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Application of Nanotechnology to Study of GPCR
Characterization of complexity of conformational states and their dynamics by single molecule spectroscopy:
Peleg, G., P. Ghanouni, B.K. Kobilka, and R.N. Zare, Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8469-74
- use of single moelcule spectroscopy: overcome the problem of ensemble averaging of other methods, ref.
1. Nie, S. and R.N. Zare, Optical detection of single molecules. Annu Rev Biophys Biomol Struct, 1997. 26: p. 567-96.
2. Nie, S., D.T. Chiu, and R.N. Zare, Probing individual molecules with confocal fluorescence microscopy. Science, 1994. 266(5187): p. 1018-21.
3. Dickson, R.M., A.B. Cubitt, R.Y. Tsien, and W.E. Moerner, On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature, 1997. 388(6640): p. 355-8.
4. Xie, X.S. and J.K. Trautman, Annu Rev Phys Chem, 1998. 49: p. 441-480.
- beta2-adrenergic receptor
- fluorescence tag in detergent micelles at position Cys265 (Fluorescein-5-maleimide) at the end of TM VI
- of 13 cysteines, Cys265 is the only accessible to the Fluorescein-5-maleimide (5 in TM region, 4 in disulfide bonds in EC domain, 1 in C-term is palmitoylated, remaining 2 C-terminal Cys form a disulfide bond during purification)
- at least two distinct substates for the native receptor, "is a conformation ally flexible molecule", 2 predominant, several minor ones possibly represented by different burst intensities
- full agonist ISO (which has higher binding affinity KI~10 microM and higher biological efficacy than adrenaline) stabilizes confrmational substates that are rare in the native receptor
- conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes
- conformational heterogeneity of GPCR in the presence and absence of a bound agonist
- various ligands (agonists and antagonists) change both the shape of the entire distribution and the populations of the conformational substaes
Immobilization of GPCR on solid support:
1. delta opioid receptor Salamon, Z., S. Cowell, et al. (2000). "Plasmon resonance studies of agonist/antagonist binding to the human delta-opioid receptor: new structural insights into receptor-ligand interactions." Biophys J 79(5): 2463-74. orientation not known
- first prepare membrane film on solid support, then add concentrated receptor in 30 mM OG detergent to the aqueous compartment, thereby diluting OG to under CMC (25 mM) -> spontaneous integration into preformed membrane bilayer
- spread small amount of lipid across Teflon sheet that separates the thin dielectric film (SiO2) from the aqueous phase
- hydrophilic surface of hydrated SiO2 attracts the polar groups of the lipids, inducing an initial orientation of the lipid molecules with the hydrophobic carbon chains pointing toward to droplet of excess lipid solution
- add aqueous buffer to sample compartment, and a plateau-Gibbs border of lipid will anchor the membrane to the Teflon spacer
- Result: PC/POPG lipid bilayer
- also see GPCRactivation
2. rhodopsin Niu, L., J. M. Kim, et al. (2002). "Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes." Proc Natl Acad Sci U S A 99(21): 13409-12 orientation known
SPR of rhodopsin:
1. Salamon, Z., Y. Wang, J.L. Soulages, M.F. Brown, and G. Tollin, Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films. Biophys J, 1996. 71(1): p. 283-94.
2. Salamon, Z., Y. Wang, M.F. Brown, H.A. Macleod, and G. Tollin, Conformational changes in rhodopsin probed by surface plasmon resonance spectroscopy. Biochemistry, 1994. 33(46): p. 13706-11.
3. Salamon, Z., Y. Wang, G. Tollin, and H.A. Macleod, Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy. Biochim Biophys Acta, 1994. 1195(2): p. 267-75.