Introduction and Future of Site-Directed Spin Labeling of Membrane Proteins

Abstract

STRUCTURAL BIOLOGY AND PROTEOMICSOne of the most important challenges in the field of structural biology and structural proteomics is the study of membrane proteins. These proteins reside in cell and organelle membranes and govern many important cellular functions, including cell signaling, membrane potential stabilization, energy transduction, pH and cell volume regulation, all of which play critical roles in cancer, heart disease, osteoporosis, cystic diseases, and neurological disorders. These proteins represent more than 50% of all present and future drug targets.As the field of structural proteomics continues to grow, new insights into biochemical processes often result from a combination of structural and biophysical/biochemical information. Therefore, the three-dimensional structures of proteins are essential for understanding their biological functions. However, despite the fact that membrane proteins account for approximately 30-40% of all expressed sequences, very little is known about their structures (Arora and Tamm 2001;Bowie 2001;Smith et al. 2001). In fact, it can be calculated from the most recent state for membrane proteins of known structure (White 2006) that less than 1% of the known protein structures are for membrane proteins. Also, the molecular mechanisms by which lipids and membrane proteins act together to accomplish these tasks are largely unknown."Membrane proteins represent one of the most significant frontiers in the fields of structural biology and structural proteomics. While 30-40% of all proteins are membrane proteins, yet less than 1% of the known protein structures are for membrane proteins".

An Iron-Sulfur Cluster in the C-terminal Domain of the p58 Subunit of Human DNA Primase

“…The signal is highly rhombic with g 1 ϭ 2.087, g 2 ϭ 2.040, and g 3 ϭ 2.013. Simulation of the EPR spectrum with SIMPOW6 software (40) (39,42,43).…”

Rotamer libraries of spin labelled cysteines for protein studies

“…Although spin labelling is a well established biochemical procedure [15][16][17] , introducing nitroxide radicals into specific sites of a protein is not a trivial task. Trying to place spin labels into spatially restricted positions might lead to narrow distance distributions, but may cause local distortion of the protein structure or result in failure of the labelling.…”

SDSL-ESR-based protein structure characterization

“…The ability to track the protein structure and dynamics in a native environment and at physiological temperature is one of the great advantages of the proposed method. Another advantage is the higher sensitivity of ESR than NMR, which means that much lower concentrations of protein samples are needed to perform the experiment (Hemminga 2007).…”

“…One alternative technique is sitedirected spin labelling (SDSL) electron spin resonance (ESR). This technique enables both structural and dynamic characterization of the local conformations of a membrane protein (or any other protein) in its native environment (Alexander et al 2008;Fanucci and Cafiso 2006;Hemminga 2007;Stopar et al 2005Stopar et al , 2006. Site-directed spin labelling at multiple sites of proteins has been widely used for protein structure characterization (Huang and Cafiso 2008;Hubbell et al 1998;Jao et al 2008;Li and Fung 2009;Pistolesi et al 2006).…”