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

A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E.coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Designer Lipid-Like Peptides: A Class of Detergents for Studying Functional Olfactory Receptors Using Commercial Cell-Free Systems

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Cell contraction forces in scaffolds with varying pore size and cell density

The contractile behavior of cells is relevant in understanding wound healing and scar formation. In tissue engineering, inhibition of the cell contractile response is critical for the regeneration of physiologically normal tissue rather than scar tissue. Previous studies have measured the contractile response of cells in a variety of conditions (e.g. on two-dimensional solid substrates, on free-floating tissue engineering scaffolds and on scaffolds under some constraint in a cell force monitor). Tissue engineering scaffolds behave mechanically like open-cell elastomeric foams: between strains of about 10 and 90%, cells progressively buckle struts in the scaffold. The contractile force required for an individual cell to buckle a strut within a scaffold has been estimated based on the strut dimensions (radius, r, and length, l) and the strut modulus, E[subscript s]. Since the buckling force varies, according to Euler's law, with r[superscript 4]/l[superscript 2], and the relative density of the scaffold varies as (r/l)[superscript 2], the cell contractile force associated with strut buckling is expected to vary with the square of the pore size for scaffolds of constant relative density. As the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease. Here we model the contractile response of fibroblasts by analyzing the response of a single tetrakaidecahedron to forces applied to individual struts (simulating cell contractile forces) using finite element analysis. We model tetrakaidecahedra of different strut lengths, corresponding to different scaffold pore sizes, and of varying numbers of loaded struts, corresponding to varying cell densities. We compare our numerical model with the results of free-floating contraction experiments of normal human dermal fibroblasts (NHDF) in collagen-GAG scaffolds of varying pore size and with varying cell densities.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Training Grant T32-EB00634)Matoula S. Salapatas Professorship in Materials Science and Engineerin