Magnetic relaxation studies of self-associating and membrane proteins

Detta är en avhandling från Biophysical Chemistry, Lund University

Sammanfattning: Magnetic relaxation dispersion measurements have been performed on aqueous protein solutions to study the dynamics of waters in the proton transport channel of bacteriorhodopsin and the self-association of the proteins BPTI, lysozyme and beta-lactoglobulin. The measurements have focused on the bulk relaxation rates, R1 and R2, of three water nuclei 1H, 2H and 17O that are coupled to the protein environments by exchange and therefore report on properties such as the protein tumbling and dynamics of internal waters. The relaxation measurements have been performed using standard inversion-recovery and spin-echo sequences on constant field super-conducting magnets and field-variable iron-core magnets as well as a specialised fast field cycling spectrometer using field-sequences for relaxation measurements. The internal waters of bacteriorhodopsin were found to exchange on a time scale fast compared to the rate-limiting step of the photo cycle. It was found that the formation of the BPTI decamer is promoted by both increased pH and addition of salt as the charged groups of the central part of the decamer are neutralised. The self-association is most sensitive to the nature of anion. Lysozyme was found to form dimers as well as larger polymers upon addition of salt and increase of pH. The protein was found to contain large numbers of waters with nanosecond residence times. Beta-Lactoglobulin displays a pH and salt-dependent monomer-dimer equilibrium as well as a larger aggregate around iso-electric pH, which is an octamer. For both lysozyme and beta-lactoglobulin the appearance of the relaxation dispersions is seriously influenced by absence of protein purification, which may cause both quantitative and qualitative errors in the interpretation.

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