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Research Interests Research in this lab revolves around the mechanisms and roles of molecular recognition in biological systems. The apolipoproteins are responsible for shuttling lipoproteins to the low-density lipid (LDL) receptor family. There are four classes of apolipoproteins: apoA, apoB, apoC and apoE. ApoE has three alleles (ApoE-2 through 4) that can be described as having two domains: a 22 kDa N-terminal domain responsible for interacting with the LDL receptor and a C-terminal domain (10 kDa) that has a high affinity for lipoproteins. By itself, the N-terminal domain of ApoE will not bind to the LDL receptor in the absence of lipoprotein particles (or lipoprotein mimics). However, the N-terminal domain of apoE will bind to the LDL receptor in the presence of lipoprotein (and lipoprotein mimics) which argues for a lipoprotein-induced conformational rearrangement as a requirement for ApoE binding to the LDL receptor. The N-terminal domain of ApoE is a four-helix amphipathic bundle whose hydrophobic core is postulated to become buried in the interior of thelipoprotein particle upon binding (see figure). Details at atomic resolution of this structural rearrangement and the time-scale of the rearrangement are being elucidated in this lab using a combination of molecular biology and magnetic resonance techniques. Site-directed mutagenesis allows cysteines to be substituted at key positions throughout the protein. In turn, the sulfhydryl group of cysteine provides an anchor forattaching a stable nitroxide radical via a disulfide linkage. Using electron paramagnetic resonance (EPR) methods, the nitroxide radical is being used to probe theconformational changes of the apoE-3 allele. Incorporating single nitroxides and then using relaxing agents that partition between the lipid and aqueous phases allows the orientation of the helices to be probed. Doubly incorporated nitroxides provide quantitative distance information based upon the spin-spin interaction between the nitroxide unpaired electrons. Utilizing a variety of double-labeling strategies, sufficient distance constraints are being developed to provide a three-dimensional representation of the helices in the absence/presence of lipids. As the model of apoE-3 conformational rearrangement emerges, the other forms of ApoE are scheduled to be examined and the results compared with those obtained from ApoE-3. The resulting structural details will be used to understand the mechanisms that direct the function of the differing ApoE alleles. Another route of exploration is Nature’s apparent use of 'building blocks' of secondary structure to design proteins. Do the helices of the N-terminal portion of ApoE -3 represent the optimized sequences/structures or are there other sequences that would be as effective in organizing the lipoprotein particle? To explore this question, combinatorial libraries of the N-terminal helices of ApoE-3 are being screened for their ability to affect the same organizational rearrangements of lipoprotein particles. Peptides successful at these rearrangements will then be screened for their ability to bind to the LDL receptor. The results of screening these peptide libraries will provide insights into the sequence/structure/function relationships that serve as the cornerstones of molecular recognition.
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Selected Publications Pardi, A., et al., Determining global conformation of nucleic acids in solution. Biophysical Journal, 2001. 80(1): p. 54. Hansen, M.R., P. Hanson, and A. Pardi, Pf1 filamentous phage as an alignment tool for generating local and global structural information in nucleic acids. Journal of Biomolecular Structure & Dynamics, 2000: p. 365-369. Hansen, M.R., P. Hanson, and A. Pardi, Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions, in Rna-Ligand Interactions Pt A. 2000. p. 220-240. Anderson, D.J., et al., Solution structures of TOAC-labeled trichogin GA IV peptides from allowed (g approximate to 2) and half-field electron spin resonance. Journal of the American Chemical Society, 1999. 121(29): p. 6919-6927. |
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