Langmuir monolayer characterization of metal chelating lipids for protein targeting to membranes

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Abstract

Targeting and organization of proteins on lipid membranes led to applications in both biological and materials sciences. Coordination of membrane-bound metal ions by surface histidine residues provides a general method for targeting of proteins to membrane surfaces. Here we report the Langmuir monolayer properties of a new class of metal-chelating lipids. The lipids utilize the metal chelator iminodiacetate (IDA) as the hydrophilic headgroup, allowing display of divalent transition metal ions on the aqueous side of the membrane. Changes in surface pressure-molecular area isotherms were used to observe metal binding, and an association constant for Cu2+ binding to the IDA lipids of 107–8 M−1 was estimated. The ability to control binding site density is important for many applications. The IDA lipid was found to be miscible with both distearoylphosphocholine (DSPC) and 1-stearoyl-2-oleoyl-phosphocholine (SOPC) at most compositions and surface pressures.

Introduction

Materials with functionalized surfaces are of importance in both materials and biological sciences for such diverse applications as construction of sensors and devices 1, 2, synthesis of novel organic [3]and inorganic 4, 5, 6materials with defined, highly-ordered nano-scale architectures [7], catalysis [8], targeted drug delivery systems 9, 10, 11and model studies of biological membrane recognition, organization and function 12, 13. Protein immobilization on artificial lipid membrane surfaces provides several important advantages for the synthesis of these materials. Naturally occurring proteins provide a host of functions applicable to a wide variety of problems. Nature has endowed lipids and many proteins with the ability to self-assemble into organized structures and arrays. Membranes can be composed of mixtures of lipids easily allowing the ability to tailor the density of functional sites or combine multiple functional groups on the same surface. Finally, membranes are dynamic structures capable of lateral mobility and reorganization, allowing pattern formation, enhanced binding of substrates, generation of functions and signal transduction.

One method of targeting proteins to membrane surfaces utilizes the specific interaction between the side chain of the amino acid histidine and divalent transition metal ions immobilized on the membrane surface. This approach has been used by this group 14, 15, 16, 17and others 18, 19, 20for the attachment of proteins and peptides to lipid monolayers. For instance, we have shown that myoglobin, containing five surface-accessible histidines, is targeted with moderate affinity (Ka>106 M−1) to copper-chelating liposomes and Langmuir monolayers [15]. Cytochrome b5 engineered to contain a hexa-histidine tag at the C-terminus was targeted to metal-chelating monolayers with somewhat higher affinity (Ka>2×107 M−1) [16]. Poly-l-histidine was found to bind mixed IDA-Cu lipid/phosphocholine monolayers, induce aggregation of the initially well-mixed metal-chelating lipids and, subsequently, generate an optical signal due to changes in the emission properties of the fluorescent IDA-Cu lipid [21]. Finally, the protein streptavidin was immobilized and crystallized in two-dimensions on a Langmuir monolayer via coordination of 2 of its 8 naturally occurring histidines to copper-chelating lipids [17].

Each of these studies has relied upon synthetic lipids containing a metal-chelating moiety as the polar headgroup. The chelator immobilizes the metal ions at the aqueous surface of the membrane where it is accessible for protein binding. Several chelating lipids that can form ternary lipid-metal-protein complexes have been designed and studied in our laboratory: 1,2-distearyl-rac-glycero-3-(8-(3,6-dioxy)octyl-1-amino-N,N-diacetic acid) (DSIDA) [15]; 1,2-dipalmityl-rac-glycero-3-(8-(3,6-dioxy)octyl-1-amino-N,N-diacetic acid) (DPIDA) and 1-octadecyl-2-(9-(1-pyrene)nonyl)-rac-glycero-3-(8-(3,6-dioxy)octyl-1-amino-N,N-diacetic acid) (PSIDA) [16]are shown in Fig. 1. An analogous lipid displaying di-unsaturated tails synthesized specifically for two-dimensional protein crystallization [17]will be described elsewhere. The variation of the ether-linked, hydrophobic tails allows desired properties to be engineered into the lipid structures (vide infra). The hydrophilic, tridentate metal-chelating iminodiacetate (IDA) is attached to the glycerol backbone via a long (ca. 11.5 Å) tri(ethylene oxide) spacer which ensures that the metal ion is accessible for protein binding. IDA forms charge-neutral complexes with divalent transition metal ions. With Cu2+ bound by IDA, two strong coordination sites on the metal remain available for ligand (protein) binding. The IDA-Cu complex is suitable for targeting proteins through their native surface histidines as well as through His-tags and engineered metal chelating sites. IDA can also chelate Ni2+, which is useful for binding His-tagged proteins.

Proteins and peptides have also been targeted via metal-ion coordination to membranes of nitrilotriacetate (NTA) lipids 18, 23. NTA-derivatized supports are widely used with Ni2+ for purification of recombinant His-tagged proteins [24]. Because the NTA-Ni complex is negatively charged, high salt concentrations must be employed to prevent non-specific electrostatic interactions. In addition, nickel binds imidazole ligands with lower affinity than does copper [25], and NTA-Ni is therefore not suitable for targeting proteins that lack a high affinity metal binding group such as a His-tag.

In the present work, the Langmuir monolayer properties of metal-chelating IDA lipids have been characterized at the air-water and air-buffer interfaces. Changes in surface pressure-molecular area isotherms have been used to observe chelation of Cu2+ from the aqueous subphase by the lipid headgroups. Epifluorescence microscopy of the Langmuir monolayers showed domain formation during compression of the DSIDA and DPIDA lipids and changes in the domain formation in the presence of copper. In addition, the miscibility of the fluorescently labeled PSIDA with two common phosphocholine matrix lipids has been examined.

Section snippets

Materials

The metal chelating lipids DSIDA [15]and PSIDA [16]were synthesized as described previously. The synthesis of DPIDA followed the same scheme as DSIDA with substitution of the palmityl group where appropriate. The phosphocholines 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-acyl-2-(6-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino)caproyl)-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL)

Results

Surface pressure-molecular area (π-A) isotherms are sensitive indicators of the molecular organization of amphiphiles at the air-water interface. The shapes of the isotherms can provide information on ligand binding and binding-induced changes in the structure or packing of the amphiphile. We have utilized π-A isotherms to probe metal ion binding to the IDA lipids.

Monolayer properties of metal-chelating lipids

Surface pressure-molecular area isotherms have been used to characterize the film-forming and metal binding properties of three new, iminodiacetate-containing lipids. The lipid headgroups and spacers are identical, and differences in the lipid properties must be interpreted in terms of the effect of their alkyl tails. In each case, the shapes of the isotherms are generally consistent with those of other lipids with similar tails. The long, saturated chains of DSIDA produce a condensed,

Acknowledgements

The DPIDA lipid was kindly synthesized by Dr. Chao-Tsen Chen. This work was supported by the Office of Naval Research (N00014-92-J-1178). D.W.P. was a Landau fellow and is supported by a training fellowship from the National Institute of General Medical Sciences, NRSA Award 1 T32 GM 08346-01.

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