Olfactory-related receptors : methods towards enabling structural and functional studies

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Mammalian noses can detect and distinguish an inestimable number of odors at minute concentrations. Four classes of G protein-coupled receptors (GPCRs) are responsible for this remarkable sensitivity: olfactory receptors (ORs), vomeronasal receptors (VNRs), trace amine-associate receptors, and formyl peptide receptors. Structural knowledge of these receptors is necessary to understand the molecular basis of smell. However, no structure exists for three main reasons. First, milligrams of protein are needed for crystallization screens, but most are expressed at low levels endogenously or in heterologous expression systems. Second, detergents capable of solubilizing and stabilizing these proteins in aqueous solution must be found. Third, the flexible nature of GPCRs can inhibit crystal lattice formation. Methods for overcoming each obstacle were developed. Milligrams of a VNR were expressed in HEK293 cells, and milligrams of 13 GPCRs were expressed in a cell-free system. All could be purified to >90%. The purified receptors had correct secondary structures, and could bind their ligands. The HEK293 and cell-free receptors had nearly identical structures and binding affinities, demonstrating that cell-free expression can be used for GPCR production and mutational studies. To demonstrate this, six variants of mOR103-15 with single amino acid substitutions were expressed. Ligand-binding measurements indicated which residues were involved in ligand recognition. The choice of detergent used in the cell-free system was critical, and significantly affected expression levels. A class of amphiphilic peptide detergents was designed and tested with the receptors. These detergents could be used to express milligrams of functional receptors. The peptide tail and head group properties did not significantly affect their function, suggesting that they may be a class of surfactants usable with multiple olfactory-related receptors, and even other membrane proteins. Lastly, the protein T4 Lysozyme (T4L) was fused in the 3rd intracellular loop of two receptors to increase potential crystal lattice contact points. Purified T4L variants had correct secondary structures, and could bind their ligands and initiate intracellular signaling. The methods described generated sufficient quantities of pure receptors for crystal screens. The large number of functionally expressed GPCRs indicates that these techniques can be applied to other olfactory-related receptors, and even other membrane proteins.by Karolina Corin.Ph.D

Inhibition of myofibroblast contraction

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2005.Includes bibliographical references (p. 46-49).Although current medical procedures cannot restore complete function of a transected nerve, inserting both of its ends in a tube helps it regenerate. The regenerate is inferior to the uninjured nerve: it has a smaller diameter and poorer electrical conduction. Layers of contractile cells known as myofibroblasts have been observed around regenerated nerve portions. An inverse relationship between the layer thickness and the quality of the regenerate has also been observed. These findings suggest that the cells are exerting contractile forces which prevent the regenerating nerve from fully developing. Inhibiting this contraction should thus improve the quality of nerve regeneration. Alpha smooth muscle actin ([alpha]-SMA) is a critical contractile protein. Its expression can be upregulated by the growth factor TGF-[beta]1, and blocked by the pharmacological agent PP2. To investigate whether blocking SMA expression alone can inhibit myofibroblast contraction, NR6 wild type fibroblasts were seeded into short cylindrical collagen-GAG matrices, and administered either media alone, media with TGF-[beta]1 (3ng/ml), or media with TGF-[beta]1 and PP2 (10 [mu]M). Non-seeded matrix samples were also prepared. The matrix diameters were measured every day for 12 days, after which the matrices were digested and the number of adhered cells were counted. The daily change in matrix diameter was calculated. The results showed that the cells contracted the matrices. TGF-[beta]1 increased cell contractility, while PP2 inhibited it..(cont.) Normalizing the Day 12 diameter change measurements to cell number and the original matrix diameter showed that TGF-[beta] increased the strain generated by each cell ... relative to ... for untreated cells), and that PP2 counteracted this effect (...). Using the linear elastic constitutive relations, the average force exerted per cell was calculated for the untreated cells (...), TGF-[beta]1 stimulated cells (...), and TGF-[beta] + PP2 stimulated cells (...). The cell counts after Day 12 indicate that PP2 interferes with cell adhesion to the matrices. After 6 hours in culture, 21% of untreated cells, 25% percent of cells treated with TGF-[beta] 1, and 25% of cells treated with TGF-[beta]1 and PP2 had adhered. By Day 12, only 12% of the seeded untreated cells, 14% of cells treated with TGF-[beta] I, and 3.2% of cells treated with both TGF-[beta]1 and PP2 remained adhered. This study thus indicates that PP2 inhibits cellular contraction, possibly by preventing cell-substrate adhesionby Karolina A. Corin.S.M