High resolution field cycling NMR methods will be developed as robust tools for studying the dynamics and headgroup orientation of phospholipids in model membranes. Initial work will test assumptions made in data reduction and interpretation of preliminary 31P studies. This will require synthesis of phospholipids where different segments have been deuterated, as well as comparisons of field cycling NMR data for model systems to molecular dynamic simulations to identify bilayer motions contributing to the relaxation rates. Field cycling relaxation results will also be compared to those from standard fixed field T1r measurements. Once validated, the field cycling methods will be used to examine lipid headgroup conformation and lateral interactions in multicomponent vesicles and aggregates with additives such as Ca2+ or cholesterol, characterize the specific interactions of two peripheral proteins, annexin V and Streptomyces chromofuscus phospholipase D, that exhibit kinetic or binding specificity for a given headgroup phospholipid in multicomponent vesicles, and assess the effect of a transmembrane helical peptide, WALP16, on phosphate conformation and dynamics. While examination of these diverse systems should provide a critical evaluation of the usefulness of the methodology, the results themselves will provide detailed information on how the two peripheral proteins and the transmembrane helix affect the dynamics and conformation of the phosphate moiety of phospholipids in multicomponent systems.
Both undergraduate and graduate students will be trained in NMR theory and experiments. One-on-one contact in the laboratory with the PI and collaborators will help produce the next generation of researchers and science educators. The dissemination of the results of this research will introduce the NMR community to a powerful new method, easy to implement with conventional spectrometers, that is optimal for characterizing diverse phospholipid aggregates and phospholipid/protein interactions. The quantitative information on the dynamics and conformation of the phosphate moiety of phospholipids in these multicomponent systems will be useful to researchers carrying out molecular dynamic simulations of membrane motions by providing experimental tests of computational results.
