Success of selection of binders for gold powder (a proof of concept)

The screening result of selecting positive binders against gold powder resulted in great success; we obtained nearly 50% of positive clones (refer result section 2.1.2, D). Many control experiments ensured the specificity of gold binding antibodies (binders). After

sequencing, we identified 32 distinct sequences which showed binding with gold surface (this is a huge number in comparison to number of binders obtained against biological targets, they result in maximum 10 different clones, usually 2 or 3 ).The gold binding scFvs are proof of concept for our ability to select biological binder for inorganic entities.

The selection of many positive binders provided us data to do statistical analysis. We observed clear selection of arginine (R) at many positions. In particular at the VH and VL

CDR3 regions (see figure 3.1a). These regions are the most variable and are directly involved in contacting the target antigens. The selection of Arg by many positive binders, clearly indicate role of this amino acid for gold binding. This predominant selection made us explore about specific interaction of arginine with gold surface (similar evidences are mentioned in the following text).

Interestingly we were not aware of the specificity of Arg for gold surface before our phage display screen. We started our screen with unbiased library of phage displaying scFvs. Our result showing abundance of arginine in gold binding antibody agrees to studies performed by other group using sophisticated tools. The result of selecting binders against gold powder strongly supports our case of using the strategy for other inorganic target, whose surface characteristics are unknown.

Evidence of Arginine showing affinity for gold surface

The Amino acid sequence of CDR in gold binding antibody reported by Kumagai group (Watanabe et al., 2008) is shown below.

Arginine is present in the VH CDR3 and VL CDR3, as well as at two other CDRs in their anti gold antibody. But they did not draw any conclusion for role of Arginine for gold binding because they had only one antibody sequence; so they do not have statistics to support role of arginine in gold binding.

Sarikaya group studied absorption behaviour of gold binding peptide (Hnilova, Oren, Seker, Wilson, Collino, Evans, Tamerler, & Sarikaya, 2008b). They compared the amino acid composition of strong, moderate, and weak binder (peptide) for gold selected using FliTrx random peptide display library. They calculated the relative abundances of amino acids among them (see figure 3.1b).

Figure 3.1b: Relative abundance of amino acids in strong- and weak binding groups of gold-binding peptides. The residues are coloured as blue (basic), red (acidic), green (hydrogen-bonding donor/acceptor), and grey (nonpolar). Source - (Hnilova, Oren, Seker, Wilson, Collino, Evans, Tamerler, & Sarikaya, 2008b)

Their result suggested that amino acids, Arg, Cys, Trp, and Tyr were over represented and Asn, Lys, Pro, and Thr under represented among strong binder sequences for gold. Arg was over represented not only among the strong gold-binding peptides but also among the weak- binding peptides.

Simulations carried out by Martin Hoefling and his group (Hoefling, Iori, Corni, & Gottschalk, 2010) on adsorption behaviour of 20 amino acids on gold surface (111) using molecular dynamics showed strong dependence for the binding affinities on the chemical character of the amino acids. Interaction free energy in KJ/mol of non covalent association of amino acids with gold surface (111) shown in table 3.1c.

Table 3.1c: Interaction free energy in KJ/mol of non covalent association of amino acid with gold surface (111) Source - (Hoefling et al., 2010)

The detailed insight of the energy landscape of the amino acids shown in above table provide hints for their behavior when they are in contact with gold surface. During their simulation, they observed that Arg made one of the first contacts, due to length of its side chain. This observation leads them to conclude that Arg may be important for adsorption of protein on gold. However, cooperative effect of all amino acids in particular conformation might lead to other conclusion.

In extension to the above mentioned question, Hoefling et al showed interaction of polypeptides with a gold surface (111) using molecular dynamics simulations (Hoefling, Monti, Corni, & Gottschalk, 2011). This study was performed in polarisable gold model in explicit water. They used two kinds of polypeptides: peptide having beta sheet conformation and biologically important protein having two beta sheet domains. They summarised their result as adsorption occur in stepwise mechanism for both kind of polypeptides. The initial contact formation is initiated by positive charged amino acid Arginine (Arg), while overall charge on polypeptide was neutral. These results also open one debate on selectivity of any peptide on gold due to charge on gold surface as electrostatic interaction could be one reason for strong affinity of peptide to any inorganic surface. Recently group of Sarikaya explored this question (Donatan, Sarikaya, Tamerler, & Urgen, 2012), and their finding suggests that under each charged condition, binding behaviour of gold binding peptide demonstrated quantitative differences in terms of adsorbed peptide amount, surface coverage ratio. This finding can be used as a potential tuning parameter of peptide adsorption.

Another article published in soft matter in 2010, where they studied adsorption mechanism of single amino acid and other surfactant molecules (which are essential for stabilisation of gold nanoparticles) on gold surface (111) in aqueous solution. Their result also favored role of arginine for gold binding (Feng et al., 2011). The amino acid and surfactant molecule which were most attracted by gold surface were large molecules and with planar sp² hybridized group (e.g. Arg, Trp, Gln,Met, Asn,and PPh3) , whereas short aliphatic molecule with sp³ hybridized group exhibit weak attraction (see figure 3.1d).

Figure 3.1d: Computed (free) energy of adsorption of single amino acids and single surfactant molecules on an even Au {111} surface in dilute aqueous solution at pH ¼ 7 using the CHARMM–

METAL force field. Large molecules with planar sp2-hybridized groups show stronger adsorption and short aliphatic molecules with sp3-hybridized groups exhibit weaker adsorption. HDAC - hexadecylammonium chloride, CTAB - hexadecyltrimethylammonium bromide. Source - (Feng et al., 2011)

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Insight of how Arginine might favor gold binding

Work of Feng et al suggested that strong affinity of arginine for gold can be attributed to guanidium group present in arginine structure. Strongly binding amino acids moved by a hoping mechanism in which surface attached group (guanidinium group of Arg ), moved from one favorable coordination pattern on the surface to another in intervals on the order of one hundread picoseconds (Feng et al., 2011). Favorable binding condition of Guanidinium group of Arg with gold is shown in figure 3.1e.

Figure 3.1e: Representative snapshot of Arg on the Au {111} surface. The guanidinium group is found most of the time in a favorable coordination pattern with numerous epitaxial sites which leads to strong adsorption (pink highlights). Diffusion on the surface occurs by stepwise hopping of the guanidinium group to similar epitaxial environments. Source - (Feng et al., 2011)