CD2.7E15 (also referred to as EEDDD in this study) variants were engineered using the classical PCR method from CD2.7E15 DNA in the vector pGEX-2T. The mutations were confirmed using automated DNA sequencing at the Biology Core Facility of Georgia State University. The protein expression, purification, and concentration estimation were carried out as previously reported 32. The background Ca²⁺ was minimized by incubating the sample with EGTA first, followed by a pH gradient separation using a HiTrap SP HP column (GE Healthcare) with chelexed (Chelex-100 resin, BioRad) buffers.

Trp emission spectra over 300-400 nm, with excitation at 282 nm, of proteins (4 μM) in 10 mM Tris, pH 7.3 in the presence of 10 mM Ca²⁺ or 1 mM EGTA were collected using a PTI fluorimeter and a cuvette with a 1 cm pathlength at room temperature.

Cation binding was done in 20 mM PIPES-10 mM KCl, pH 6.8. The Tb³⁺-FRET fluorescence signals were monitored using a PTI fluorimeter following the procedure described previously. The Tb³⁺ binding affinity was measured by direct Tb³⁺ titration and the La³⁺ binding affinity was derived using the competitive binding of La³⁺ against Tb³⁺. The Ca²⁺-binding affinity was measured using ¹H-¹⁵N HSQC NMR spectra by titrating Ca²⁺ into the protein sample with 40-50 μM EGTA at the initial point and monitoring the chemical shift changes versus the Ca²⁺ concentrations. The K_d of each CD2.7E15 variant was calculated using the average of results from multiple resonances and the uncertainty represented the different responses of these resonances to Ca²⁺ binding.

Far-UV CD spectra were collected on a Jasco-810 spectropolarimeter coupled with a Peltier temperature controller. The signal was monitored over 260-200 nm with four repeat scans. The proteins (25 μM) were in 10 mM Tris-10 mM KCl, pH 7.3 with either 1 mM EGTA, 10 mM Ca²⁺, or 0.05 mM Tb³⁺, placed in a 1 mm pathlength cell with a sealed top. Buffer signals were subtracted from the spectra. For thermal titration measurements, CD spectra were taken at 2-5 degree increments with four to six repeat scans over a temperature range from 15 to 85°C. Five minutes were allowed for equilibration at each temperature before the scans were taken. The signal change at 225 nm was plotted using KaleidaGraph and fitted using a two-state transition model,

where ΔS and ΔS_max are the signal changes at temperature T point and the final temperature, respectively; T_m is the transition temperature; and k is a fitting parameter measuring the steepness of the thermal transition. The reported T_m and the uncertainty are the average and standard deviation of three runs, respectively.

The model structures of EEDDD with bound Ca²⁺ were generated by the design program and the structures of the other variants were generated from EEDDD using the program SYBYL (Tripos Co.). Hydrogen atoms were added using SYBYL. The bound Ca²⁺ was removed for the apo-form structures, and the Ca²⁺ was replaced by a Tb³⁺ at the same location to construct the Tb³⁺-loaded form structures. All Asp and Glu residues were assumed to be unprotonated (and all Lys and Arg protonated); while pK_a values are prone to be perturbed when charges are clustered 34, the assumed protonation states seem appropriate for the neutral pH where melting temperatures and ion binding affinities were measured.

The Poisson-Boltzmann (PB) equation was solved to calculate the electrostatic free energies of the protein variants in bound- and apo-forms using the UHBD program35. A salt concentration of 10 mM in the solution was used. Electrostatic contributions of mutational effects on the Ca²⁺ binding affinity and folding stability of CD2.7E15 variants were calculated following previously-published protocols 436. The effect of a mutation on the folding stability was calculated as the change in the electrostatic folding free energy:

In our case, the wild-type protein was identified as the original 7E15 (i.e., EEDDD), and the mutant was any of the variants introduced here. The unfolded state of proteins was modeled as individual residues separately dissolved in the solvent. Therefore, in the unfolded state, residues other than the one under mutation make the same contribution to the electrostatic free energies of the wild-type protein and mutant, and do not affect ΔΔG_f. Neglecting this contribution, the electrostatic folding free energy of either the wild-type protein or the mutant is:

where G_el(X) is the electrostatic free energy of molecule X; "protein" refers to the folded protein; and "residue" refers to a residue, the one under mutation, that is carved out of the folded structure. G_el has both a Coulombic component and a solvation component.

Calculations of ΔΔG_f were done on the apo forms only. A basic assumption underlying the present study is that the metal ions bind to the designed proteins only when they are in the folded state, since only then coordination residues come together to form the binding site. Under this assumption, a state in which the protein is both unfolded and metal ion-loaded does not exist, and it would not be appropriate to apply the procedure for calculating ΔΔG_f to the metal ion-loaded forms. We note that preferential binding of the folded state by metal ions shifts the folding-unfolding equilibrium towards the former.

The contribution of a point mutation to metal ion binding affinity can be expressed as the binding free energy difference between the mutant and the wild-type complex:

where ΔG_b is the binding free energy, calculated as

The calculated result is to be compared with the counterpart from experimentally determined binding affinity:

where k_B is Boltzmann's constant.

In a previous publication 32, CD2.7E15 referred to a rationally designed Ca²⁺-binding protein with an engineered Ca²⁺-binding site at the B, E, and D β-strands of the host protein CD2 (Fig. 1), with coordination residues Glu, Glu, Asp, Asp, and Asp occupying positions 15, 56, 58, 62, and 64, respectively. Here CD2.7E15 is referred to as EEDDD; other variants of EEDDD are referred to using a similar notation, consisting of single-letter codes for the residues occupying the five coordination sites. The EEDDD variant was chosen as the template to investigate the effects of charge and coordination residue type on Ca²⁺ binding based on the following considerations. First, this metal binding site presented the possibility to study the relationship between Ca²⁺ binding and protein stability. We have previously shown that the cluster of five negatively charged residues at this location did not unfold the protein and metal binding did not alter the global conformation of the protein 32. Second, this variant retains two wild-type residues, E56 and D62. Third, using the EEDDD template, a series of variants with local net charges ranging from -5 to -2 by replacing Asp or Glu with Asn or Gln (Table 1) could be generated. Variants with local net charges of -1 or 0 were not generated since they were not expected to bind Ca²⁺ with a reasonable affinity. Finally, using the EEDDD template, pairs of variants, such as EEDDD/EEDDE or EEDDN/EEDDQ, which possess identical net charges but differ by one methylene group, could also be generated, presenting the opportunity to investigate the size effects of coordination residues.

At 25°C the far-UV CD spectra for all of the variants displayed a single negative maximum at ~216 nm, nearly identical to that of wild-type CD2, indicating the maintenance of wild-type β-sheet secondary structure. Moreover, the Trp fluorescence emission spectra of the variants displayed a single maximum at 327 nm, identical to that of the wild-type protein, suggesting that the mutations did not alter the native fold. The CD and the Trp fluorescence spectra remained unchanged with the addition of cations, suggesting the absence of global conformational changes upon metal binding (Fig. 2).

Taking advantage of aromatic residues such as Trp-32 in the host protein, we used aromatic sensitized fluorescent resonance energy transfer (FRET) to analyze metal binding in the EEDDD variants (Fig. 3). As negative control, the addition of wild-type CD2 into a Tb³⁺ solution resulted in negligible change in the Tb³⁺ fluorescence emission at 545 nm. In contrast, the addition of EEDDD, EEDDE, EEDDN, and EEDDQ into the Tb³⁺ solution resulted in more than 30-fold increases in Tb³⁺ fluorescence emission. The addition of NENDN only resulted in a slight enhancement relative to CD2, suggesting that the variant bound Tb³⁺ at most with a weak affinity. The Tb³⁺-binding affinities were obtained by directly titrating Tb³⁺ into the protein variants (Table 1). The variants with -5 charges showed the strongest affinities with K_d < 1 μM. The variants with -4 charges show affinities at the μM level. The Tb³⁺ binding affinity of EENDN was not accurately measured due to the limitation of solubility of the protein in high concentration of Tb³⁺. La³⁺ binding affinities were obtained by competitively titrating La³⁺ into Tb³⁺-protein mixtures and monitoring the decrease in Tb³⁺ fluorescence intensity. The trend of La³⁺-binding affinities for the 7E15 variants was the same as that for Tb³⁺. That is, the -5-charged variants showed the strongest affinities, followed by the -4, -3 and -2 charged variants. The addition of Ca²⁺ into Tb³⁺-protein mixtures led to only a small decrease in the Tb³⁺ fluorescence, suggesting inefficient competition by Ca²⁺ due to weaker Ca²⁺ binding.

The Ca²⁺-binding affinity of EEDDD was previously determined to be 100 ± 50 μM from the chemical shift changes of the residues proximate to the metal binding position using ¹H-¹⁵N HSQC spectra of ¹⁵N labeled proteins 32. In this study, similar Ca²⁺ titrations on the other variants were performed. The resonances in the spectra for the other variants were partially identified by comparing them to the spectrum of EEDDD. For the -4 and -5 charged variants, Ca²⁺ specifically perturbed several resonances while other resonances maintained their positions throughout the titration process. Fig. 4A shows the ¹H-¹⁵N HSQC spectra of EEDDQ as an example of data obtained. Perturbed resonances were assigned to coordination residues or their neighbours, including G61, L63, Q64, E58, E56, K66, and Q22. The Ca²⁺-binding affinities of EEDDE, EEDDQ, and EEDDN were obtained by analyzing the chemical shift perturbations (Fig. 4B and Table 1). Unlike Tb³⁺ and La³⁺ binding, the -4 charged EEDDQ showed a stronger Ca²⁺ binding affinity than the -5 charged EEDDE. Additionally, the chemical shift perturbations induced by Ca²⁺ binding to EEDDQ were greater than those to EEDDE. The -3 charged variant EENDN displayed small but significant chemical shift perturbations at high Ca²⁺ concentrations, while the -2 charged variant NENDN did not undergo any significant changes with the addition of up to 13 mM Ca²⁺ (Fig. 4C). Hence EENDN possessed only weak Ca²⁺ binding ability, while NENDN did not show observable Ca²⁺ binding. The null binding results of EENDN and NENDN confirmed that the chemical shift perturbations observed on the other variants were due to specific Ca²⁺ binding and not to other nonspecific processes such as salt effects.

The thermal transition temperatures (T_m) of all the variants were obtained using far-UV CD (Fig. 5). At 90°C, EEDDD and the other variants were found to be fully unfolded, just like wild type CD2. Compared with CD2, which possesses a T_m of 61 ± 1°C 19, the clustered negative charges at the β-strands decreased the T_ms of EEDDD (41 ± 1°C) and EEDDE (45 ± 3°C) in the absence of metal. Under the same conditions, the T_ms of the -4 charged variants were significantly higher, with values of 53 ± 2°C for EEDDN and 56 ± 2°C for EEDDQ. In the absence of metal, T_m values of EENDN (61 ± 1°C) and NENDN (62 ± 1°C) were similar to that of wild-type CD2 (Table 2).

A consequence of the assumption that metal ions can only bind to the folded proteins is that the folding stability would be increased by metal binding. Upon binding of Ca²⁺ or Tb³⁺, the T_m values of the -4 and -5 charged variants indeed increased significantly (Table 2). Ca²⁺-binding increased the T_m of -5 variants EEDDD and EEDDE by ~10°C and Tb³⁺ binding increased the T_m of EEDDE by ~20°C. Additionally, Ca²⁺ binding increased T_mof -4 variants EEDDN and EEDDQ by ~5°C, and Tb³⁺ binding increases the T_m of both variants by ~2-4°C. Neither Ca²⁺ nor Tb³⁺ was observed to cause significant changes in the T_m values of EENDN and NENDN, consistent with the weak or non-observable metal binding of these variants.

The order of T_ms of the variants in the absence of metal binding can be summarized as follows: CD2 (-1) ~NENDN (-2) ~EENDN (-3) > EEDDQ (-4) > EEDDN (-4) > EEDDE (-5) > EEDDD (-5). Upon binding Ca²⁺, the order T_ms was largely the same, except that T_m values of EEDDQ and EENDN were the same. Tb³⁺ binding introduced another alteration in the ordering of T_ms: the T_m of EEDDE now exceeded that of EEDDQ.

Upon folding, a cluster of like charges is expected to decrease protein stability due to the individual loss of solvation and the collective repulsion among the charges. Upon metal ion binding, the protein and metal ion simultaneously experience unfavorable desolvation and favorable charge-charge attraction. These electrostatic effects were modeled using the Poisson-Boltzmann (PB) equation, from which the electrostatic contributions to folding stability and binding free energy were calculated. Since the CD, fluorescence, and NMR results all showed that the overall tertiary and secondary structures of the variants were similar to those of wild-type CD2, the structures of the variants on the structure of wild-type CD2 were modeled with minimal changes (i.e., all residues other than the one under mutation were fixed). In addition, it was assumed that the metal binding did not perturb the binding pocket or the global conformation of the protein. The calculations of mutational effects on the folding (binding) free energy ΔΔG_f (ΔΔG_b) are described in Materials and Methods.

The calculated results for ΔΔG_f correlated well with the experimentally observed T_ms of the apo forms, with R² = 0.91 (Fig. 6A). Both the calculations and the experimental data indicated that the folding stability of the variants is ordered in the following way: EEDDE ~EEDDD < EEDDQ ~EEDDN < EENDN ~NENDN. Apparently, the variants with lower net charges experienced less charge repulsion around the binding pocket, thereby increasing folding stability and T_m.

According to our electrostatic calculations of ΔΔG_b, the Ca²⁺ binding affinities of the variants were ordered as follows: EEDDD > EEDDE > EEDDQ > EEDDN > EENDN > NENDN. That is, binding is more favorable at sites with more negative net charge. Compared with the experimental measurements, the order in the Ca²⁺ binding affinities of EEDDE and EEDDQ was reversed, which may be an indication of non-electrostatic effects not included in our modeling. Nevertheless, there appeared to be good overall agreement between the electrostatic calculations and the experimental measurements regarding the mutational effects on metal-binding affinity (Fig. 6B), suggesting that electrostatic interactions indeed play a major role in the Ca²⁺ binding affinities of these protein variants.