Spectroscopic and calorimetric studies of the 17% N-terminal domain of apolipoprotein B-100.

摘要

Apolipoprotein B-100 (apo B) is the sole protein component of normal human low density lipoprotein (LDL). Elevated levels of LDL have been correlated with atherosclerosis and other coronary artery diseases. The large size of apo B (4536 a.a.) necessitates that it be studied in pieces corresponding to its structurally organized domains. This work concentrates on the conformation and stability properties of the 17% N-terminal domain, B17.; Mass expression of B17 was achieved via two different cell systems, mammalian-derived murine C127 cells that were transfected with a bovine papilloma virus-based expression vector, and insect-derived Sf-9 cells that were transfected with a baculovirus-based expression vector. The protein yields from both systems were compared for purity and homogeneity.; Structural characterization was carried out using far- and near-UV circular dichroic (CD) spectroscopy, where secondary and tertiary structures were studied as a function of temperature and pH. Mild acidic conditions that correlate with histidine residue protonation invoked a change in the α-helix and random coil contents of the protein, with no apparent change in the β-sheet structural content. Specific changes in the structure of the protein that occur in response to temperature were also investigated to understand the stability and conformational changes of B17. Far- and near-UV CD, as well as differential scanning calorimetry (DSC), were used to probe the thermal changes in the protein.; Structural modeling pinpointed key amino acids that may contribute to the changes seen in both far- and near-UV CD spectroscopy. The protonation of some histidine residues was attributed to underlie the increase in the helical content of the protein. The positions of some tyrosine and tryptophan residues may explain the degree of thermal stability of both the α-helices and β-sheets of B17.