tailieunhanh - Báo cáo khoa học: Replacement of helix 1¢ enhances the lipid binding activity of apoE3 N-terminal domain

The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational adapta-tion is required for manifestation of LDLR binding ability. | iFEBS Journal Replacement of helix 1 enhances the lipid binding activity of apoE3 N-terminal domain Katherine A. Redmond1 Conrad Murphy1 Vasanthy Narayanaswami1 Robert S. Kiss2 Paul Hauser1 Emmanuel Guigard3 Cyril M. Kay3 and Robert O. Ryan1 1 Lipid Biology in Health and Disease Research Group Children s HospitalOakland Research Institute CA USA 2 Lipoprotein and Atherosclerosis Group University of Ottawa Heart Institute Ottawa Ontario Canada 3 Department of Biochemistry and Protein Engineering Network of Centres of Excellence University of Alberta Edmonton Canada Keywords apolipoprotein E fluorescence spectroscopy low-density lipoprotein low-density lipoprotein receptor phospholipids Correspondence R. O. Ryan Children s HospitalOakland Research Institute 5700 Martin Luther King Jr. Way Oakland CA 94609 USA Fax 1 510 450 7910 Tel 1 510 450 7645 E-mail rryan@ Received 3 November 2005 revised 1 December 2005 accepted 5 December 2005 doi The N-terminal domain of human apolipoprotein E apoE-NT harbors residues critical for interaction with members of the low-density lipoprotein receptor LDLR family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change the short helix connecting helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG introducing a b-turn. Recombinant helix-to-turn HT variant apoE3-NT was produced in Escherichia coli isolated and characterized. Stability studies revealed a denaturation transition midpoint of M guanidine hydrochloride for HT apoE3-NT vs. M for wild-type apoE3-NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and .