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Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.

Titanium (Ti) and its alloys are among the most successful implantable materials for dental and orthopedic applications. The combination of excellent mechanical and corrosion resistance properties makes them highly desirable as endosseous implants that can withstand a demanding biomechanical environment. Yet, the success of the implant depends on its osteointegration, which is modulated by the biological reactions occurring at the interface of the implant. A recent development for improving biological responses on the Ti-implant surface has been the realization that bifunctional peptides can impart material binding specificity not only because of their molecular recognition of the inorganic material surface, but also through their self-assembly and ease of biological conjugation properties. To assess peptide-based functionalization on bioactivity, the present authors generated a set of peptides for implant-grade Ti, using cell surface display methods. Out of 60 unique peptides selected by this method, two of the strongest titanium binding peptides, TiBP1 and TiBP2, were further characterized for molecular structure and adsorption properties. These two peptides demonstrated unique, but similar molecular conformations different from that of a weak binder peptide, TiBP60. Adsorption measurements on a Ti surface revealed that their disassociation constants were 15-fold less than TiBP60. Their flexible and modular use in biological surface functionalization were demonstrated by conjugating them with an integrin recognizing peptide motif, RGDS. The functionalization of the Ti surface by the selected peptides significantly enhanced the bioactivity of osteoblast and fibroblast cells on implant-grade materials.

Recently, phage and cell-surface display libraries have been adapted for genetically selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported and their bionanotechnological utility as synthesizers and molecular linkers, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of genetically designing these peptides, understanding the binding kinetics and thermodynamics, and using the peptides as molecular erectors, in this report we discuss molecular structural constraints imposed upon the quantitative binding characteristics of peptides with an affinity for inorganics. Specifically, we use a high-affinity seven amino acid Pt-binding sequence, PTSTGQA, as we reported in earlier studies and build two constructs: one is a Cys-Cys constrained "loop" sequence (CPTSTGQAC) that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, a septapeptide, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2-trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the integral Pt-binding septapeptide sequence and that the presence or absence of constraints could be used to tune the adsorption and structural features of inorganic binding peptide sequences.