Team:Stanford-Brown-RISD/Measurement

When researching metal binding and interactions between metals and peptides, we realized that few teams or researchers focused on isolating the specific manner of interaction and determining the thermodynamic parameters pertaining to the affinity. We utilized a method called Isothermal Titration Calorimetry (ITC) to characterize this, and quantitively understand the specifics of how our rationally designed peptides and fusion proteins bound copper. The dual approach of understanding the molecular underpinnings of our filter in addition to more macroscopic effects (such as the Phen Green SK copper assay for bulk surface adsorption) allowed us to definitely conclude that our filter behaves according to our hypothesis and supports the claims pertaining to copper binding that we subsequently make What follows is a summary of how ITC (and QUARK ab initio modeling) was used in our study.

ITC Specifications

Isothermal titration calorimetry (ITC; MicroCal iTC200) was employed to determine the association equilibrium constant (K_a), enthalpy (ΔH), and the number of ions bound per ligand (n). K_a describes the affinity of a ligand for its substrate, and we used it to quantitatively characterize the interaction between our peptide and copper [21]. All binding parameters for the test were within the specifications determined by the manufacturer. We used 10 mM, 2-(N-morpholino)-ethanesulfonic acid (MES) buffer for testing because it does not cause metal ion interference, and has a stable pKa over a wide temperature and pH range. Experiments were conducted at pH 5.5 to prevent copper precipitation, and pre-made copper stock solutions of known concentration were used.

Peptides were prepared for ITC by dissolving lyophilized protein (powder) in MES buffer, and ITC experiments were run at 25°C and set to deliver 20, 0.5 – 1 µL injections of Cu at 150 second intervals. The metal solution in the syringe was titrated into the peptide solution in the cell, and interactions were measured. Raw data were corrected by subtracting the heats of dilution, and collected data were fit with a one-site binding model using the Origin-7™ software.

ITC Results

The results from the ITC experiment to determine binding parameters for peptides 1x-, 2x-, and 3x-HHTC-Re alongside the predicted structure (QUARK Ab Initio program) follows [2]. This program uses Monte Carlo simulations and knowledge of atomic force fields to construct the most probable structural conformation of a protein from just the linear amino acid sequence.

Figure 3: ITC data for the three HHTC-Re peptides. The binding affinity (K_a) was assessed to determine the strength of the interaction, and the results follow: 1x-HHTC-Re K_a = (1.55 ± 0.21) x 10⁶ M^-1; 2x-HHTC-Re K_a = (3.73 ± 0.53) x 10⁵ M^-1; 3x-HHTC-Re K_a = (1.50 ± 0.05) x 10⁵ M^-1. Figure design credits to Jesica Urbina.

The binding affinity values in Figure 3 are largely comparable to one another, and we do see a linear trend between the number of HHTC-Re repeats and injections taken to reach saturation, which means that each peptide can bind a linearly increasing amount of copper proportional to the HHTC-Re repeats. This validates our hypothesis underlying our construct design for our fusion proteins, allowing us to bind multiple metals on the same biomolecule. In Figure 4, we can see how the specific subunits maintain proper folding (predicted by QUARK), supporting the notion that they would retain their original functions.

Figure 4: Quark ab initio model of 2x-HHTC-Re with chitin binding domain. Domains within the fusion protein have been annotated to display the conformation and spatial orientation.

After confirming the copper binding of the individual HHTC-Re x n peptides, we then needed to assess whether our fusion protein could bind copper and chitin, and whether it could do so when already saturated with the other substrate. Figure 5 depicts two experiments a) Raw data and b) isotherm for 2x-HHTC-Re-CBD + NaDg and Cu. In this experiment, N-acetyl D-glucosamine (“NaDg”; analogous to a chitin monomer and widely used in the literature for assessing chitin binding) was first titrated into 2x-HHTC-Re-CBD and no isotherm was calculated because binding sites were not saturated by the ligand. Cu was then titrated into the 2x-HHTC-Re-CBD + NaDg complex and this resulted in a Cu affinity at K_a= 7.61 +/- 1.49x 10⁶ M^-1 that is largely comparable to 2x-HHTC-Re (no CBD) and lowerby an order of magnitude than 2x-HHTC-Re-CBD without bound NaDg. Data show 20 1 µL injections. 2x-HHTC-Re-CBD was selected as the candidate for testing because it displayed the most consistent and strong results during protein purification procedures, and seemed most promising for downstream applications (such as our filter). Work is in progress on optimizing the 3x and 6x fusion proteins and creating prototypes (thus far, protein production as been successful).

Figure 5: Isotherm and data produced by ITC for assessing the binding affinity of our fusion protein (2x-HHTC-Re-CBD) for chitin (represented by N-acetyl D-glucosamine) and Cu.