La Ciber-cultura características ··································································································

As stated in one of the criteria in section 1.5, it is important that the host protein can provide a stable environment for the inserted EF-loop to interact with metal ions. For both CaM-CD2-III-5G and CaM-CD2-IV-5G, the HN chemical

81

shifts of the La(III) or Ca(II) loaded forms are comparable to the wild type CD2 (Figure 3.5). There are no major changes observed for the host protein residues, suggesting that the host protein has maintained its native conformation in the presence of La(III) or Ca(II). The goal of the grafting approach was to bypass the conformational change of the native calcium binding protein but also provide a stable environment that allows the EF-loop to form a proper calcium binding geometry. Based on the results of these titration studies, we are able to further confirm that the host protein has also maintained the native conformation in the presence of La(III) or Ca(II). In addition, the direct observation of residues from grafted EF-loop III and IV of CaM involved in Ca(II) binding suggest that they maintain the original Ca(II) binding properties. These results are in good agreement with the original design plan.

The metal binding studies in section 3.1.4 have shown that the EF-loop III of CaM-CD2-III-5G has a stronger metal binding affinity than the EF-loop IV of CaM-CD2-IV-5G. The charge-ligand-balance model suggests that the inclusion of positively charged residues at position 2 of the loop enables EF-loop III to have a stronger metal binding affinity than EF-loop IV. Our metal binding studies led us to propose the following working models with two determinants that

contribute to the different site specific calcium binding affinities of the C-terminal domain of calmodulin.

First, it is possible that the conformational differences between the apo and Ca(II) loaded forms of site IV are larger than site III of calmodulin.

82

Therefore, more energy is required for the formation of the metal binding pocket. In the absence of metal, we have observed that the resonances of the calcium binding ligands at position 1, 3, 5, 7, and 12 of EF-loop III in CaM-CD2-III-5G have different chemical shifts than the calcium binding ligands at the

corresponding positions of EF-loop IV in CaM-CD2-IV-5G (Figure 3.13). These data indicate that EF-loop III and IV of calmodulin have different structural properties in the apo form. This possibility is further supported by the structural comparison between the apo and loaded forms of the EF-loops from CaM.

Figure 3.22 shows that the RMS deviation between the Cα atoms of the apo form to the calcium loaded form for site IV is significantly greater than that of site III of calmodulin (1CFD compared to 3CLN) (46, 62). On the other hand, both sites

are very similar with small RMS deviations in the presence of Ca(II).

Second, the dynamic properties of the two EF-loops are different, which can also affect the metal binding affinity due to a difference in conformational entropy. While it is widely accepted that dynamic properties will contribute to the Ca(II) binding, the direct demonstration of such an effect is complicated by the coupled EF-hand motifs and domain-domain interaction in the intact Ca(II) binding proteins. Elegant studies on the dynamic properties of the EF-hand proteins were previously studied by several research groups, such as Bax, Ikura, and Linse, using the intact calmodulin and calbindinD9k (60, 65, 139). The

binding of the first EF-hand motif in the same domain was shown to affect the dynamic properties of the second EF-hand motif and vice versa. The advantage

83

of our grafting approach to engineer a single calcium binding site is that the dynamic properties can be obtained without the interference of other metal binding site. Our La(III) titration studies on both CaM-CD2-III-5G and CaM-CD2- IV-5G reveal that the binding processes of these two EF-loops are different. The resonances for the EF-loop III disappeared from the spectrum after the addition of 230 µM La(III). The addition of La(III) to CaM-CD2-IV-5G caused the

resonances of the EF-loop IV to shift to different regions but remained visible in the spectrum. The results from the NMR spectra suggest that the residues for EF-loop IV are in a fast exchange time scale, while the residues for EF-loop III are in the medium exchange time scale. The disappearance of crosspeaks observed in EF-loop III at higher La(III) concentrations is a result of extensive line broadening. Since, EF-loop III and EF-loop IV were grafted into CD2 with two glycine linkers, it is reasonable to state that the changes observed in the NMR spectra are solely from the inserted EF-loop.

To understand the dynamic properties of CaM-CD2-III-5G and CaM-CD2- IV-5G, we have carried out detailed structural studies on CaM-CD2-III-5G using heteronuclear NMR experiment in chapter 4. In addition, we have also employed hydrogen exchange and 15N relaxation methods to study the dynamic properties of CaM-CD2-III-5G and CaM-CD2-IV-5G as is shown in chapter 5.