Entire Thesis (13 Mb)

Title_and_Contents

Chapter_1_Introduction

Chapter_2_Ethidium

Chapter_3_Azioct

Chapter_4_Progestin

Summary_and_References

Figure 1-1. Proposed subunit topology and receptor oligomerization.

Figure 1-2. 9 Å structure of nAChR from electron microscopy

Figure 1-3. Agonist and benzodiazepine binding sites of the nAChR family

Figure 1-4 M2 and M1 segments of the nAChR

Figure 1-5. M3 and M4 segments of the nAChR

Figure 1-6. NCAs of the nAChR

Figure 1-7. The binding site of ethidium localized by FRET

Figure 1-8. Proposed binding sites of alcohols on the nAChR and GABA_AR

Figure 1-9. Formation of photoreactive intermediates from aryl azides and diazirines

Figure 1-10. Photoaffinity probes of the nAChR

Figure 2-1. Binding of [³H]ethidium in the presence and absence of various cholinergic drugs.

Figure 2-2. Photoincorporation of [³H]ethidium diazide into integral and peripheral membrane proteins of nAChR-rich membranes in the presence or absence of carbamylcholine or PCP.

Figure 2-3. Photoincorporation of [³H]ethidium diazide into integral and peripheral membrane proteins of nAChR-rich membranes in the presence of oxidized glutathione.

Figure 2-4. Photoincorporation of [³H]ethidium diazide into integral and peripheral membrane proteins of nAChR-rich membranes in the presence of various cholinergic drugs.

Figure 2-5. Proteolytic mapping of sites of [³H]ethidium diazide incorporation into the nAChR a -subunit using S. aureus V8 protease.

Figure 2-6. Reverse-phase HPLC purification of [³H]ethidium diazide labeled fragments from EndoLysC digest of a V8-20.

Figure 2-7. ³H and mass release upon N-terminal sequence analysis of HPLC fractions of EndoLysC-digest of [³H]ethidium diazide labeled a V8-20

Figure 2-8. Proteolytic mapping of the sites of [³H]ethidium diazide incorporation in the nAChR d -subunit using V8 protease.

Figure 2-9. EndoLysC digest of d V8-14 fragment labeled with [³H]ethidium diazide resolved by Tricine SDS-PAGE.

Figure 2-10. Reverse-phase HPLC purification of [³H]ethidium diazide labeled fragments from EndoLysC digest of d V8-20.

Figure 2-11 Sequence Analysis of d V8-20 EndoLysC digest fragments.

Figure 2-12. Model of ethidium and a M2 helix

Figure 3-1. Photoincorporation of [³H]3-azioctanol into integral and peripheral membrane proteins of nAChR-rich membranes in the presence or absence of carbamylcholine.

Figure 3-2 Time course of incorporation of [³H]3-azioctanol into integral membrane proteins of nAChR-rich membranes in the presence of carbamylcholine.

Figure 3-3. Dependence of [³H]3-azioctanol incorporation into a -subunit on the concentration of carbamylcholine.

Figure 3-4. Photoincorporation of [³H]3-azioctanol into nAChR-rich membranes in the presence of various cholinergic agonists and competitive antagonists.

Figure 3-5. Photoincorporation of [³H]3-azioctanol into nAChR-rich membranes in the presence and absence of various cholinergic drugs.

Figure 3-6. Effect of [³H]3-azioctanol concentration on the incorporation into a -subunit.

Figure 3-7. Proteolytic mapping of sites of [³H]3-azioctanol incorporation into the nAChR a -subunit using S. aureus V8 protease.

Figure 3-8. Reverse phase HPLC purification of [³H]3-azioctanol labeled fragments from an EndoLysC digest of a V8-20 and sequence analysis of HPLC fractions

Figure 3-9. Reverse phase HPLC purification of [³H]3-azioctanol labeled fragments from S. aureus V8 protease digest of a V8-20.

Figure 3-10. Reverse phase HPLC purification of [³H]3-azioctanol labeled a V8-18 and sequence analysis of HPLC fraction.

Figure 3-11. Reverse phase HPLC purification of [³H]3-azioctanol labeled fragments from trypsin digestion of a V8-10 and sequence analysis of HPLC fractions.

Figure 3-12. Model of 3-azioctanol and a M2 helix

Figure 4-1. Structure of progesterone and photoaffinity derivatives

Figure 4-2. Photoincorporation of [³H]progestin aryl azide into integral and peripheral membrane proteins of nAChR-rich membranes in the presence or absence of various cholinergic drugs.

Figure 4-3. Proteolytic mapping of sites of [³H]progestin aryl azide incorporation into the nAChR a -subunit using S. aureus V8 protease.

Figure 4-4. Reverse phase HPLC purification of [³H]progestin aryl azide labeled fragments from trypsin digest of a V8-10

Figure 4-5. HPLC purification of [³H]progestin aryl azide labeled fragments from trypsin digest of b -subunit

Figure 4-6. HPLC purification of [³H]progestin aryl azide labeled fragments from trypsin digest of g -subunit

Figure 4-7. HPLC purification of [³H]progestin aryl azide labeled fragments from EndoLysC digest of d -subunit