NMR study of CyaY and YFHJ, two proteins involved in iron metabolism

Abstract

From the Summary: In this thesis we apply Nuclear Magnetic Resonance to gain insights into the structures and functions of the two proteins CyaY and YFHJ. The overall material is organised in eight chapters, an introduction and a conclusion. Chapter 1: In this chapter the basis of Nuclear Magnetic Resonance are provided, with reference to its application to the study of biological macromolecules Chapter 2: This chapter describes the 2D and 3D NMR experiments that allow the assignment of a protein\u2019s spectra. Chapter 3: Here a short description of the bioinformatic tools and software packages available to extract information on macromolecules is provided. The material is organised following the proteins\u2019 level of structure it refers to: first there is a short description of the web databases and the programs that can be used to extract information from the primary sequence of a protein, then an elucidation of the programs useful to analyse the secondary structure and some details on the one applied in this study to calculate the 3D structure; finally, the available tools for structure comparison and refinement are reported. Chapter 4: This chapter summarises what is known about Friedreich\u2019s ataxia and frataxin. Chapter 5: Here the study of CyaY iron binding site by means of NMR is presented. In the first part of the chapter the problems and advantages of using paramagnetic probes in NMR are briefly discussed. Chapter 6: This chapter reviews the systems involved in iron-sulphur clusters\u2019 biosynthesis with particular attention to the proteins encoded by the bacterial Iron Sulphur Cluster operon. Chapter 7: An elucidation of the 3D structure determination process of the protein YFHJ is reported. In the first part circular dichroism and its application in biomolecular studies are discussed. Chapter 8: This chapter illustrates the functional characterisation of the protein YFHJ by means of NMR, mass spectrometry and fluorescence polarisation. The techniques used are elucidated in the initial paragraphs