Characterization of Membrane Protein Preparations: Measurement of Detergent Content and Ligand Binding After Proteolipos
The study of membrane proteins is a difficult task due to their natural embedding in hydrophobic environment made by lipids. Solubilization and purification from native membranes or overexpressed system involves the use of detergent to make them soluble w
- PDF / 1,828,846 Bytes
- 16 Pages / 504 x 720 pts Page_size
- 42 Downloads / 177 Views
1. Introduction The quantity and distribution of naturally expressed membrane protein is a limiting step for functional and structural studies, which usually require important amounts of highly purified and concentrated protein (1). Along the past decades, various Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_1, © Springer Science+Business Media, LLC 2010
3
4
Ostuni et al.
strategies have been developed to either purify or overexpress membrane proteins. These strategies include the isolation and enrichment of naturally abundant proteins from their original tissues or the heterologous overexpression production systems and purification protocols (2). In this latter case, several vectors, constructions, and expression systems, including eukaryotes organism and cells, prokaryotes, and acellular systems (see further chapters), have been developed to optimize both expression and purification yields. Although there are exceptional cases of naturally abundant proteins, which can be highly enriched in a few steps (e.g., sarco– endoplasmic reticulum calcium ATPase (SERCA) from rabbit fast skeletal muscle or adenine nucleotide transporter (ANT) from heart liver mitochondria), membrane protein production protocols often include numerous purification steps mostly in the presence of a detergent (3). The choice of the detergent is complex since, on one hand, the membrane protein should be solubilized but remaining functional, (4) and on the other hand, the detergent should be compatible with biochemical and structural studies. For instance, noncharged detergents are a requisite for ion desorption experiments (MALDI-TOF), and deuterated detergents are needed for nuclear magnetic resonance (NMR) studies. Furthermore, depending on structural studies performed, additional steps might be needed in order to eliminate, diminish, or replace the detergent used to purify the membrane protein. The other major component of a membrane protein preparation is the lipid environment. Lipids could be present from extraction, or added or even exchanged, depending on the purification process. Characterization of the various components present in the final membrane protein preparation is required to assure not only the reproductibleness of experiments, but also the knowledge of the structural and the functional states of the protein. Indeed, any additional step in purification protocols implies the possibility of introducing an unexpected structural modification that may lead to misfolding and/or loss of function. This characterization is important to avoid the loss of time and money used in the analysis of improper samples and it may include several measurements: (1) the assessment of protein purity on gels and the characterization of contaminant proteins as well as the polymeric state of the protein of interest; (2) the analysis of the secondary and tertiary folding not only by spectroscopic (circular dichroism (CD)) techniques
Data Loading...
