Brian D. Sykes

The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.

The backbone resonance assignments have been completed for the apo (¹H and ¹⁵N) and calcium‐loaded (¹H, ¹⁵N, and ¹³C) regulatory N‐domain of chicken skeletal troponin‐C (1–90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical‐shift information, along with detailed NOE analysis and ³J_HNHα coupling constants, permitted the determination and quantification of the Ca²⁺‐induced secondary structural change in the N‐domain of TnC. For both structures, 5 helices and 2 short β‐strands were found, as was observed in the apo N‐domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761–779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca²⁺ state solution structure with the apo one. The major conformational change observed is the straightening of helix‐B upon Ca²⁺ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca²⁺‐induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca²⁺ binding. The present study shows that there is virtually no change in α‐helical content associated with the transition from apo to the 2Ca²⁺ state of the N‐domain of TnC. Therefore, the Ca²⁺‐induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.