|M.Sc Student||Cohen-Avni Sharon|
|Subject||Self-Association Behavior and Nanostructure of the Protein|
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Dganit Danino|
|Full Thesis text|
k-Casein is a small amphiphilic milk protein (~19 kDa, 169 amino acids) containing two cysteine residues.
k-Casein can be referred on two ways: its native from and its reduced form.
Native k-casein assembles into spherical oligomers composed of smaller multimeric subunits, created by disulfide bonds. These oligomers associate further, via electrostatic, hydrophobic and hydrogen-bonding interactions, to form micelles. In contrast reduced k-casein forms self-associating micelles in equilibrium with monomers similar to a classic copolymer.
At physiological pH and temperature both reduced and native k-casein forms fibrils that suspected as amyloid.
This study deals with the self-association behavior and structural characteristics of k-casein. Here we present k-casein nanostructures in its native and reduced forms with emphasize on two main self-associations: the micellization and fibrillization processes of k-casein.
Native k-casein micelles morphology and size were directly revealed by cryogenic transmission electron microscopy (Cryo-TEM) and dynamic light scattering (DLS). The micelles appear to be oblate and spherical with a diameter of ~ 18±2 nm. Native k-casein micelles were stable and didn't influence by various parameters such as concentration and temperature. The thermodynamics of micellization were studied using isothermal titration calorimetric (ITC) technique. The calorimetric data showed a complex non-monotonic titration with various energetic changes.
We also showed that k-casein readily forms fibrils at 37 °C, particularly following reduction of its disulfide bonds. Generally the fibrillation process is a gradual process where the fibrils evolve from short rigid fibrils to long semi-flexible fibrils. We found that k-casein exhibits a multifaceted association behavior, a result of competition between the micellization and the fibrillization processes. This competition can be adjusted and to some degree controlled by concentration, temperature, protein reduction level and incubation time.
We also investigate the self-association and interaction of k-casein with the addition of β-casein, a known member in the casein family known for its chaperon like-activity character. The combination of calorimetry and direct-imaging experiments verified the formation of the mixed micelles which depend on temperature, incubation time and k/β-caseins molar ratio. Since both nano-structures (k-casein fibrils and mixed k/β-caseins micelles) compete on the same hydrophobic region of k-casein, a competition between k-casein fibrils and the mixed k/β-caseins micelles with different kappa-casein content occur. k-Casein fibrils are non-degradable assemblies therefore the amount of k-casein monomers decreases with time and β-casein monomers tends toward the creation of individual micelles.
The inhibition of native k-casein by β-casein was verified by microscopic and calorimetric methoud and succeeded through combination of k/β molar ratio and system temperature.
Although k-casein is not considered a disease-related protein, study of this protein self associations and its fibrils inhibition by β-casein may assist in clarifying the fibril formation phenomena in numerous degenerative diseases.