• sma
• sav1866
• purification
• protein
• pgp
• membrane
• exporter
• epr
• drug resistance
• abc transporter
• smalp
• spinl label

One of the greatest challenges in the field of modern science is based on the study of membrane proteins, which thanks to the evolution in the field of physics, chemistry and engineering, has made it possible to obtain tools that have enabled the study of these proteins. Membrane proteins contribute to the 20-30% of the proteome in all genomes but they are not only numerous, they play a crucial role in the life of the cell, as they take part in various and important functions such that they have earned the nickname of “cellular gate-keeper”. Membrane proteins (MP) are involved in complex process of mediating between cells and their environment. They undertake neurotransmission, sensing, transport of nutrients and drugs in and out of the cell. Many MPs are also enzymes, they are also constituents of large dynamic assemblies, intervening in cellular processes such as endocytosis, cell division and migration. In contradiction to their abundance and importance, only a few MP proteins have been purified and characterized both from the functional and structural point of view. Their importance of this class of protein is underlined by the fact that various diseases are connected with defects in MP and they are also involved in multidrug resistance and failure in chemotherapy.
This project focuses its attention on the study of a particular class of membrane proteins, ATP binding cassette (ABC), one of the largest superfamilies of membrane transporters present in eukaryotes and prokaryotes. The choice to study these transporters does not arise only from their abundance, but also for the role they play within the cell and their involvement in drug resistance, one of the greatest challenges in the medical field and in diseases like cystic fibrosis, retinal degeneration and adrenoleukodystrophy. The viability of new techniques to investigate the structure and function of these transporters may constitute a new starting point to fully understand their involvement in these pathologies and the mechanism of import-export of drugs. The ABC transporter chosen for this project is Sav1866, a trans-membrane protein of Staphylococcus aureus, involved in drug resistance. The choice is not due only to the task performed at the level of microorganism, but because studies have shown the similarity of the architecture of this MP to the human multidrug transporter P-glycoprotein. A well characterization of Sav1866 could provide a general and a cheaper model for predict P-glycoprotein structure and behavior. One of the first difficulty encountered in the study of MP is obtaining the proteins themselves. MP are usually present at low levels in biological membranes and it is rare for a single membrane protein to be expressed at levels such as to be a major component of the protein composition of cellular membrane, hence the need to optimize the protocols that lead to overexpression of the proteins of interest. Another difficulty is that MPs are generally not soluble in aqueous solution and need an environment that satisfies their high hydrophobicity. For this purpose, one of the major approaches to isolating MP uses detergents. The amphipathic nature of detergents (polar head and hydrophobic tails) meant that they were widely used for the solubilization of membranes, mimicking the natural environment. However, the use of these surface-active agents is not sufficient to provide an environment that reflects the complexity of lipid bilayer. This derives from the fact that the lipid membrane is made up of a large number and variety of lipids, which contribute to creating a unique environment of its kind. The complexity of the native environment, which has an impact on MP, is lost with the use of detergents, but is maintained for a restricted area if a simple organic copolymer, styrene-maleic acid (SMA) is used to solubilize membrane. SMA extract the proteins from the membrane into nano disks consisting of a central lipid supported by a SMA polymer anulus, this structure is called SMALP.
For the characterization of the proteins, the site-directed spin labeling (SDSL) technique was used, coupled with EPR spectroscopy. SDSL is a technique used to investigate the structure and local conformation of proteins using electron spin resonance (EPR). Spin labels are paramagnetic molecular reporters (they have an unpaired electron) and in this technique the aminoacids, in particular a cysteine residue, acts as probe attachment site. Unique cysteine residues can be introduced as recombinant protein by site-directed mutagenesis. At neutral pH, the amino acid thiol group reacts with the functional group of the probe forming a covalent bond. thanks to their sensitivity to motion, it is exploited for EPR spectroscopy. SDSL with EPR can be used to understand the structure of SMALP encapsulated membrane proteins and measure the distance between these probes to determine the conformation assumed by MPs in their native lipid environment.