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Solid phase membrane mimetics: Immobilized artificial membranes
Solid phase membrane mimetics: Immobilized artificial membranes Charles Pidgeon School of Pharmacy, Purdue Uniw'rsity, West Lafayette, Indiana vironment9 of the membrane. Measuring Kd under several solution conditions (e.g. different pH values) and different membrane lipid conditions requires substantial experimental effort. Equilibrium dialysis is usually used to obtain Kd, but nuclear magnetic resonance (NMR) ~° and electron paramagnetic resonance (EPR) 11'12 have also been used. All of these methods have long measurement times and substantial data manipulation is required before Kj can be obtained. A rapid method to measure K~ using H P L C - I A M techniques will reduce the experimental effort associated with binding studies and permit substantially more experimental conditions to be evaluated.
Introduction The biological activity of membrane proteins is critical in evaluating cell growth, drug-receptor interactions, and several important cellular events. Consequently, membrane proteins are frequently purified with the intent of elucidating the biological activity of the membrane protein. However, after membrane proteins are isolated, reconstituted membrane systems (i.e. liposomes or vesicles) are necessary to study the function of membrane proteins. Reconstituted membrane systems provide the fluid lipid environment necessary to study membrane protein function. We have taken a different approach and are attempting to provide a lipid environment on a solid support similar to the lipid environment of vesicles or cell membranes. We denote solid supports containing immobilized monolayers of membrane lipids as "solid phase membrane mimetics" or Immobilized Artificial Membranes (IAM). ~Our purpose for designing and synthesizing solid phase membrane mimetics is to develop solid phase techniques to quantitate binding events between membrane lipids and biomolecules or membrane proteins and biomolecules in a natural environment. For this purpose, we have established a data base of transmembrane proteins to obtain model compounds that are biologically importantfl High performance liquid chromatography (HPLC) columns packed with IAM solid phases have the potential to rapidly obtain binding constants between membranes using retention times instead of the long, tedious, time-consuming method of Scatchard analysis. IAM-HPLC columns are also useful in purifying membrane proteins.
Solute-membrane interactions
Significance Measuring binding constants,Kd, to membranes is critical in evaluating metal-induced membrane fusion) amino acid transport through membranes, 4 drug transport through membranes, 5 pH-dependent catecholamine uptake into vesicles, 6 the accumulation of toxic products in membranes, 7 viral-induced membrane fusion, 8 etc. Frequently the binding of molecules to membranes is pH dependent 8 or depends on the lipid enAddress reprint requests to Dr. Pidgeonat The Schoolof Pharmacy, Purdue University, West Lafayette,IN 47907 © 1990 Butterworth Publishers
Biologically important solutes are frequently drugs or peptides. Figure 1 shows a few of the processes occurring when a drug/peptide interacts with a membrane. Process (a) involves electrostatic binding to lipid headgroups; process (b) involves partitioning into the membrane; process (c) involves transport through the membrane; process (d) involves membrane adsorption followed by receptor binding. Several experiments have demonstrated the importance of process (d) in the biological activity of peptide hormones. Membrane protein receptors occupy a small area on the cell surface, and consequently a drug is more likely to interact with membrane lipid before interacting with the receptor. Studies with adrenocorticotropin-(1-24)-tetracosapeptide (ACTH: 1-24) and dynophin-(1-13)-tridecapeptide (dynorphin: 1-13) have shown that these polypeptide hormones interact specifically with the membrane before the receptor. 13-18These peptides bind to the membrane through electrostatic interactions [i.e. headgroup interactions, process (a) in Figure 1 ], which causes a conformational change in the peptide. The membrane-induced conformational change causes part of the peptide to enter the hydrophobic region of the bilayer. Once membrane associated, ACTH: 1-24 or dynorphin : 1-13 binds to the appropriate receptor embedded in the membrane. In this example, the membrane was a catalyst for a specific peptide-receptor interaction, and catalysis does not require transport through the membrane [process (c)] or merely partitioning into the interior of the membrane [process (b)]. Rapid methods to study the binding of these peptides
Enzyme Microb. Technol., 1990, vol. 12, February
149
Literature Survey
al ~ b
c1 ~TJ'~ ~ ~ Pihn°dS Phg°':Pi:s
cleavagesite
) C
~
cleavages i t e ~ membraneprotein Figure 1 A few drug/peptide membrane interactions. (a) Headgroup binding; (b) partitioning into membrane; (e) transport through membrane; (d) transport of solute to a membrane protein by partitioning into the membrane followed by binding to the protein
cZx-->C~qc
Figure 2 Schemic of TGF-~x modified from ref. 19. The palmitate addition is not shown because the cys (*) residue that accepts the palmitoyl group is unknown
Acknowledgement We wish to thank Becky Winger for help in preparing this manuscript. This work was supported by NIH Grant IUOIA1CA25712-01. References I "~ 3 4 5 6 7 8 9 10
Summary and conclusions
II
The studies discussed demonstrate the importance of developing rapid methods to purify membrane proteins and also quantitate binding events between cell membranes and biomolecules. Traditional equilibrium methods are experimentally very difficult because of long equilibration times, peptide aggregation, and the need to make several measurements to obtain a single binding constant. We are developing chromatographic methods to measure binding constants between membranes and biomolecules by using solid phase membrane mimetics. Solid phase binding assays are well established for reactions that typically occur in solution, whereas for reactions that require a membrane environment, no solid phase assay exists. Solid phase membrane mimetics have the potential of filling this gap.
12 13
150
Enzyme Microb. Technol., 1990, vol. 12, February
site
(35))@J:l/ C--_t~CZ>
receptor
and peptide analogs to different membranes would be a significant advancement in the field of membrane science. In addition to these peptide hormone-membrane studies, several biological events also require a rapid method to study peptide-membrane interactions. For example, several proteins are palmitoylated via cysteine residues through thiol ester linkages. ~9-23Palmitoylation is a posttranslational modification of the protein that facilitates localizing proteins to the plasma membrane. Figure 2 showing the palmitoylated protein TGF-e, exemplifies the significance of studying Cyscontaining amino acid sequences. TGF-c~ is synthesized as a precursor protein, and the extracellular side of the protein is cleaved at least twice (arrows) to generate soluble TGF whereas the intracellular side contains palmitoyl groups attached to Cys. The C-terminal side contains 7 Cys residues (two at the membrane interface) and the Cys residue accepting the palmitoyl group from palmitoyl-CoA is unknown. The C-terminal peptide may have amino acid sequences that fold over and bind to membrane lipids to facilitate the palmitoylation event. The sequence specificity for palmitoylation of Cys residues is unknown. A rapid method to obtain binding constants between cysteine peptides and membranes would greatly enhance the development of inhibitors of this process and perhaps determine if there is sequence specificity to the reactions.
~_._.cleavage
14 15 16 17 18 19 20 21 22 23
t'idgeon, ('. and V e n k a t a r u m . U. V. Anal. Bioch. 1989, 176, 36-47 Pidgeon. C., Williard, R. L. and Schroeder, S. 1,. Pharm. Re,s. 1989, 6, 779-786 Bentz, J., ()uzqune, N. and Schlomo, N. d i o c h c m i s t o ' 1983, 22, 3320-3330 Naoi, M., Naoi,M., Shimizu, T., Malviya, A. N. and Yagi, K. Biochcm. Biophy.~. Acta 1977, 471, 305-310 Susi, H.. S a m p u g n a , J., H a m p s o n , J. W. and Ard, ,I. S. Biochemist O' 1979, 18, 297-301 Nichols. J. and Dctlmer, D. W. Biochem. Biophys. Acta 1976, 455, 269-27 I Eriksen, E. F., Dunielsen, H. and Broadersen, R. J. Biol. Chem. 1981. 256, 4269-4274 Maeda, T., Kawasuki, K. and Ohnishi, S. Proc. Natl. Acad. Sci. USA 1981, 78, 4133-3147 Dufi'ane. S.P., Poss, A., G o o r m a g h t i g h , E. and R u y s s c h a e r l , ,I. M. I:'xpericlt/ia 198 I, 37, 524-525 Grasdalen, H., Eriksson, L, E. G., W e s t m a n , .I. and Ehrenberg, A. Biochem. Biophys. Acta 1977, 469, 151-162 Griffilh, O. H., Baumeister, J. C. and Jost, P. C. Biophys. J. 1982, 37, 152-154 Paskin, J. S. ,/. Memhr. Biol. 1977.35, 39 55 Sargent. D. F. and Sehwyzer, R. Proc. Natl. Acrid. Sci. USA 1986, 83, 5774-5778 Schwyzer, R. Biochemi,stry 1986, 25, 4281-4286 Schwyzer, R., Ernc, D. and Rolk~l, R. lh'h:. Chim. Acta 1986, 69, 1789-1797 Erne, D.. Rolka, K. and Schwyzer, R. Heir. Chim. Acta 1986. 69, 1807-1816 Ro[ka, K., Erne, D. and Schwyzer, R. Heh,, Chim. Acta 1986, 69, 1798-1806 Schwyzer, R. Biochemisto' 1986.25, 6335-6342 Bringman, T. S., I,inquist, P. B. and l)erynek, R. Cell 1987, 38, 429-440 Buss, J. E. and Set'ton, B. M. Mol. Cell. Biol. 1986, 6, 116 122 Chen, Z.-Q., Ulsh, L. S., Dubon, G. and Shih, 'I'. Y. ,I. Vir,d. 1985, 56, 607-612 Sefton, B. M., Trowbridge, 1. S., Cooper, J. A. ~md Scolnick, E. M. Cell 1982, 3 1 , 4 6 5 - 4 7 4 Schmidt. M. F. G. CIII'I'. Top. Microhiol. Immlt//o[. 1983, 102, 101-124
Introduction The biological activity of membrane proteins is critical in evaluating cell growth, drug-receptor interactions, and several important cellular events. Consequently, membrane proteins are frequently purified with the intent of elucidating the biological activity of the membrane protein. However, after membrane proteins are isolated, reconstituted membrane systems (i.e. liposomes or vesicles) are necessary to study the function of membrane proteins. Reconstituted membrane systems provide the fluid lipid environment necessary to study membrane protein function. We have taken a different approach and are attempting to provide a lipid environment on a solid support similar to the lipid environment of vesicles or cell membranes. We denote solid supports containing immobilized monolayers of membrane lipids as "solid phase membrane mimetics" or Immobilized Artificial Membranes (IAM). ~Our purpose for designing and synthesizing solid phase membrane mimetics is to develop solid phase techniques to quantitate binding events between membrane lipids and biomolecules or membrane proteins and biomolecules in a natural environment. For this purpose, we have established a data base of transmembrane proteins to obtain model compounds that are biologically importantfl High performance liquid chromatography (HPLC) columns packed with IAM solid phases have the potential to rapidly obtain binding constants between membranes using retention times instead of the long, tedious, time-consuming method of Scatchard analysis. IAM-HPLC columns are also useful in purifying membrane proteins.
Solute-membrane interactions
Significance Measuring binding constants,Kd, to membranes is critical in evaluating metal-induced membrane fusion) amino acid transport through membranes, 4 drug transport through membranes, 5 pH-dependent catecholamine uptake into vesicles, 6 the accumulation of toxic products in membranes, 7 viral-induced membrane fusion, 8 etc. Frequently the binding of molecules to membranes is pH dependent 8 or depends on the lipid enAddress reprint requests to Dr. Pidgeonat The Schoolof Pharmacy, Purdue University, West Lafayette,IN 47907 © 1990 Butterworth Publishers
Biologically important solutes are frequently drugs or peptides. Figure 1 shows a few of the processes occurring when a drug/peptide interacts with a membrane. Process (a) involves electrostatic binding to lipid headgroups; process (b) involves partitioning into the membrane; process (c) involves transport through the membrane; process (d) involves membrane adsorption followed by receptor binding. Several experiments have demonstrated the importance of process (d) in the biological activity of peptide hormones. Membrane protein receptors occupy a small area on the cell surface, and consequently a drug is more likely to interact with membrane lipid before interacting with the receptor. Studies with adrenocorticotropin-(1-24)-tetracosapeptide (ACTH: 1-24) and dynophin-(1-13)-tridecapeptide (dynorphin: 1-13) have shown that these polypeptide hormones interact specifically with the membrane before the receptor. 13-18These peptides bind to the membrane through electrostatic interactions [i.e. headgroup interactions, process (a) in Figure 1 ], which causes a conformational change in the peptide. The membrane-induced conformational change causes part of the peptide to enter the hydrophobic region of the bilayer. Once membrane associated, ACTH: 1-24 or dynorphin : 1-13 binds to the appropriate receptor embedded in the membrane. In this example, the membrane was a catalyst for a specific peptide-receptor interaction, and catalysis does not require transport through the membrane [process (c)] or merely partitioning into the interior of the membrane [process (b)]. Rapid methods to study the binding of these peptides
Enzyme Microb. Technol., 1990, vol. 12, February
149
Literature Survey
al ~ b
c1 ~TJ'~ ~ ~ Pihn°dS Phg°':Pi:s
cleavagesite
) C
~
cleavages i t e ~ membraneprotein Figure 1 A few drug/peptide membrane interactions. (a) Headgroup binding; (b) partitioning into membrane; (e) transport through membrane; (d) transport of solute to a membrane protein by partitioning into the membrane followed by binding to the protein
cZx-->C~qc
Figure 2 Schemic of TGF-~x modified from ref. 19. The palmitate addition is not shown because the cys (*) residue that accepts the palmitoyl group is unknown
Acknowledgement We wish to thank Becky Winger for help in preparing this manuscript. This work was supported by NIH Grant IUOIA1CA25712-01. References I "~ 3 4 5 6 7 8 9 10
Summary and conclusions
II
The studies discussed demonstrate the importance of developing rapid methods to purify membrane proteins and also quantitate binding events between cell membranes and biomolecules. Traditional equilibrium methods are experimentally very difficult because of long equilibration times, peptide aggregation, and the need to make several measurements to obtain a single binding constant. We are developing chromatographic methods to measure binding constants between membranes and biomolecules by using solid phase membrane mimetics. Solid phase binding assays are well established for reactions that typically occur in solution, whereas for reactions that require a membrane environment, no solid phase assay exists. Solid phase membrane mimetics have the potential of filling this gap.
12 13
150
Enzyme Microb. Technol., 1990, vol. 12, February
site
(35))@J:l/ C--_t~CZ>
receptor
and peptide analogs to different membranes would be a significant advancement in the field of membrane science. In addition to these peptide hormone-membrane studies, several biological events also require a rapid method to study peptide-membrane interactions. For example, several proteins are palmitoylated via cysteine residues through thiol ester linkages. ~9-23Palmitoylation is a posttranslational modification of the protein that facilitates localizing proteins to the plasma membrane. Figure 2 showing the palmitoylated protein TGF-e, exemplifies the significance of studying Cyscontaining amino acid sequences. TGF-c~ is synthesized as a precursor protein, and the extracellular side of the protein is cleaved at least twice (arrows) to generate soluble TGF whereas the intracellular side contains palmitoyl groups attached to Cys. The C-terminal side contains 7 Cys residues (two at the membrane interface) and the Cys residue accepting the palmitoyl group from palmitoyl-CoA is unknown. The C-terminal peptide may have amino acid sequences that fold over and bind to membrane lipids to facilitate the palmitoylation event. The sequence specificity for palmitoylation of Cys residues is unknown. A rapid method to obtain binding constants between cysteine peptides and membranes would greatly enhance the development of inhibitors of this process and perhaps determine if there is sequence specificity to the reactions.
~_._.cleavage
14 15 16 17 18 19 20 21 22 23
t'idgeon, ('. and V e n k a t a r u m . U. V. Anal. Bioch. 1989, 176, 36-47 Pidgeon. C., Williard, R. L. and Schroeder, S. 1,. Pharm. Re,s. 1989, 6, 779-786 Bentz, J., ()uzqune, N. and Schlomo, N. d i o c h c m i s t o ' 1983, 22, 3320-3330 Naoi, M., Naoi,M., Shimizu, T., Malviya, A. N. and Yagi, K. Biochcm. Biophy.~. Acta 1977, 471, 305-310 Susi, H.. S a m p u g n a , J., H a m p s o n , J. W. and Ard, ,I. S. Biochemist O' 1979, 18, 297-301 Nichols. J. and Dctlmer, D. W. Biochem. Biophys. Acta 1976, 455, 269-27 I Eriksen, E. F., Dunielsen, H. and Broadersen, R. J. Biol. Chem. 1981. 256, 4269-4274 Maeda, T., Kawasuki, K. and Ohnishi, S. Proc. Natl. Acad. Sci. USA 1981, 78, 4133-3147 Dufi'ane. S.P., Poss, A., G o o r m a g h t i g h , E. and R u y s s c h a e r l , ,I. M. I:'xpericlt/ia 198 I, 37, 524-525 Grasdalen, H., Eriksson, L, E. G., W e s t m a n , .I. and Ehrenberg, A. Biochem. Biophys. Acta 1977, 469, 151-162 Griffilh, O. H., Baumeister, J. C. and Jost, P. C. Biophys. J. 1982, 37, 152-154 Paskin, J. S. ,/. Memhr. Biol. 1977.35, 39 55 Sargent. D. F. and Sehwyzer, R. Proc. Natl. Acrid. Sci. USA 1986, 83, 5774-5778 Schwyzer, R. Biochemi,stry 1986, 25, 4281-4286 Schwyzer, R., Ernc, D. and Rolk~l, R. lh'h:. Chim. Acta 1986, 69, 1789-1797 Erne, D.. Rolka, K. and Schwyzer, R. Heir. Chim. Acta 1986. 69, 1807-1816 Ro[ka, K., Erne, D. and Schwyzer, R. Heh,, Chim. Acta 1986, 69, 1798-1806 Schwyzer, R. Biochemisto' 1986.25, 6335-6342 Bringman, T. S., I,inquist, P. B. and l)erynek, R. Cell 1987, 38, 429-440 Buss, J. E. and Set'ton, B. M. Mol. Cell. Biol. 1986, 6, 116 122 Chen, Z.-Q., Ulsh, L. S., Dubon, G. and Shih, 'I'. Y. ,I. Vir,d. 1985, 56, 607-612 Sefton, B. M., Trowbridge, 1. S., Cooper, J. A. ~md Scolnick, E. M. Cell 1982, 3 1 , 4 6 5 - 4 7 4 Schmidt. M. F. G. CIII'I'. Top. Microhiol. Immlt//o[. 1983, 102, 101-124