Assembly of Gold-Binding Proteins for Biomolecular Recognition

Research Article

Austin J Biosens & Bioelectron. 2015;1(1): 1005.

Assembly of Gold-Binding Proteins for Biomolecular Recognition

Zareie HM1,2* and Sarikaya M3

1Department of Material Science and Engineering, Izmir Institute of Technology, Turkey

2Microstructural Analysis Unit, School of Physics and Advanced Materials, Australia

3Materials Science and Engineering & GEMSEC, University of Washington, USA

*Corresponding author: Hadi M. Zareie, Department of Material Science and Engineering, Izmir Institute of Technology, Urla, Izmir, 35430 Turkey.

Received: October 23, 2014; Accepted: February 04, 2015; Published: February 09, 2015


Controlled binding and assembly of proteins onto inorganics is at the core of biological materials science and engineering with wide ranging applications. Here we demonstrate ordered assembly of genetically-engineered inorganicbinding polypeptides on an atomically-flat solid surface. We used a 3-repeat, 14 amino acid Gold-Binding Protein (GBP1) that forms a monolayer-thick film with nano structured domains on the gold surface. The protein film conforms into 3-fold symmetry, commensurate with Au (111) lattice, suggesting crystallographic recognition. The GBP1 was selected for its specific affinity to gold by using cell-surface display. The engineered proteins could have significant potential impact by providing self-assembled functional molecular substrates in nanoand bio-technologies. We demonstrate directed assembly of ferritin (target) onto streptavidin (probe) conjugated to biotinylatedGBP1, and quantitatively analyze the results.

Keywords: Gold-Binding Protein (GBP); Self-assembly; Atomic Force Microscopy (AFM)


GBP1: Gold-Binding Protein; SAMs: Self-Assembled Monolayers; MD: Molecular Dynamics; SPR: Surface Plasmon Resonance; bio- GBP1: biotinylated GBP1; SA: Streptavidin; GEPI: Genetically Engineered Polypeptides for Inorganics


The attachment of biomolecules, in particular proteins, onto solid supports is fundamental in the development of advanced biosensors, bioreactors, and many diagnostics such as those used in cancer therapeutics [1-5]. The realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between macromolecules and biominerals or human-made inorganic materials [3]. Macromolecular interactions at solid surfaces play key roles in implants and hard-tissue engineering [4], and proteins adsorbed onto substrates are used to build protein microarrays suitable for modern proteomics [5]. With a new approach, we show self-assembly of a genetically engineered Gold-Binding Protein (GBP1) as a monomolecular film forming self-patterned domains, immobilize a probe protein onto it, and show its utility in target recognition. Our results demonstrate unique advantages offered by molecular biomimetics [6] including surface recognition, self-assembly, and genetic engineering, in creating functional molecular substrates for potential nanobiotechnological applications.

Combinatorial genetic techniques (both cell-surface display and phage display) permit isolation of specific recognition elements for surfaces, including those not recognized by natural proteins, in the absence of a priori prediction of necessary structures [7-8]. Recently, a number of inorganic binding polypeptide sequences have been selected using display technologies [9-13]. In our experiments, GBP1, a gold-binding protein, was used with an amino acid sequence MHGKTQATSGTIQS, repeated three times to increase its affinity [9,11]. Selection of gold binders was designed to produce modular, independently folding metal recognition sequences. Gold-binding sequences had been isolated as extracellular loops of maltoporin [9]. Many proteins bind tightly to gold at low salt concentrations; however the engineered proteins were selected to bind at higher salt concentrations [9,11]. To improve the binding activity, multiple repeats (up to 11) of this sequence was generated. The binding motif does not contain cysteine which is known to form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs) [14]. In our experiments, strong binding activity required at least 3 repeats and most of our current experiments were carried out using this protein.

Material and Methods

GBP1 self-assembly

The gold sample was prepared following the well-known protocol frequently used in the preparation of Self-Assembled Monolayer (SAM). Atomically-flat 0.5 to 1.0 μm diameter gold grains were produced, textured about Au [111] axis (Figure 1A and B). For the assembly of the proteins, the gold substrates were immersed into 1 mg/ml GBP-1 solution for a period from a few minutes to few hours and days. The samples were rinsed with the buffer solution first and then with de-ionized water to remove physisorbed multilayers to ensure that the remaining molecules were strongly bound to the surface (see SPR results below). After a sufficient period of incubation, the gold surfaces were examined using an AFM in air.