**4. Conclusions**

*Homology Molecular Modeling - Perspectives and Applications*

AuNCs modified with ATP linker (positively charged NH3

+

group (~ 2 Å to the AuNCs NH3

surface functional group) (**Figure 7B**).

enzyme than the positively charged.

negatively charged surface derived from the presence of 12 exposed acidic residues. Each electrostatic potential was plotted as isosurface over each HRP conformation. The free energy calculations between negatively charged isosurface of HRP and

of 60 Kcal · mol−1, with a minimum distance of 22 Å from the Fe(III) of the heme

the positively charged isosurface of HRP showed higher coupling energies of ~120 Kcal · mol−1 at a minimum distance of ~21.5 Å to the same AuNCs functional group. For the AuNCs modified with MBA, the coupling energy of the negatively charged isosurface of HRP was 130 Kcal · mol−1 at 33 Å distance, (~3 Å from surface functional groups), while the positive HRP surface showed free coupling energies of ~75 Kcal · mol−1 and minimum distances of 32.5 Å from Fe(III) heme group (3.5 Å from

The difference between both HRP isosurfaces with the positively charged surface imposed by ATP was ~70 Kcal · mol−1 which means that the enzyme electrostatic potential imposed a clear effect for the coupling to AuNCs. However, an overall difference of 15 Kcal · mol−1 between both linkers showed that the coupling assays were energetically more stable for the negative isosurfaces of the HRP

+

functional group) (**Figure 7A**). On the other hand,

) showed coupling values

**110**

**Figure 7.**

*Coupling free energy profiles of HRP isosurfaces and AuNCs. (A) Free energy profile of HRP coupled to 6 AuNCs-ATP, (B) molecular interaction model of negative isosurface of HRP and 6 AuNCs-ATP, (C) free energy profile of HRP coupled to 6 AuNCs-MBA, (D) positive isosurface of HRP and 6 AuNCs-MBA.*

The findings of this study on HRP coupled to gold nanoclusters, indicate that the polarized electrostatic isosurface potentials are key factors to select the most efficient linker for coupling. The resulted interaction energy and distance between HRP and AuNCs-ATP are adequate to promote the formation of covalent bonds between acidic residues and amino functional groups. The evidence from this study points towards the idea that molecular simulation methods, such as homology modeling and molecular dynamics are valuable tools to take into account for design of BEI. Our previous multidisciplinary work of VP6 capsids has led us to conclude that molecular dynamics simulations elucidate structural determinants to understand the behavior of biomolecules on BEI. An implication for using homology models coupled to molecular dynamics, is the possibility of widely sample the conformational space of biomolecules probes before electrochemical experimentation. Further theoretical and experimental studies are necessary to describe the interaction with other functional group linkers and validate by electrochemical techniques the real effect of the charge difference between AuNCs-ATP and AuNCs-MBA on the redox response of this enzyme, respectively.
