CD2 and excellent host system

The host system for our design approach is domain 1 of the rat cell surface adhesion receptor CD2 (Figure 1.20). The β-sheet architecture of CD2 is similar to the extracellular Ca2+ _{dependent cell adhesion molecule cadherin (Nagar et al., 1996) (Figure}

1.19). Therefore, information obtained by designing Ca2+ binding sites in CD2 can provide insight into Ca2+ dependent cell adhesion properties. Previous studies on CD2

shows that the protein is amenable to protein engineering with over 40 single mutations being generated, which do not exhibit significant effects on the structure of the protein (Arulanandam et al., 1993a). CD2 is stable against a wide range of pH, temperature, and salt concentrations (Yang et al., 2000a). These characteristics of CD2 meet the criteria for an excellent host protein, therefore CD2 is an ideal model system to understand the effect of protein environment on calcium binding affinity and molecular recognition. 1.11 Objective and significance of this study

Long range electrostatic interactions have been shown to be important for Ca2+ binding affinity (Linse et al., 1988). However their role in site specific binding has not been elucidated due to the prevalence of proteins with multiple Ca2+ binding sites. The cooperativity between the multiple Ca2+ ions hampers the study of site specific binding (Linse and Forsen, 1995). In addition, many natural Ca2+ binding proteins undergo large conformational changes upon Ca2+ binding which makes it difficult to distinguish the binding energy from the energy of conformational change. A structured host protein will provide the stability for both Ca2+ free and Ca2+ bound forms which will allow for a focus on local factors including the effect of charge distribution around the Ca2+ _coordination

shell on Ca2+ binding affinity. The goals of this study are to examine the effect of electrostatic interactions within 5-15 Å of the Ca2+ coordination shell on Ca2+ binding affinity. This study will help gain insight into the factors that control Ca2+ binding affinity. An establishment of rules for Ca2+ binding affinity will aid in understanding the mechanisms of Ca2+ _{affinity related diseases. In addition, the structural similarities}

understanding of other Ca2+ dependent processes including cell adhesion. The knowledge obtained is valuable for the development of therapeutic treatments of diseases caused by the abnormal functions of cell adhesion proteins.

Understanding the factors which control Ca2+ binding affinity will allow for the modulation of Ca2+ binding affinities through design. The ability to design Ca2+ binding proteins with varying affinities will be useful for the development of Ca2+ sensors. Different compartments of the cell and the extracellular matrix can be targeted based upon their levels of Ca2+ concentrations. It will aid in the development of designed Ca2+ binding proteins as contrast agents and useful reagents for diagnostic tests.

In this study, the role of electrostatic interactions in Ca2+ binding affinity will be approached from two different directions. First, several proteins were designed with Ca2+ binding sites in different regions of CD2. The first generation proteins 206, 5606, and 6775, have Ca2+ binding sites that are in varying electrostatic environments. However, although these proteins bind Ca2+ and its analogs Tb3+ and La3+, they have a conformation that is different from wild type CD2 due to mutations of residues that were involved in the formation of the hydrophobic core of the protein. Therefore, it became necessary to design second generation proteins with refined design criteria. The second generation protein CD2.6D79 required two solvent exposed residues to be mutated to form the metal binding site and also utilizes two wild type residues as ligands. CD2.6D79 has a conformation similar to wild type CD2 and displays a strong metal binding affinity as well as metal selectivity. The electrostatic environment of this Ca2+

1.2 shows each designed protein that is studied in this research and the rationale for their design.

Chapter 2 of this dissertation lists all of the materials and methods used in this study including the molecular cloning, expression, and purification of designed proteins, and the techniques used for monitoring the conformation and metal binding of these proteins.

Chapter 3 focuses on the electrostatic calculations used to predict Ca2+ binding affinity and calculate pKa values of CD2 and calbindin D9k as well as to predict the

charge distribution variants that will have the greatest effect on Ca2+ binding affinity. Chapter 4 focuses on the conformation and metal binding affinity of the first generations proteins 206, 5606, 6775, and N80/89.

Chapter 5 focuses on the conformational, metal binding, and molecular recognition properties of the second generation designed protein 6D79.

Chapter 6 discusses the effect of close and long range electrostatic interactions on protein stability through analysis of the charge distribution variants of 6D79.

Chapter 7 discusses the effect of charge around the coordination shell on Ca2+

binding affinity through the analysis of the charge distribution variants of 6D79.

Chapter 8 focuses on the pH dependent folding of the R34 charge distribution variants.