BINDING AND SPECIFICITY OF ENGINEERED GOLD-BINDING POLYPEPTIDES
USING SPR AND QCM
Memed Duman1,2; Eswaranda Venkatasubramanian1, Turgay Kacar2,3, Daniel Heidel3,
E. Emre Oren3, Candan Tamerler2,3, Mehmet Sarikaya2,3.
1
Bioengineering, Hacettepe University, Beytepe, Ankara, Turkey,
Molecular Biology and Genetics, Istanbul Technical University, Maslak, Istanbul, Turkey
3
Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
2
In biological hard tissues, proteins control inorganic materials assembly, morphogenesis and
formation through molecular recognition and specific binding. Instead of natural proteins which
may be large and complex, hard to isolate and purify and difficult to use in reconstruction of the
hybrid structures, one can use molecular biology technologies, such as directed or forced
evolution to obtain inorganic-binding polypeptides. Combinatorial techniques such as cell-surface
and phage display have been used to select short polypeptide sequences (7-12 amino acids) that
bind to noble metals (Au, Pt, and Pd) and oxide semiconductors (ZnO, Cu2O, and Al2O3) [1].
These polypeptides are used as molecular erectors in assembly or immobilization of
nanoparticles, and as agents in bionanofabrication. The understanding of the nature of molecular
recognition and binding characteristics of these engineered polypeptides need to be well
understood to further engineer their molecular structure and, thereby, utilize them more
effectively as molecular biomimetic building blocks in nano- and bionanotechnology. Here we
present the binding characteristics of gold binding protein (GBP) that was selected using cell
surface display and post-selection engineered to assemble and form ordered nanostructured
molecular films. We use a combination of surface plasmon resonance (SPR), quartz crystal
microbalance (QCM), and atomic force microscopy (AFM) quantitative binding, assembly and
formation on oriented gold surfaces The GBP was used in single repeat and 3, 5, 6, 7, and 9
repeats of the single sequence of MHGKTQATSGTIQS. While AFM provided molecularly
ordered structural information, both QCM and SPR were used to examine adsorption processes
and provided quantitative binding information under controlled solution environments (such as
composition, pH, temperature) as well as kinetic parameters such as binding rates, equilibrium
constants and binding energies. The experiments, e.g., in the case of 3repeat-GBP, were carried
out in the concentration range of 0.5 to 4 microgm/mL (QCM) and 0.2 to 0.43 microgm/mL
(SPR). The data was fit to a simple Langmuir 1:1 adsorption model to obtain kon and koff for
both techniques. The kinetics was well described by a single exponential in the case of QCM
while distinct biexponential kinetics was observed for the data from SPR. The two-step
adsorption in SPR is explained as being due to surface heterogeneity of the substrates. The
binding energies obtained using the two techniques were comparable within the range of
experimental error and were much higher than those for thiol adsorption to gold from an ethanolic
solution. The results show promise for using such genetically engineered protein as molecular
scaffolds and also as an alternative to thiols with significant advantages as discussed in specific
applications discussed in this presentation.
Research was supported by ARO-DURINT of USA.