DEVELOPMENT AND APPLICATION OF ELECTRON SPIN RESONANCE (ESR) SPECTROSCOPY TO STUDY INTERACTIONS IN BIOMEMBRANES
Electron Spin Resonance (ESR) spectroscopy has been extensively used to study membrane dynamics and structure. ESR has been shown to be sensitive to a wide range of molecular motions (105 to 1012 s-1). The use of multi-frequency ESR has helped analyze the complex modes of motions typically observed for spin probes attached to biomolecules. Further, the use of pulsed two dimensional ESR techniques (e.g. 2D-ELDOR) has increased the sensitivity to detect molecular motions and has helped to better resolve spectral features. The use of 2D-ELDOR at high frequencies has been particularly challenging due to relatively short dead times required (T2 minimum ~4 ns). We have developed a 95 GHz high power spectrometer which can be operated at room temperatures to study ‘lossy” aqueous samples. We have used this technique to study the partitioning of TEMPO into DMPC. We show that 95 GHz 2D-ELDOR can resolve the spectra of TEMPO in water and the lipid phase. Further, for the first time, we have observed the exchange dynamics of TEMPO between these phases and estimate it to have an exchange rate of 0.5 µs-1. We have then used multi-frequency ESR to study the formation of nanoscopic, co-existing Lo & Ld domains in ternary lipid mixtures. We find that formation of nanodomains does not affect the phase behavior, but indicates the presence of a transition “boundary” region that has intermediate order between the Lo & Ld domains. We further observe that crosslinking of biotinylated gramicidin in these nanodomains, affects the phase behavior by reducing the ordering in the system, but does not change the phase behavior in the macrodomain region. We have also studied the effect of cholesterol depletion in plasma membrane vesicles obtained from RBL cells, and observe that this increases the fraction of the Ld phase in the system. Finally we have used pulsed dipolar ESR to study the crosslinked aggregates of IgE, and have determined that inter IgE-IgE distances to be about 3 - 4 nm. All these results show the ability of ESR to address a wide range of questions related to membrane biophysics.
Physical chemistry; Biophysics; Chemistry
Freed, Jack H.
Baird, Barbara Ann; Feigenson, Gerald W.
Chemistry & Chemical Biology
PHD of Chemistry & Chemical Biology
Doctor of Philosophy
dissertation or thesis