Protein Complexity via Non-Native States: Binding, Stability, and Structural Studies of Calbindin D9k and HAMLET (Human alpha-lactalbumin Made LEthal to Tumor cells)

Abstract: Using optical spectroscopy, nuclear magnetic resonance (NMR), and differential scanning Calorimetry (DSC), I have studied two different calcium binding proteins that can form kinetically trapped altered states. Calcium is very important in numerous biological processes such as blood coagulation, signal transduction, muscle contraction and bone formation. Calcium binding to proteins regulates these processes. First, the influence of a bound water molecule on the cooperativity of calcium binding in an EF hand protein, calbindin D9k, was studied. Affinity, kinetics, stability and structure properties of two mutant proteins showed the importance of water molecules in the binding process. Surprisingly, one mutant formed a 3D domain swapped dimer upon crystallization. We showed that packing of a hydrophobic substitution in the linker region is the driving force for formation of the kinetically trapped dimer. The human genome consists of fewer genes than predicted but maintain complexity by mechanisms on different levels from DNA to proteins. In my second project, I studied a system that changes function and structure upon binding a fatty acid ligand. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1, 9 cis) that kills tumor cells by an apoptosis-like mechanism. Previously, it has been shown that only calcium-free, apo, alpha-lactalbumin can be converted to HAMLET. Apo alpha-lactalbumin is partly unfolded. A bovine version, BAMLET and D87A-BAMLET, a converted non-calcium binding, and thus permanently partly unfolded, were both able to kill tumor cells. HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. The conclusion is that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. The stability towards thermal and urea denaturation was measured for HAMLET, BAMLET and human and bovine alpha-lactalbumin. Three lines of evidence indicate that HAMLET and BAMLET are kinetic traps. I) HAMLET/BAMLET has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid. II) Its denaturation is irreversible and HAMLET/BAMLET is lost after denaturation. III) Formation of HAMLET/BAMLET requires a specific conversion protocol. NMR studies show that oleic acid is bound in a u-shaped fashion in HAMLET, but the spectra of the protein are poorly dispersed further underlining previous observations of a highly dynamic and unstructured state under physiological conditions.