Biophysical analysis of progressive C-terminal truncations of human apolipoprotein E4: insights into secondary structure and unfolding properties.
Apolipoprotein E4 (apoE4) is a risk factor for Alzheimer's disease and has been associated with a variety of neuropathological processes. ApoE4 C-terminally truncated forms have been found in brains of Alzheimer's disease patients. Structural rearrangements in apoE4 are known to be key to its physiological functions. To understand the effect of C-terminal truncations on apoE4 lipid-free structure, we produced a series of recombinant apoE4 forms with progressive C-terminal deletions between residues 166 and 299. Circular dichroism measurements show a dramatic loss in helicity upon removal of the last 40 C-terminal residues, whereas further truncations of residues 203-259 lead to recovery of helical content. Further deletion of residues 186-202 leads to a small increase in helical content. Thermal denaturation indicated that removal of residues 260-299 leads to an increase in melting temperature but truncations down to residue 186 did not further affect the melting temperature. The progressive C-terminal truncations, however, gradually increased the cooperativity of thermal unfolding. Chemical denaturation of the apoE4 forms revealed a two-step process with a clear intermediate stage that is progressively lost as the C-terminus is truncated down to residue 230. Hydrophobic fluorescent probe binding suggested that regions 260-299 and 186-202 contain hydrophobic sites, the former being solvent accessible in the wild-type molecule and the latter being accessible only upon truncation. Taken together, our results show an important but complex role of apoE4 C-terminal segments in secondary structure stability and unfolding and suggest that interactions mediated by the C-terminal segments are important for the structural integrity and conformational changes of apoE4.
Chroni
Angeliki
A
Institute of Biology, National Centre for Scientific Research Demokritos, Aghia Paraskevi, Athens 15310, Greece. achroni@bio.demokritos.gr
Pyrpassopoulos
Serapion
S
Thanassoulas
Angelos
A
Nounesis
George
G
Zannis
Vassilis I
VI
Stratikos
Efstratios
E
D053327:Apolipoprotein E4 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D053327:Apolipoprotein E4