate phase determination in X-ray crystallography is a sophisticated technique that enhances the accuracy of structural elucidation. This approach indicates a forward-thinking application of advanced methodologies to address potential obstacles in phase problem resolution, thereby improving the overall reliability of the structural data obtained.
Collaboration and resource utilization: the project's collaboration with synchrotron facilities, such as the ESRF in Grenoble, exemplifies an effective use of specialized resources and expertise. By leveraging state-of-the-art facilities and working with experienced researchers, the project maximizes the quality and precision of its X-ray diffraction analyses. This collaborative effort highlights the importance of utilizing external resources to augment internal capabilities.
Potential for broader applications: conceptually, the project's framework sets a precedent for future research in structural biology. The methodologies and insights gained can be applied to a wide range of proteins, paving the way for new discoveries in enzyme mechanisms, drug design, and therapeutic interventions. The project's approach not only addresses the immediate objectives but also lays a foundation for long-term advancements in the field.
The specific aims of this project are:
1. To determine the three-dimensional structure of the PTP-BL KIND domain;
2. Determine the three-dimensional structure of PTP-Eya;
3. Determination of the three-dimensional structure of phosphoketolase from Lactococcus lactis;
Project coordinator: Dr.Stefan Szedlacsek
Dr.Balasu Mihaela Camelia - scientific researcher
Georgiana Petrareanu - PhD student
Mihaela Pascaru - PhD student
Rodica Badea - PhD student
Impact of the project
This project has had a significant impact on the field of structural biology, providing detailed insight into the three-dimensional structures of important proteins. By successfully determining the structures of BL-KIND,
PTP-Eya and phosphoketolase, the project has had the following impacts:
· Improved our understanding of protein function: The structural models developed have deepened our understanding of the molecular mechanisms and interactions in which these proteins are involved, particularly in enzymatic processes and signalling pathways.
· Facilitated drug design and development: The high-resolution data obtained can serve as a fundamental resource for the design of new drugs targeting these proteins, which could lead to innovative treatments for diseases in which these proteins play a critical role.
· Advanced purification techniques: Advanced purification methods developed in the project can be applied to other proteins, improving the efficiency and effectiveness of future structural biology studies.
· Collaborative research fostered: The project results have paved the way for further collaborative research, encouraging the exchange of knowledge and techniques between researchers in the field.
Project objectives and achievements
1. Determination of the three-dimensional structure of BL-KIND
· Achievements:
o Successful cloning and expression of the DNA encoding the KIND domain.
o Development of an advanced purification process for BL-KIND.
o Carried out crystallisation trials and obtained X-ray diffraction data.
o Generation of three-dimensional models of the crystallised proteins.
2. Determination of the three-dimensional structure of PTP-Eya
· Achievements:
o Successful cloning and expression of recombinant PTP-Eya.
o Development of an advanced purification procedure for PTP-Eya.
o Enzyme activity assays confirmed that the proteins retained their native conformation.
o Carried out crystallisation tests and obtained X-ray diffraction data.
o Generated three-dimensional models of the crystallised proteins.
3. Determination of the three-dimensional structure of phosphoketolase (PK)
· Achievements:
o Successful expression of PK and PK-TEV-6XHis with and without fusion peptides.
o Development of an advanced purification procedure for both forms of PK.
o Establishment of the synthetic pathway for xylulose-5-phosphate (Xu5P).
o Spectral and chromatographic characterisation of Xu5P.
o Carry out crystallisation tests and obtain high quality crystals for X-ray diffraction.
o Production and crystallisation of selenomethionine-labelled proteins.
o Obtained high resolution (2.2 Å) diffraction data for PK.
Summary of achievements
· Cloning and expression of the genes for PTP-KIND, PTP-Eya, PK, and PK-TEV.
· Advanced purification of the recombinant proteins PTP-KIND, PTP-Eya, PK, and PK-TEV.
· Enzymatic activity testing confirmed the active forms of the obtained enzymes.
· High-quality crystallization tests performed for all enzymes, achieving suitable crystals for X-ray diffraction (2.2 Å resolution for phosphoketolase).
· Successful marking of phosphoketolase with selenomethionine and subsequent crystallization tests.
· Identification of PP, PYR, and C-terminal domains in the phosphoketolase structure and some amino acids involved in the enzymatic reaction through the resulting structural model.
This project has successfully met its primary objectives, providing valuable structural insights into important proteins using X-ray diffraction techniques. The high-resolution structural data obtained will contribute significantly to the understanding of the molecular mechanisms and potential regulatory roles of these proteins.
Articles:
1. „Interface Analysis of the Complex between ERK2 and PTP-SL”.; Authors: M. C. Balasu; L. N. Spiridon; S. Miron; C. T. Craescu; A.J.Scheidig; A-J.Petrescu; S.E. Szedlacsek; PLoSONE 4(5): e5432; 2009
2. “Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis”, Authors: Petrareanu, G; Balasu, MC; Zander, U; Scheidig AJ and Szedlacsek, S.E., Acta Cryst. F66, 805–807; 2010
3. “Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics“; Authors: Petrareanu G; Balasu MC; Vacaru AM; Munteanu CV; Ionescu AE; Matei I; Szedlacsek SE. Appl Microbiol Biotechnol. 98, 7855-67; 2014.
Conference:
2008: Posters (on PTPs): “Structure-function analysis of Eyes absent proteins - aspartate dependent protein tyrosine phosphatases”, poster authors: Mihaela Pascaru; Stefan E. Szedlacsek, Congress of the International Union of Crystallography, 23-31 august, Osaka, Japan
2008: Poster: “Expression, purification and crystallization of phosphoketolase from Lactococcus lactis”; poster authors: Petrareanu G.; Stefan E.Szedlacsek; Congress of the International Union of Crystallography, 23-31 august, Osaka, Japan
2009: Oral presentation Stefan Szedlacsek: “Interface Analysis of the Complex between ERK2 and PTP-SL, The International Conference Europhosphatases 2009 “Protein phosphatases in development and disease”, Egmond aan Zee, (The Netherlands), 14-18 July.
Romanian Academy