IMITACIONES MONETALES

E- and N-cadherins are the two most well studied members of the classical cadherin family. While they are similar in primary, secondary, and tertiary structure, they differ by a factor of 4 in the strength of the adhesive dimer23 and in the calcium- dependent kinetics of exchange between the monomeric and dimeric forms14. Since EC1 is the site of the adhesive interface and the critical noncovalent interactions between domains all lie in EC1, our interest is in characterizing this N-terminal module of the ectodomain of N- and E-cadherin. This study aimed to characterize the differences in stability and dimerization between EC1s of E- and N-cadherin.

The main goal of denaturation studies was to assess whether we could detect a difference in the global stability of EC1s of E- and N-cadherin. Generally in our laboratory, we employ thermal denaturation to study the global stability of protein constructs. Unfortunately, ECAD1 denatured and precipitated as the temperature was increased. Thus, we turned our attention to chemical denaturation as a way to keep the protein in solution as it unfolded. Chemical denaturant studies showed that stability differed between the constructs and that estimates of stability depended upon the type of denaturant employed. ECAD1’s ΔGu obtained by denaturant studies coincided well and confirmed our results. However, NCAD1’s denaturation profiles obtained from chemical and temperature denaturations varied from one another. Unambiguously, it can be concluded that ECAD1 is a more unstable protein compared to NCAD1.

The difference in DMU and Gdn chemical denaturation studies can be attributed to a difference in the charge on the denaturant. Gdn is a positively charged ion, whereas DMU is a neutral weak denaturant. The positive charge of Gdn may interact favorably with the anionic cadherin constructs (pIs ~ 4.5). Unfortunately since DMU is a weaker denaturant, DMU denaturation data possessed a poorly resolved unfolding baseline.

In thermal denaturation studies, we were not able to effectively study ECAD1 due to precipitate formation. ECAD1 demonstrated strong temperature dependence with a very narrow window for effective analytical studies. As reported in the Material and Methods chapter, ECAD1 had low solubility in chilled solutions, as it did in heated solutions. Thermal denaturation studies on wild type E-cadherin showed two clear transitions for the unfolding of each domain. This increases our confidence in the estimate of ΔG from the thermal denaturation studies of EC1 of E-cadherin. The transition for the unfolding of the first domain of N-cadherin was not as clear on the thermal denaturation studies. The transition drifted with no clear inflection points such that neither the intermediate nor the unfolded baselines were obvious, leading to a poor estimate of the global stability of the first domain. In thermal unfolding studies of the isolated NCAD1 domain (data not shown), the unfolding transition is cooperative, indicating that N-cadherin’s EC2 affects the stability of EC1. Variations from the two- state model could be attributed to NCAD1 having multiple transitions in its unfolding profile.

Dimerization kinetics between E-cadherin and N-cadherin have previously been studied and determined to be distinct14. In this thesis, analytical SEC studies support that the dimerization states are, in fact, different between E-cadherin and N-cadherin in both

two domain and isolated domain 1. NCAD1 was found to be in both monomer and dimer; whereas, ECAD1 was found to be what we believe to be exclusively monomer. Thus, dimerization by the isolated EC1 constructs mirrored that found in their

corresponding 2-domain constructs. Formation of dimer was not expected in the isolated EC1 constructs because dimerization is calcium dependent and isolated EC1 domains do not have an intact calcium binding site. The fact that NCAD1 forms a dimer indicated that there is an “X-dimer” interface that lies in EC1 that orients the domains so that the strands can exchange and that once they exchange, disassembly is very slow. An

alternative explanation is that the trapped dimer is formed during purification of NCAD1 as the N-terminal fusion peptide is cleaved by trypsin, and once formed, it is very slow to disassemble. This question can be addressed with additional experiments, but is beyond the scope of this thesis.

As predicted, a relatively large difference in global stability of E-cadherin and N- cadherin was observed. We believe that differences in kinetics of dimerization is due to the differences in intrinsic stabilities of the first domain. Perhaps, future areas of study will shift focus from ECAD12 to ECAD1. We will explore using a combination of denaturant and thermal denaturations to fully characterize the stability of ECAD1 using methods we have available to us in-house. Further, we believe that differential scanning calorimetry might offer an advantage in that it should be able to pick up unfolding transitions that are not represented in spectroscopic signals.

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