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. 2015 Nov 9;54(46):13555-60.
doi: 10.1002/anie.201505798. Epub 2015 Sep 18.

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Deep Chatterjee  1 , Carl Elias Eckert  2 , Chavdar Slavov  2 , Krishna Saxena  1 , Boris Fürtig  1 , Charles R Sanders  3 , Vsevolod V Gurevich  4 , Josef Wachtveitl  5 , Harald Schwalbe  6
Affiliations

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Deep Chatterjee et al. Angew Chem Int Ed Engl. .

Abstract

Continued activation of the photocycle of the dim-light receptor rhodopsin leads to the accumulation of all-trans-retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre-activated truncated bovine visual arrestin (Arr(Tr)) with rhodopsin in 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin-arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr(Tr) leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all-trans-retinal from rhodopsin.

Keywords: NMR spectroscopy; UV/Vis spectroscopy; arrestin; retinal regeneration; rhodopsin.

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Figures

Figure 1
Figure 1
Mammalian visual cycle. The concentration of all-trans-retinal (ATR) is dependent on two processes: i) retinal release during the photocycle in the rod outer segment (ROS) and ii) retinal removal by a regeneration pathway in the retinal pigment epithelium (RPE) as schematically shown on the left. Continuous exposure to light leads to the accumulation of ATR (right, top), which initiates rhodopsin deactivation by arrestin (arr). Arrestin binding slows down the retinal release, which gives ample time to retinal dehydrogenase (RDH) to remove excess ATR by converting it to all-trans-retinol (right, bottom).
Figure 2
Figure 2
a) Schematic representation of the secondary structure of bovine rhodopsin with the five tryptophan residues (W35, W126, W161, W175, and W265) used as reporter groups in the NMR experiments. b) Overlay of the indole region of 2D 1H, 15N SOFAST-HMQC spectra of α,ε-15N-tryptophan-labeled rhodopsin in DDM (black) and DHPC (gray) micelle. Overlay of 2D 1H, 15N SOFAST-HMQC spectra of α,ε-15N-tryptophan-labeled rhodopsin without (blue) and with Tr (red) (rhodopsin : Tr – 1 : 2) showing c) backbone region, d) indole region of the spectra. Overlay of 1D projection of the corresponding cross peaks of the indole region of 2D spectra is shown in inset (b and d). All spectra were recorded at 800 MHz (T = 298 K) in the following buffer: DDM spectrum – 20 mM sodium phosphate buffer pH 7.4; DHPC spectra – 25 mM Tris pH 7.5, 100 mM NaCl, 0.1 μM EDTA, 10% D2O and 1 mM 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionic acid (TSP-d4).
Figure 3
Figure 3
NMR kinetics. a) A series of 1D 1H NMR spectra of α,ε-15N-tryptophan-labeled rhodopsin recorded at different time intervals after illumination. The indole region of the spectrum is shown. The five resonances visible in the dark state correspond to the five tryptophan residues present in rhodopsin. Subsequent to illumination, 1D 1H spectra were recorded with a temporal resolution of one minute. Inset: overlay of spectrum of the jump-return-echo (gray) and 1H x-filter (black) NMR experiment of rhodopsin samples. The dashed lines connect the corresponding resonances of the five tryptophan residues. b) Extracted signal intensities from the series of 1D 1H NMR spectra of α,ε-15N-tryptophan-labeled rhodopsin in presence and absence of Tr. A mono-exponential fit was applied for the signal intensities of W35 (black curve) and W126 (gray curve).
Figure 4
Figure 4
Transient absorption spectra of rhodopsin a) without and b) with Tr. Red is positive and blue is negative change in absorption. Double difference spectra of a) and b) calculated by subtracting the absorption change recorded at the maximum delay time from the transient absorption data are depicted in c) and d) respectively.
Figure 5
Figure 5
Lifetime density analysis of transient absorption data. LDMs of rhodopsin samples with increasing Tr concentration. The reading of the LDMs is as for DAS with positive (red) amplitudes accounting for decay of absorption and negative (blue) amplitudes accounting for rise of absorption. The lifetimes and the distribution maxima of the signatures C-F are shown in table 1.
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