Glosario

To investigate the site-specific metal binding properties of each of the four EF-hand motifs in CaM, each EF-loop of calmodulin was individually inserted into the protein between S52 and G53 with a glycine linker on either side of the loop (Table 3.1).

CaM-CD2-I-5G The EF-loop I of CaM was inserted between S52 and G53.

Three glycine residues were used to connect the N-terminus of the EF-loop to S52 and two glycine residues were used to connect the C-terminus of the EF- loop to G53.

R1….S52-GGG-D-K-D-G-D-G-T-I-T-T-K-E-GG-G53….E99

CaM-CD2-II-5G The EF-loop II of CaM was inserted between S52 and G53.

64

S52 and two glycine residues were used to connect the C-terminus of the EF- loop to G53.

R1….S52-GGG-D-A-D-G-N-G-T-I-D-F-P-E-GG-G53….E99

CaM-CD2-IV-5G The EF-loop IV of CaM was inserted between S52 and G53.

Three glycine residues were used to connect the N-terminus of the EF-loop to S52 and two glycine residues were used to connect the C-terminus of the EF- loop to G53.

R1….S52-GGG-D-I-D-G-D-G-Q-V-N-Y-E-E-GG-G53….E99

The 1_{H spectra of the CD2 variants and wild type CD2 are shown in Figure 3.8.}

The well-resolved resonances of all four engineered proteins, observed at the downfield and upfield regions of the spectra, including the aromatic ring proton of W32, the backbone amide proton of the Y93, and the methyl protons of the side chains of V95, V39, and L16, are similar to wild type CD2. The chemical shifts are sensitive to the local environment. The majority of the resonances from the host protein region of CaM-CD2-III-5G do not change significantly, suggesting that the integrity and packing of the host protein frame is maintained after the insertion of the calcium binding loop from calmodulin.

The calcium binding affinities of the CD2 variants with different EF-loop insertions were determined by NMR, CD, and fluorescence (Table 3.4). The calcium binding affinities of the CD2 variants with EF-loops I to IV of CaM are 34,

65

245, 185, 814 µM, respectively. EF-loop I of CaM has the strongest calcium binding affinity followed by EF-loop III and EF-loop II. EF-loop IV of CaM has the weakest metal binding affinity.

The calcium binding affinities of the four CD2 variants with different EF- loops of CaM, however, are not in good agreement with the acid-pair hypothesis postulated by Reid and co-workers (136). The acid-pair hypothesis stated that

there are two criteria to determine the calcium binding affinity of an EF-loop: one is the total number of acidic residues, and the other is the arrangement of the acidic residues in the coordination sphere (Figure 3.9). The EF-loops I, II, and IV of CaM have four acidic residues in the coordination sphere whereas the EF-loop III of CaM only has three acidic residues, which suggests that the EF-loops I, II, and IV of CaM have more stable anionic arrangements (EF-loop III does not have paired axis charge) than EF-loop III. As long as the arrangement of the acidic residues is concerned, Reid and Hodges suggested that the ideal arrangement in the coordination sphere would be to have the acidic residues paired on the x- and z-axes to reduce the dentate-dentate repulsion (there is no paired acidic sidechain on the y-axis since the y position is chelated by a

carbonyl oxygen at position 7 of the EF-loop). As shown in Figure 3.9, the coordination sphere of CaM EF-loop I has a pair of charged residues in the z- axis. Similarly, EF-loop IV also has an acid-pair in the z-axis. Taken these two criteria together, according to the acid-pair hypothesis, the calcium binding affinities for the EF-loops in CaM would be I ≈ IV > II > III. Since EF-loop I and IV

66

of CaM have four acidic residues in the coordination sphere with paired acidic residues on the Z-axis, the metal binding affinities for these two EF-loops should be the strongest among the four EF-loops of CaM. Because EF-loop III of CaM has three acidic residues in the coordination sphere with no paired acidic axis, EF-loop III would have the weakest calcium binding affinity.

The calcium binding affinities for the CD2 variants with the CaM EF-loops insertion indicate the order of the calcium binding affinities as I > III ≈ II > IV. According to the assumptions of the acid-pair hypothesis, the calcium binding affinity of EF-loop IV should display an affinity similar to EF loop I. However; the calcium binding affinity of EF-loop IV is weaker than expected. Our studies on the CD2 variants with CaM EF-loops insertion prompted us to propose a charge- ligand-balanced model that can well define this discrepancy. The charge ligand- balanced model agrees with the acid-pair hypothesis that the number of acidic residues in the coordination sphere affects the metal binding affinity of an EF- hand motif, but further suggests that the residues adjacent to the calcium binding ligand also affect the affinity. A positively charged residue adjacent to the

calcium binding ligand balances the electron dentate-dentate repulsion in the presence or absence of calcium. The calcium binding ligands for the EF-hand motif are located at positions 1, 3, 5, 7, and 12 of the loop. Positions 4 and 6 of the EF-loop are generally Gly residues (21). So the residue types at position 2

and 11 affect the metal binding ability of the EF-loop more frequently. The EF- loop I of CaM has Lys at positions 2 and 11, which can balance out the charges

67

in the EF-loop. On the contrary, the EF-loop IV of CaM has a Glu at position 11. It is possible that the Glu at this position causes more repulsion in the absence of calcium. The structure comparisons between the calcium free and calcium loaded forms of the EF-hand motifs in the C-terminal domain of CaM have indicated that EF-loop IV exhibits a larger deviation between the apo and the loaded form than EF-loop III. EF-loop II of CaM has four acidic residues in the coordination sphere, but it does not have basic residues at position 2 and 11 of the EF-loop. EF-loop III only has three acidic residues in the coordination sphere, but it has a Lys at position 2 of the EF-loop to balance the charge repulsion in the N-terminus of the loop. The calcium binding affinity of the EF- loop III is similar to the EF-loop II because both of the EF-loops include one of the criteria described in the charge-ligand-balance model.