PEPTIDE AND PROTEIN RECONSTITUTION IN BICELLES FOR PULSED ESR STUDY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The study of membrane protein structure by ESR is challenged by the non-uniform spatial distribution of proteins in the samples. The standard way of protein reconstitution in liposomes lead to their low average concentrations and increased local concentrations. Thus heterogeneity of spin distribution is manifested directly in dipolar signals as a non-linear baseline in the logarithmic plot with a non-linearity comparable in magnitude to often unresolved signals from spin pairs. The other problem is a tendency of membrane proteins to aggregate, which makes things worse. Using detergents helps to alleviate the problem. Although very useful in pulse ESR study, the detergents may affect protein structure, oligomerization number, and functionality. We see clearly that we cannot use detergents, to study peptides. In order to place a protein or peptides at more relevant conditions we are exploring reconstitution in bicelles, which as is the case of detergents would help the study of membrane proteins and peptides in lipid bilayers, since they would be isolated from each other and with heterogeneity effects greatly reduced.