Fluorescence Characterization Of Protein And Lipid Dynamics

Fluorescence techniques were used to study the motion of protein domains in adenylate kinase, and to characterize the formation of myelin in neuron Schwann cell co-cultures. Domain motion upon substrate binding was examined in adenylate kinase using fluorescence resonance energy transfer. The process of myelin formation was examined by observing the growth and composition of myelin using a fluorescent ceramide analogue. Crystallographic evidence suggests that there is a large hinged domain motion in adenylate kinase. To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated. Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site. Mutant F86W that is located at the AMP binding site, while mutant S41W that is in the loop that closes over AMP. Energy transfer was measured between each of these tryptophans and 5-((2(-(acetyl)-amino) ethyl) amino) naphthalene-1-sulfonic acid covalently bound to the single cysteine residue at position 77. The distance between the tryptophan of the F137W mutant adenylate kinase and the AEDANS-labeled cys-77 decreased by 12.1 A upon the binding of the bisubstrate inhibitor $rm Psp1,Psp5$-$rm bis(5spprime$-$rm adenosyl)$pentaphosphate $(APsb5A).$ These results suggest that there is significant closure of the ATP binding domain upon the binding of ATP or $APsb5A.$ Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain. Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal co-cultures. The uptake of the fluorescent ceramide analogue N-(5-(5,7-dimethyl BODIPY$sp{rm TM})$-1-pentanoyl) D-erythro-sphingosine and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days three and seven after induction of myelination. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier.