New Insights into Light-Induced Deactivation of Active Rhodopsin by SVD and Global Analysis of Time-Resolved UV/Vis- and FTIR-Data

Abstract

Time-resolved Fourier transform infrared (FTIR) and UV.Vis difference spectra of light induced deactivation of the photoreceptor rhodopsin were simultaneously recorded on the same sample. The data were analyzed by a special designed combination of singular value decomposition and Global analysis to verify our recently published model of receptor deactivation. This mathematical approach enables us to obtain the pure difference spectra of the conversions between the species involved. We found two different species, which could be identified as key intermediates of the model. The new data allow deeper insights into the structural changes that come along with the formation of the deactivated state. By means of this method we can now separate and assign even strongly overlapping and highly coupled bands of the infrared difference spectra.

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin

“…Briefly, difference spectra were calculated from time-resolved RapidScan data using the SVD/ Global Analysis tools to obtain pure difference spectra without contribution of decay products as described earlier (40). Subsequently, all difference spectra were normalized to the intensities in the 1050-970 cm -1 region, where all light-activated conformations exhibit identical absorbance changes.…”

“…FTIR difference spectra were measured in the presence and absence of 20 mM ArrFL-1 peptide (∆(w/ ArrFL-1) and ∆(w/o ArrFL-1), gray and black lines in Figure 3 and 4, respectively). As for all difference spectra shown in this manuscript, data analysis was performed using a combination of SVD and Global Analysis (40), which allows determination of pure difference spectra of the light-induced transition without any contribution of photoproduct decay. This approach led to extremely high signal to noise ratio and reproducibility, thus allowing the interpretation of all bands, which appear clearly above the noise level (see Supplemental Information for assessment of signal to noise ratio).…”

The Activation Pathway of Human Rhodopsin in Comparison to Bovine Rhodopsin

“…To obtain sets of difference spectra, the whole dataset was subtracted from the average of all spectra recorded before illumination. Because a photostationary state is usually not reached due to the overlay of formation and decay processes of the different species, the difference spectra of the intermediates and their kinetics were determined by a singular value decomposition (SVD) method and global fitting procedure (13). The procedure yields the spectral characteristics of the reaction separated by their different kinetics FIGURE 1.…”

Formation and Decay of the Arrestin·Rhodopsin Complex in Native Disc Membranes

“…In cases of pure Meta II conditions, this was additionally verified by the intensity of the Meta II marker band at 1768 cm Ϫ1 . Spectra describing pure transition components (b-spectra) and the corresponding kinetic constants of these components were derived by a combination of singular value decomposition (SVD) and global fitting as described (28,29). Briefly, the measured and baseline-corrected time-dependent data sets of difference spectra D(,t) were decomposed into three matrices:…”

“…Finally, after reestablishing orthonormality by a second application of SVD, a global fit procedure utilizing the columns of the kinetics matrix was applied under the assumption of a sequential reaction scheme. This yielded b-spectra that represent the difference spectra of pure transitions from one state to the next characterized by the same kinetics (29). Calculations were performed with GNU Octave.…”