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RESEARCH PROGRAM

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Keywords
MACROMOLECULAR RECOGNITION; DPS PROTEINS; DPS PROTEINS-DNA COMPLEXES; FERROXIDASE ACTIVITY; PEF PROTEINS; SORCIN; SODIUM-CALCIUM EXCHANGER; RYANODINE RECEPTORS; CALCIUM-MEDIATED SIGNAL TRANSDUCTION

Supramolecular assemblies. The Dps (DNA-binding proteins from starved cells)- DNA and sorcin - calcium channels systems and their biological function

Università degli Studi di Roma "La Sapienza"
Abstract
The project concerns two systems differing in composition and function.

1. Dps proteins-DNA
The bacterial Dps proteins, expressed under a variety of stress conditions, protect DNA via two mechanisms, a physical one that entails sequestration of DNA in Dps-DNA complexes and a chemical one based on the Dps ferroxidase activity which inhibits formation of hydroxyl radicals from Fe(II). Escherichia coli Dps, the family prototype, interacts with DNA through the three lysine residues of the N-terminal region. In vitro experiments carried out within PRIN 2003 demonstrated that native Dps condenses plasmid DNA, i.e. forms large Dps-DNA complexes containing many Dps molecules and one or few plasmids, whereas mutants with one lysine in the N-terminus bind DNA without condensation. Moreover, unpublished data show that such lysine residues are partially methylated in Dps expressed during the stationary growth phase, but are unmethylated in the protein expressed during the exponential one. Methylation influences the mode of interaction with DNA, i.e. favors its condensation, and hence appears to act as a novel regulatory factor of potential importance in an evolutionary perspective. To define the mode of interaction between the two macromolecules at a resolution level never achieved before, the crystallization experiments which resulted in the production of X-ray quality albeit very fragile Dps-DNA co-crystals will continue; first their stability will be increased >>>

Principal Investigator
Emilia CHIANCONE Università degli Studi di ROMA "La Sapienza"
Research Objectives
The general aim of all studies on biological supramolecular structures, which involve mainly proteins and nucleic acids, is the understanding of the principles that determine the high specificity of the binding reaction, and of the energetics and dynamics of the process. The latter aspect is of importance since it allows the system to respond to the requirements of the living cell. The availability of a continuously increasing number of crystallographic structures of proteins and nucleic acids greatly facilitates the task. Indeed, the structural knowledge of the interacting surfaces allows one to plan and carry out focused experiments that permit the definition of the above mentioned parameters.
The specific aims of the project concern the structural and functional characterization of two different systems: the complexes between DNA and members of the Dps (DNA-binding Proteins from Starved cells) family and those between sorcin (Soluble Resistance-related Calcium-bINding protein) and Ca2+ channels. The proponent's group has already contributed to the analysis of their properties.
Dps proteins are expressed by bacteria under nutrient depletion in order to protect DNA from the attack of damaging factors (e.g. oxidants, alkylating agents or radicals). They represent one of the most important defence mechanisms during starvation and contribute to the enhancement of bacterial resistance during this and other stress conditions. The protective action of Dps >>>

Timescale
24 months
National and international background
The text of the B model is reported below since the proposed project comprises only one Research Unit.

The study of supramolecular structures is central to modern biology because the biological world is rich in such ordered molecular assemblies whose role is of fundamental physiological relevance. Examples that exemplify their nature, complexity and efficiency are oligomeric proteins and the extended actin and tubulin polymers of the cytoskeleton, the lipid bilayers of membranes, the multi-enzymatic complexes involved in cell respiration, and the protein-nucleic acids complexes of chromosomal structures. Supramolecular assemblies are stabilized by diverse weak interacting forces that contribute to the specific recognition of the different components and permit their dynamic assembly and disassembly in response to cellular cues; moreover, interactions with small molecules and ions may be of great structural and functional relevance.
The research project addresses the study of two different supramolecular systems, chosen on the basis of the knowledge acquired by the proponents thanks to grants from PRIN (1999, 2001 and 2003) and the National Research Council. The conceptual approach provides the unifying element, while the methodology differs in part; for this reason the two systems are presented separately.
The interest in the experimental systems chosen is proven by the numerous papers published in prestigious international journals, as apparent >>>