ESR data obtained from spin-labeled animal cell membranes supports a vertical asymmetry model in which the inner and outer monolayers of the cytoplasmic membrane and the intracysternal and extracysternal monolayers of the endoplasmic reticulum bilayer have different physical states. Thus, changes in the physical state in the monolayer which faces the cytosol occur at 22 and 37 C; changes in physical state in the othermonolayer occur at 15 and 30 C. These results were obtained by comparing the temperature-sensitive partitioning of surface restricted and non-restricted ESR spin labels in right-side out, inside out, and disrupted membrane preparations. Future experiments are designed to develop and characterize other surface monolayer restricted fatty acid probes. Membrane impermeant spin destroying agents, model vesicle systems, and whole cells will be used to establish the kinetics with which probes "flip" or become internalized. Information thus obtained will be exploited to predict the behavior of photoreactive counterparts which we have begun to synthesize for high resolution mapping of membrane protein. 14C and 125I-labeled probes containing an azido function at various positions along an alkyl chain will be used to map the depth of proteins within the bilayer, the dynamics of protein intercalation into membranes, and the transmembrane movement of proteins in response to hormones, drugs, etc. Preliminary studies will involve systems where predictions can be made, e.g., Newcastle disease virus and Sendai virus (protein locations are known) and phospholipid vesicles containing phage M13 coat protein asymmetrically displayed across the bilayer (amino acid sequence is known). ESR spectral analysis of spin-labeled proteins will be performed to elicit complementary information. Data obtained from this concerted attack on membrane protein dynamics can be utilized to formulate tailored approaches to several clinically relevant problems such as (1) the dynamics of cholera toxin, (2) the mechanism whereby complement mediates cell lysis, and (3) the trans-membrane distribution of proteins in normal vs malignant cells.
NIH ExPorter
NIH ExPorter