Absorbance Changes by Aromatic Amino Acid Side Chains in Early Rhodopsin Photointermediates

Lewis, James W.; Jäger, Stefan; Kliger, David S.

doi:10.1111/j.1751-1097.1997.tb03218.x

Cited by 8 publications

(5 citation statements)

References 36 publications

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“…The 233 nm probe lies on the steep red side of the deep UV absorption curve (see Figure 1), so a blue shift would be sufficient to explain the negative Raman difference features. While we are not aware of UV absorption difference spectra of bathorhodopsin which extend to the probe wavelength of 233 nm, a combination of an absorption blue shift and perhaps reduced hyperchromism has been reported in the later intermediates (38,40,41). Our data support a picture in which the UV spectral blue-shift, arising from no more than two tryptophans (38), occurs as rapidly as the all-trans photoproduct is formed, in the photorhodopsin stage.…”

Section: Discussionsupporting

confidence: 76%

Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

2003

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The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the β-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsinto-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.Knowledge of the initial response of a protein to an activating agent or event is required to derive a mechanistic understanding of protein function. In the case of the visual pigment rhodopsin, absorption of a single visible photon by the antagonist 11-cis retinal chromophore results in the formation of a highly energetic activated all-trans photo-product (1). This stored energy is transduced into global protein conformational changes at the Meta I-Meta II stages that trigger the G-protein visual cascade (2). The mechanism by which the photoisomerization reaction induces protein conformational changes is not understood, largely due to the lack of high temporal resolution structural tools that can elucidate these early events. Here, we report the first structural study of the protein conformational changes associated with the primary photochemical event in vision using picosecond time-resolved UV resonance Raman vibrational spectroscopy.Vibrational structural studies of biomolecules have greatly advanced since the introduction of UV resonance Raman (UVRR) 1 spectroscopy (3-5). When the excitation wavelength is tuned between ∼195 and 260 nm, strong resonance Raman scattering from the peptide backbone and aromatic amino acids provides vibrational information on local protein structure and environmental changes. This technique has been used, for example, to study rhodopsin protein structure (6), protein folding (7, 8), and protein-ligand interactions (9, 10). In addition, time- † We dedicate this paper to the memory of Helena (Ilona) Anna Palings (1956Palings ( -2002. This work was supported by grants from the National Institutes of Health (EY-02051) and the National Science Foundation (CHE-98-01651 (6,27,28), there remains a lack of understa...

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Section: Discussionsupporting

confidence: 76%

Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

2003

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“…The batho to lumi transition in bovine rhodopsin is also associated with changes in the protein backbone (42)(43)(44)(45), although these changes have not been well characterized. UV absorbance spectroscopy (46)(47)(48), linear dichroism (49), and site-specific mutagenesis (50) have shown that the environments of two tryptophan residues, W126 and W265, are perturbed during the formation of the active state.…”

Section: Discussionmentioning

confidence: 99%

Phototransduction by Vertebrate Ultraviolet Visual Pigments: Protonation of the Retinylidene Schiff Base following Photobleaching

et al. 2002

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The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.

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“…Sci. USA 94 (1997) are similar, as required from time-resolved spectral measurements in the UV (55). The Lumi Intermediate.…”

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confidence: 80%

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

Jäger

Lewis

Zvyaga

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Rhodopsin is a prototypical G proteincoupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin's transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.Rhodopsin is the light-triggered membrane receptor in rod outer segments responsible for dim-light vision. Absorption of light transforms rhodopsin (rho) into an activated state, which interacts with a heterotrimeric G protein to initiate an enzyme cascade leading to visual transduction. To reach this active state, called metarhodopsin (meta) II (1, 2), rho passes through a number of intermediates that have been characterized by UV-visible, Fourier-transform infrared (FTIR), Raman, and NMR spectroscopies, in addition to spin-labeling and biochemical studies (3-10). One way to characterize the intermediates is to trap them at low temperatures. This approach led to the classical ''bleaching'' scheme ( max values in nanometers): rho (498) 3 bathorhodopsin (batho) (542) 3 lumirhodopsin (lumi) (497) 3 meta I (480) 3 meta II (380) Scheme I However, at more physiologically relevant temperatures, time-resolved measurements up to 1 msec after photolysis reveal a new intermediate and a different kinetic scheme (3, 11). The following reaction scheme was proposed to occur during the nanosecond-to-microsecond time regime (3): rho (498) 3 batho (529) ª bsi (477) 3 lumi (492) 3 . . . Scheme II The new intermediate, blue-shifted intermediate of rho (bsi), is entropically favored and thus not trapped at low temperatures (11).The role of various structural features of the retinal chromophore in early rho photointermediates has been studied through time-resolved spectral measurements of artificial rho, in which the native chromophore is replaced by synthetically modified retinals (12). In studies of bacteriorhodopsin, timeresolved absorption spectroscopy has been shown to be particularly valuable when combined with site-directed mutagenesis to probe the roles of specific protein residues in different intermediates (13). Unti...

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Absorbance Changes by Aromatic Amino Acid Side Chains in Early Rhodopsin Photointermediates

Cited by 8 publications

References 36 publications

Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

Picosecond Dynamics of G-Protein Coupled Receptor Activation in Rhodopsin from Time-Resolved UV Resonance Raman Spectroscopy

Phototransduction by Vertebrate Ultraviolet Visual Pigments: Protonation of the Retinylidene Schiff Base following Photobleaching

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

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