Protein Targeting to Metal-Chelating Lipid Monolayers: Toward New Materials Inspired by Nature

Author: Pack, Daniel W.

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Arnold, Frances Hamilton

Committee Members: Arnold, Frances Hamilton; Baldeschwieler, John D.; Hubbell, Jeffrey A.; Kornfield, Julia A.; Wang, Zhen-Gang

Option: Chemical Engineering

DOI: 10.7907/s3n0-5169

Abstract

Biological membranes are functional structures responsible for controlling numerous cellular functions. Many membrane functions (for example, transmembrane signaling and endocytosis) are the direct result of the dynamic nature of the membrane. The capacity to mimic biomembranes can lead to applications such as the production of biosensors and targeted drug delivery systems. In this work, a synthetic membrane-based recognition system capable of reproducing important features of biological targeting and assembly was developed. This system may open new routes to controlling molecular architecture in materials and devices, as well as produce new, functional materials.

Proteins are targeted to membranes through the affinity of surface histidine side chains for transition metal ions immobilized in the lipid headgroups. A set of metalchelating lipids for this purpose was previously synthesized in our laboratory. Through measurement of the lipid surface pressure-molecular area (π-A) isotherms, the capability of the iminodiacetate (IDA) headgroups to chelate Cu2+ was demonstrated; the apparent association constant for the IDA lipid-Cu complex is 1.3-1.8 x 108 M-1. In addition, to tailor the density of metal sites on the membrane, mixing of a pyrene-labeled IDA lipid with two phosphocholine lipids was investigated.

The binding of horse heart myoglobin to IDA-Cu lipid monolayers by naturally occurring histidines was observed through changes in the lipid π-A isotherms. In addition, targeting of a recombinant rat liver cytochrome b5, engineered to display a hexa-histidine affinity tag at the C-terminus, to IDA-Cu lipid monolayers was demonstrated. The protein binding was measured as a function of the type of IDA-Cu lipid composing the monolayer, the concentration of protein in the aqueous subphase, and the fraction of IDA-Cu lipids in the monolayer. The hexa-histidine tag was found to provide an apparent affinity for IDA-Cu lipid binding of at least 2 x 107 M-1. A Frumkin-type adsorption isotherm was developed to describe the binding of proteins to lipid monolayers.

The inherent two-dimensional order of biological membranes 1s also capable of promoting ordered growth of organic and inorganic biomaterials. Synthetic lipid monolayers can likewise induce growth of 2D protein crystals at the air-water interface. For the first time, growth of 2D protein crystals on metal-chelating lipid monolayers via coordination of naturally-occurring surface histidines is demonstrated. Two-dimensional crystals of the protein streptavidin, grown on IDA-Cu lipid monolayers, were observed using Brewster angle and fluorescence microscopies. The IDA-Cu-histidine interaction may provide a general route to the growth of 2D protein crystals for structure determination, as well as the production of functional materials based on the properties of the proteins, themselves.

Finally, many biological membrane functions are produced by ligand-induced assembly of membrane components (e.g. transmembrane signaling). Mimicking ligand-induced assembly and subsequent signaling can produce functional materials such as the IDA lipid-based metal ion sensor (Sasaki, et al. Angew. Chem. Int. Ed. Engl. 34, 905- 907, 1995). In order to probe similar ligand-induced reorganization of a pyrene-labeled IDA lipid in Langmuir monolayers, a UV-vis fluorescence microphotometer has been constructed. As a demonstration of its utility, the instrument was used to quantitate fluorescence from a pyrene IDA lipid monolayer and estimate a diffusion coefficient for the lipids. In addition, reorganization of the IDA lipids by binding of a multivalent ligand, poly(histidine), was observed via the pyrene lipid fluorescence.

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