New strategies for the study of membrane protein structure and dynamics with SDSL and NMR.
Site Directed Spin Labeling
In SDSL, a nitroxide probe is introduced to a unique site within a protein. In most cases, a cysteine mutant is introduced and subsequently reacted with a sulfhydryl-reactive nitroxide reagent. The resulting nitroxide side chain, the most commonly used shown in Fig. 1a, is sensitive to the molecular environment, which allows the determination of structure as well as site-specific dynamics. One of the major attributes of the SDSL technique is the ability to investigate membrane proteins in the native lipid environment.
Currently membrane protein investigations rely on a qualitative understanding of the nitroxide side chain dynamics. The EPR lineshapes of the commonly used nitroxide label on helical surface sites in soluble proteins is different from that observed on membrane proteins (Fig. 1). Quantifying dynamic modes requires an understanding of the dynamics of the nitroxide in the lipid environment. Using x-ray crystallography, EPR spectral simulations, and nitroxide side chain derivatives we are investigating the molecular determinants of the dynamics of lipid exposed α-helical sites.
Figure 1. (a) X4/X5 model for aqueous exposed α-helical proteins. (b) Motional model for the intermediate regime observed for aqueous exposed helical sites. (c) Typical EPR lineshapes of aqueous exposed α-helical nitroxide side chains. A spectral simulation is shown in red and offset from the experimental spectrum (d) Typical EPR lineshapes of lipid exposed α-helical nitroxide side chains.
