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P-C4-53 Phospholipid asymmetry in photoreceptor disc membranes
C4 Lipid-protein interactions and dynamics P-C4-53 PHOSPHOLIPID ASYMMETRY IN PHOTORECEPTOR DISC MEMBRANES HESSELE.,‘HERRMANN A.:HOFMANN K.P.’ ’ Inst.Med.Physics& Biophysics,CharitC,Berlin * Inst. Biology/Biophysics,Humboldt University, Berlin (Germany) Purpose: Investigations of the transmembrane distribution of phospholipids in photoreceptor rod outer segment(ROS) disc membraneshave led to controversial results. Current work indicates a dependenceon the intactnessof native membranes. Up to now the role of lipid asymmetry in signal transductionis not clear. Methods: Disc membranes of freshly prepared ROS from bovine retinae were used. The transmembranemovement of lipids was assessed by spin-labeled phospholipids (phosphatidylcholine PC, phosphatidylserinePS)using either the back exchangemethod or the ascorbatereduction method. Results: A rapid transmembranediffusion of PC and PS was found, which did not depend on ATP. At equilibrium, PC was more enriched than PS in the inner monolayer. Conclusion: The rapid transmembrane diffusion occurs in ROS disc membranes is not driven by ATP-consumingprotein(s) (Translocasen).
P-C4-54 ANIONIC PW&OL~ kR42 DETERMINANTS O@ lb@l%$&kANE PROTEIN TOPOLOGY VAN KLOMPENBURG W,’ NILSSON IM,2 VON HEIJNE G,2 DE KRUIJFF B.’ *Instituteof Biomembranes, UtrechtUniversity @IL) *ArrheniusLaboratory. StockholmUniversity(S) Purpose: Positively charged amino acid residuesare most often enriched in the untranslocatedloops of membrane proteins. We tested whether interactionsof these residueswith negatively charged phospholipids can control the orientation of membrane proteins. Methods: Constructs derived from the membrane protein Lep with different charge distributions acrossthe transmembranesegments were expressedin E.coli strains with controlled lipid headgroup compositions. The topologies of the constructswere determined by proteolytic and enzymaticassays. Results: The topologies of several constructs were dependent on the lipid composition; when lessanionic phospholipids were present, positively charged residuestranslocated more efficiently. Conclusion: Anionic phospholipids prevent positive chargesfrom translocation and are therefore strong determinants of membrane protein orientation.
P-C&56 INTERACMON OF HBV pm!3 DOMAINS wIm~-~vEsacLm ‘NirREZ, E.. ‘GbMEZ-CiUTl8kRE2, ‘GbMEZ-CiUT@kREZ, J., ‘Y6LAMOS, B., ‘PACHECO, B., *PETERSON, D.L. and ‘GAVILANES, F. ‘Dpto.Bicqulmica,Fat. Qufmicas,UCM, Madrid(Spin) and ‘Dept. Biochemistry. MCV-VCU, Richmond, Va.
P-C%55 SEIiECTIVE LYSIS OF BACTERZA BY’ DIASTEREOMERS OF PARDAXIN OREN 2. SHAI
Y.
*Department of Membrane Research Biophysics,Weizmann Inst.of Science, Rehovot (IL)
and
Purpose: The cytolysin pardaxin can lyse both mammalian cells and bacteria, but its diastereomers can lyse only bacteria. To determine the molecular basis for the observed cell specificity, diastereomers of pardaxin and its truncated forms were synthesized, fluorescently labelled, and structurally and functionally characterized. Methods: Secondary structures of the peptides were determined using circular dichroism spectroscopy,cytotoxicity was examined against bacteria and human red blood cells (hRBC), and their mode of action with model membranes was studied using fluorescencespectroscopy. Results: The diastereomers did ,not retain a-helical structures,lost their hernolytic activity towards hRBC, but retained the high antibacterial activity of native oardaxin. Furthermore, they c&d permeate iegatively charged phospholi id (PS/PC) vesicles bet?er than zwitterionic &c ) ones, and were bound onto the surface of the membrane. Conclusions: Diastereomers of pardaxin lyse bacteria in a detergent like (“carpet-like”) manner rather than via the format&n of transmembiane pores, in which the u-helical structure is not prerequisite.
(USA).
Purpose: The Hepatitis B virus (HBV) envelope contains three viral proteins: major, middle and large, which contains the external preS domains. Their roles in the adsorption and penetration of the virus have not yet been elucidated. Methods: Recombinant preS domains (subtype ayw) were obtained. Fluorescencemeasurements of NBD-labeled preS and CD spectra were used to monitor the interaction of the protein with phospholipid vesicles. Resuits~ Binding isotherms were obtained from the increase in the fluorescenceemission yietd of NBD from which a surface par&km coefficient can be calculated. As indiited by CD the interaction is accompanied by a change in the secondary struchtre of the protein. Conclusions: The interaction between preS and vesicles seems to be specific for negatively charged phosphoJ&idslud increasesat ai$dic PH. These m&s sttppmt 3% &@a6% preS can be important in the first steps of the viral cycle. 130
P-C4-54 ANIONIC PW&OL~ kR42 DETERMINANTS O@ lb@l%$&kANE PROTEIN TOPOLOGY VAN KLOMPENBURG W,’ NILSSON IM,2 VON HEIJNE G,2 DE KRUIJFF B.’ *Instituteof Biomembranes, UtrechtUniversity @IL) *ArrheniusLaboratory. StockholmUniversity(S) Purpose: Positively charged amino acid residuesare most often enriched in the untranslocatedloops of membrane proteins. We tested whether interactionsof these residueswith negatively charged phospholipids can control the orientation of membrane proteins. Methods: Constructs derived from the membrane protein Lep with different charge distributions acrossthe transmembranesegments were expressedin E.coli strains with controlled lipid headgroup compositions. The topologies of the constructswere determined by proteolytic and enzymaticassays. Results: The topologies of several constructs were dependent on the lipid composition; when lessanionic phospholipids were present, positively charged residuestranslocated more efficiently. Conclusion: Anionic phospholipids prevent positive chargesfrom translocation and are therefore strong determinants of membrane protein orientation.
P-C&56 INTERACMON OF HBV pm!3 DOMAINS wIm~-~vEsacLm ‘NirREZ, E.. ‘GbMEZ-CiUTl8kRE2, ‘GbMEZ-CiUT@kREZ, J., ‘Y6LAMOS, B., ‘PACHECO, B., *PETERSON, D.L. and ‘GAVILANES, F. ‘Dpto.Bicqulmica,Fat. Qufmicas,UCM, Madrid(Spin) and ‘Dept. Biochemistry. MCV-VCU, Richmond, Va.
P-C%55 SEIiECTIVE LYSIS OF BACTERZA BY’ DIASTEREOMERS OF PARDAXIN OREN 2. SHAI
Y.
*Department of Membrane Research Biophysics,Weizmann Inst.of Science, Rehovot (IL)
and
Purpose: The cytolysin pardaxin can lyse both mammalian cells and bacteria, but its diastereomers can lyse only bacteria. To determine the molecular basis for the observed cell specificity, diastereomers of pardaxin and its truncated forms were synthesized, fluorescently labelled, and structurally and functionally characterized. Methods: Secondary structures of the peptides were determined using circular dichroism spectroscopy,cytotoxicity was examined against bacteria and human red blood cells (hRBC), and their mode of action with model membranes was studied using fluorescencespectroscopy. Results: The diastereomers did ,not retain a-helical structures,lost their hernolytic activity towards hRBC, but retained the high antibacterial activity of native oardaxin. Furthermore, they c&d permeate iegatively charged phospholi id (PS/PC) vesicles bet?er than zwitterionic &c ) ones, and were bound onto the surface of the membrane. Conclusions: Diastereomers of pardaxin lyse bacteria in a detergent like (“carpet-like”) manner rather than via the format&n of transmembiane pores, in which the u-helical structure is not prerequisite.
(USA).
Purpose: The Hepatitis B virus (HBV) envelope contains three viral proteins: major, middle and large, which contains the external preS domains. Their roles in the adsorption and penetration of the virus have not yet been elucidated. Methods: Recombinant preS domains (subtype ayw) were obtained. Fluorescencemeasurements of NBD-labeled preS and CD spectra were used to monitor the interaction of the protein with phospholipid vesicles. Resuits~ Binding isotherms were obtained from the increase in the fluorescenceemission yietd of NBD from which a surface par&km coefficient can be calculated. As indiited by CD the interaction is accompanied by a change in the secondary struchtre of the protein. Conclusions: The interaction between preS and vesicles seems to be specific for negatively charged phosphoJ&idslud increasesat ai$dic PH. These m&s sttppmt 3% &@a6% preS can be important in the first steps of the viral cycle. 130