RICERCA ITALIANA     

Role of molecular interactions in the acquisition of the functional structure of model proteins

Università degli Studi dell'Insubria Varese-Como

Abstract

The project is proposed as a concerted and intertwined action of three groups with experience in structural biology (protein chemistry, molecular biology, and trafficking of peroxisomal proteins). The target is the definition of molecular recognition properties in well-characterized experimental models with large evolutionary distance (Archaea, Bacteria, and Eukarya). The main goal is the understanding at the molecular level of the specificity of some particular protein-ligand and protein-protein interactions, and their effects on protein conformation and functionality. Furthermore, we aim to identify the role of the interactions between these biomolecules in establishing a physiologically active protein in a number of prototype models representative of relevant protein classes and involved in important metabolic functions such as cell protection and/or diseases.
The effects of the binding protein G72 on the tertiary structure, dynamics and functionality of the human flavoprotein DAAO (hDAAO) will be studied. The main goal of this study, besides its contribution to our understanding of the correlation between coenzyme binding, protein folding, and modulation of the enzymatic activity in flavoproteins, is to clarify structural and functional effects of G72 binding on human DAAO in order to investigate its involvement in schizophrenia at the molecular level. Interaction of protein components in the isc (iron sulfur cluster assembly) operon of E. coli will be studied >>>

Principal Investigator

Mirella PILONE Università degli Studi INSUBRIA Varese-Como

Research Objectives

The biological functions of proteins almost invariably depend on their direct, physical interaction with other molecules. Of crucial importance is the specificity of such interactions, the general properties of binding interactions and the relationship between protein conformation and binding. This is an interdisciplinary project with the aim to provide a substantial contribution to the understanding at the molecular level of the mechanism of interactions of proteins with other macromolecules, and of their significance to physiological or pathological processes.
To this purpose, the three Units involved in the project will use their experience in protein chemistry, molecular biology, and inorganic biochemistry, in combination with a broad array of sophisticated techniques to develop a direct approach to the most important aspects above cited of this topic using selected experimental models. The three Units in this project are intertwined and complement each other in the proposed study, that spans molecular biology, protein functionality and structural biology. The large evolutionary distance between the different proteins under study appears well suited to elucidate common features in processes where protein-dependent molecular recognition is of paramount relevance.
The Varese Unit will focus on the effects of the binding protein G72 on the tertiary structure, dynamics and functionality of the human flavoprotein DAAO (hDAAO). Efforts of the Unit of Milano >>>

First Results

Phase 1

Work-package 1:
- Defining the catalytic properties of recombinant hDAAO, including its substrate specificity.
- Acquiring information on the high-order structures of recombinant, purified G72 and hDAAO, and preparing samples for preliminary crystallization attempts.
- Definition of the stability under different conditions of the overall structure of hDAAO and of structural regions relevant to its functionality.
- Framing the folding/unfolding process for hDAAO by establishing equilibria, rates, and intermediates of relevant structural transitions in the native protein.

Work-package 2:
- Establishing the role of non-isc-encoded sulfurtransferases in sulfur transfer to IscA and in subsequent cofactor assembly on IscA.
- Assessing the thermodynamic and kinetic stability of transient clusters on holoIscA and holoIscU, also in dependence of their redox state.
- Information as for the "substrate specificity" of IscA and IscU in transferring inorganic cofactors to different acceptor proteins.
- Establishing whether IscA and IscU, in the absence of other protein(s), are capable of catalysing the synthesis/transfer of inorganic cofactors under turnover conditions.

Work-package 3:
- Establishing a possible correlation between addition of aromatic aldehydes to the growth medium and expression of ADH2536 through transcriptional analysis of the Bald16 gene.
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Timescale

24 months

National and international background

In the challenging time that biosciences are now living, the understanding of structure and function in proteins at the molecular level represents the most stimulating field of structural biology. In this field, the topic of mechanisms of molecular recognition and ligand binding in macromolecules is a cutting-edge area. The increasing number of structural data now available for proteins in complexes with other biomolecules, hypothesis-driven experiments, and molecular modeling approaches, all provide important tools for understanding the fine points of molecular recognition.
We propose an interdisciplinary project aimed at contributing in a substantial way to the understanding the mechanisms of interactions of proteins with other biomolecules at the molecular level. To this purpose, the three Units involved in this project will use a multidisciplinary (and largely shared) experimental approach to study the most important aspects of this topic, by using selected experimental models. The three Units are intertwined and complement each other in the proposed study, that spans molecular biology, protein functionality, and structural biology. The large evolutionary distance between the different proteins under study is suitable to elucidate common features in molecular recognition by proteins. The experimental systems belong to the three domains of life: higher eukaryotes (in Varese), Bacteria (in Milano), and Archea (in Napoli). Archaea are phylogenetically distinct from >>>