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Effects of different environments on...

Epps, Jacqueline Jeannelle.

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Effects of different environments on rhodopsin photochemistry.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Effects of different environments on rhodopsin photochemistry./
作者:	Epps, Jacqueline Jeannelle.
面頁冊數:	141 p.
附註:	Adviser: Eugene Switkes.
Contained By:	Dissertation Abstracts International69-02B.
標題:	Biophysics, General. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3301332
ISBN:	9780549465263

Effects of different environments on rhodopsin photochemistry.
Epps, Jacqueline Jeannelle.

Effects of different environments on rhodopsin photochemistry. - 141 p.

Adviser: Eugene Switkes.

Thesis (Ph.D.)--University of California, Santa Cruz, 2008.

The investigations presented in this body of work were performed to explore the behavior of the photointermediates of rhodopsin in differing environments. The environments included that of two-dimensional crystal, detergent solubilized (5% dodecyl maltoside) and native membrane suspensions of rhodopsin. The presence or absence of the native membrane environment is well documented to affect the kinetics associated with rhodopsin's photointermediates. The goal of these studies was to characterize the deviation from wild-type kinetics that occurs with a change in rhodopsin environment.

ISBN: 9780549465263Subjects--Topical Terms:

1019105
Biophysics, General.

Effects of different environments on rhodopsin photochemistry.
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100 1 $a Epps, Jacqueline Jeannelle. $3 1264742
245 1 0 $a Effects of different environments on rhodopsin photochemistry.
300 $a 141 p.
500 $a Adviser: Eugene Switkes.
500 $a Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0994.
502 $a Thesis (Ph.D.)--University of California, Santa Cruz, 2008.
520 $a The investigations presented in this body of work were performed to explore the behavior of the photointermediates of rhodopsin in differing environments. The environments included that of two-dimensional crystal, detergent solubilized (5% dodecyl maltoside) and native membrane suspensions of rhodopsin. The presence or absence of the native membrane environment is well documented to affect the kinetics associated with rhodopsin's photointermediates. The goal of these studies was to characterize the deviation from wild-type kinetics that occurs with a change in rhodopsin environment.
520 $a Bovine rhodopsin photointermediates formed in two-dimensional crystal suspensions were studied by measuring the temperature-dependent absorbance changes produced after excitation at 15, 25 and 35°C. The crystalline environment favored the Meta I480 photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membrane suspension at 35°C and its decay to a 380 nm absorbing species being less complete than it is in the native membrane at all temperatures. In addition to the temperature-dependence of this environment, the pH dependence was performed. At pH 5.5, the 380 nm absorbing product of Meta I480 decay did not display the anomalous pH dependence of classical Meta II in the native membrane suspension. Furthermore, crystal suspension bleached at 35°C and quenched to 19°C showed that a rapid equilibrium existed on the &sim;1 second time scale, which suggests that the unprotonated predecessor of Meta II in the native membrane environment forms in two-dimensional rhodopsin crystals but that the non-Schiff base proton uptake completing classical Meta II formation is blocked there. Thus, the 380 nm absorbance arises from an on-pathway intermediate in G protein-coupled receptor (GPCR) activation and does not result from early Schiff base hydrolysis.
520 $a The study continued with the characterization of the Lumi I → Lumi II transition in both membrane and detergent solubilized suspensions of bovine rhodopsin at physiological temperature (20°C). Time-resolved absorbance difference spectra collected from 1 to 128 mus post-photolysis under both conditions fit well to two-exponential decays plus a constant. A similar fast process was observed (11 mus in membrane and 12 mus in detergent solubilized suspensions) with a small but similar spectral change. This demonstrates that the Lumi I → Lumi II process, previously characterized in detergent suspensions, has similar properties in membrane without significant effect of detergent. The slower exponential process detected in the data was quite different in membrane compared to detergent solubilized samples, showing that the pronounced effect of detergent on the later photointermediates begins fairly abruptly at 20 mus. Besides affecting the late processes, the data collected here show that detergent induces a small blue shift in the Lumi-minus-rhodopsin difference spectrum. This blue shift may indicate that the ionization state of Glu181 is affected by the presence of detergent.
520 $a Upon confirmation that the Lumi I → Lumi II transition was intrinsic to the rhodopsin photointermediate cascade, the temperature-dependence of this transition was explored in order to accurately account for its presence in the square scheme kinetic model. At 15, 20 and 35°C, native membranous rhodopsin showed effects on the kinetics as a result of the alterations in temperature. In membrane, the increase in temperature is accompanied by a loss in membrane rigidity which is shown to modify the kinetic behavior of the photointermediates. Because detergent disturbs the structure supplied by the membrane, detergent solubilized rhodopsin displayed no changes in the kinetics as a result of increasing temperature. The temperature-independent spectra obtained from the detergent solubilized rhodopsin were used to produce three mathematical approaches derived on the assumption that the observed kinetics follow a classical thermodynamic dependence.
520 $a The final approach to characterization of rhodopsin's photointermediates was by the use of a mutant preparation of rhodopsin, S186A, which was detergent solubilized. The role of this Serine located in the retinylidene chromophore-binding pocket of rhodopsin was investigated to determine its role in the mechanism of receptor photoactivation. While time-resolved absorbance difference spectra showed a normal Batho, the microscopic rate constant for the back reaction of the S186A BSI to Batho and the forward reaction of the S186A BSI to Lumi were both significantly reduced. Furthermore, the UV-Vis absorption maximum of S186A BSI was red-shifted by almost 20 nm relative to that of wild-type BSI, and the deprotonation of the Schiff base was unusually rapid and was complete in microseconds. The observed large mutagenic perturbations to the kinetics and photointermediate spectra suggest that the hydroxyl group of Serine 186 interacts with the protonated Schiff base and/or its counterion after photoexcitation.
590 $a School code: 0036.
650 4 $a Biophysics, General. $3 1019105
650 4 $a Chemistry, Biochemistry. $3 1017722
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791 $a Ph.D.
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