Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

Abstract

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.The two-dimensional (2D) crystallization of proteins on lipid monolayers has become a powerful technique with diverse applications in fields ranging from structural biology to materials science. Protein 2D crystals have been used for structure determination via electron diffraction (1, 2) and for the seeding and epitaxial growth of three-dimensional protein crystals (3, 4). Furthermore, 2D protein crystals could form the basic structure of biosensors and opto-electronic devices or act as a template for the synthesis of novel organic and inorganic materials. However, the range of soluble proteins that might be crystallized in two dimensions has been limited due to the lack of a general method for addressing proteins to lipid interfaces.A number of proteins have been crystallized at interfaces by targeting to surface-bound affinity ligands (1, 5), by electrostatic interactions (6), and by metal coordination by polyhistidine-tagged proteins (7). The first approach requires synthesis of a lipid displaying an appropriate affinity ligand (8-10). Furthermore, for many interesting proteins no convenient affinity ligand is known. Electrostatic.attraction may be successful when the protein's surface charge distribution promotes adsorption with a unique orientation at the interface, but this approach is not general, and the protein orientation is difficult to predict. Kubalek et al. (10) recently described the 2D crystallization of polyhistidine-tagged HIV-reverse transcriptase on a monolayer of chelating lipids complexed to Ni2+.Polyhistidine fusion peptides, widely used for purification of recombinant proteins by immobilized metal-affinity chromatography (IMAC) (11), provide a convenient mechanism for targeting recombinant proteins to lipid interfaces (12, 13). While this approach represents a valuable move toward more general crystallization methods, the peptide tag at the N or C terminus of the engineered protein imposes restrictions on the 2D crystal morphology and limits application to recombinant prote...

Direct reagentless detection of the affinity binding of recombinant His-tagged firefly luciferase with a nickel-modified gold electrode

“…Self-assembled monolayers (SAMs) of metal-chelating lipids [12][13][14][15][16][17][18] or thiolfunctionalized chelating agents [10,11,[19][20][21][22][23][24][25] on gold supports have been widely investigated for the oriented tethering of His-tagged proteins because of the compatibility of gold supports with surface plasmon resonance, which was used for studies of kinetics and thermodynamics of specific binding between the immobilized protein and a target molecules [7,10,20,23,[26][27][28][29][30].…”

Synthetic Polypeptide Adsorption to Cu-IDA Containing Lipid Films: A Model for Protein−Membrane Interactions

“…The use of metal ion coordination to target the adsorption of proteins to lipid membranes has received much attention previously. 1,[7][8][9][10][11][12][13][14][15][16][17][18][19] Mixtures of metal-chelating lipids with other lipids are being investigated for protein detection schemes, as protein adsorption can lead to in-plane rearrangements of the lipids detectable by fluorescence techniques. 12,13 The present study explored adsorbed peptide structures involving the α-helix motif.…”

Reversible, site‐specific immobilization of polyarginine‐tagged fusion proteins on mica surfaces

“…Several efforts have been made to immobilize proteins with controlled orientation either covalently utilizing single reactive thiol groups of cysteine residues [1], or non-covalently, but specifically via immobilized antibodies [2,3], the biotin/streptavidin system [4], or metal-chelating Langmuir-Blodgett films [5][6][7][8] and metal-chelating self-assembled monolayers [9,10] for binding of polyhistidine fusion proteins.…”