Elsa Yan


Postdoctoral Fellow

Ph. D. Columbia University, New York, 2000
M. Phil. Columbia University, New York, 1999
M. A. Columbia University, New York, 1996
B. Sc. Chinese University of Hong Kong, 1995

Elsa's CV



I am investigating the activation mechanism of G protein-coupled receptors (GPCRs) by studying protein and chromophore structures of visual pigments along the photoactivation pathway using a multidisciplinary approach that integrates Raman spectroscopy, theoretical calculation, molecular biology and bioinformatics. Accomplishment includes the elucidation of the counterion switch in the activation of rhodopsin and determination of the mechanism of energy storage in the primary photoproduct.

(1) Counterion Switch in the Photoactivation of the GPCR Rhodopsin

We have explored the biological functions of Glu181 in the photoactivation process of rhodopsin through spectroscopic studies of site-specific mutants. The crystal structure of bovine rhodopsin surprisingly revealed that extracellular loop 2 (E2) folds deep into the transmembrane region, thereby placing Glu181, a potentially charged and highly conserved residue, only 4.7 Å from C12 of the 11-cis retinal (Figure 1).


Figure 1. Proximity of Glu181 to retinal chromophore.

Our earlier work(2) showed that E181Q is the only mutant among the full set of Glu181 mutants we expressed that gives a significant spectral shift in the dark (~5 nm in the presence of Cl ion and ~10 nm in the absence of Cl ions). We also showed that the absorption spectrum of E181Q is insensitive to the pH in the dark. These results indicate that the interaction between the chromophore and Glu181 is not strong, and that Glu181 is neutral (protonated) in the dark state of rhodopsin. In our more recent work1, preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are found to be nearly identical to spectra of the native (pMT4) pigment (Figure 2), supporting the view that Glu181 is uncharged (protonated) in the dark state.


Figure 2. Raman spectra of Glu181 mutants.

The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with that of pMT4 (Figure 3). This result is most consistent with the assignment of Glu181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu113 in the dark state to Glu181 in Meta I.



Figure 3. Titration of Schiff base in metarhodopsin I.


Base on these data, we propose a model where the counterion switch occurs by transferring a proton from Glu181 to Glu113 through an H-bond network in the chromophore-binding pocket (Figure 4). This H-bond network is composed mainly of the residues on EII such as Glu181, Ser186, Cys187, and Tyr192. It is likely that the E2 loop residues are utilized because the loop is more flexible and can better accommodate the structural changes during isomerization and the counterion switch process.

Figure 4. Mechanism for the counterion switch.


The counterion switch model also has profound implications for understanding the role of the disulfide bond (Cys110-Cys187) that links E2 and helix 3 (H3) in G protein-coupled receptor activation (Figure 5). Glu113 is one helical turn away from Cys110; and Glu181 is on the same loop as Cys187. Considering the high conservation of Glu113, Glu181, and the disulfide bond in visual pigments, we anticipate that the E2 and H3 movements triggered by the counterion switch and coordinated by the disulfide bond could be a general activation mechanism in GPCRs.


Figure 5. A highly conserved disulfide bond linking E2 and H3 plays a critical role in the conformational changes that leads to the GPCR activation.  
(2) Energy-Storage and Chromophore Distortion in the Primary Photoproduct of Rhodopsin

To understand how rhodopsin stores the light energy subsequently used for driving conformational changes in visual signal transduction, we studied the vibrational structure of chormophore in the primary photoproduct, batorhodopsin (Batho). In particular, the origin of the decoupled hydrogen-out-of-plane (HOOP) modes across C11=C12 of retinal (Figure 6), a marker of highly energetic and structurally distored chromophore, was investigated in detail.



Figure 6. (a) The cis-trans isomerization of retinal.
(b) The decoupled C11=C12 HOOP modes.

We obtained low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin (Figure 7). The spectra show mild mutagenic perturbations to the decoupling suggesting that the dipole or electrostatic perturbation from these residues does not cause the decoupling.


Figure 7. Raman Batho spectra of Glu181 Mutants.

Reviewing Batho Raman spectra and the protein sequence of different visual pigments, we identified the electrostatic interaction of the protonated Schiff base with its Glu113 counterion and the steric interactions at 9- and 13-methyl group of retinal with its surrounding residues correlated to the HOOP decoupling. These results demonstrate that the chromophore is anchored to the opsin through these interactions that twist the ethylenic chain in all-trans retinal in Batho leading to the decoupling of the C11=C12 HOOP modes. Density functional theory (DFT) calculations is currently used to explore the effect of chromophore geometry on the HOOP decoupling.



References and Publications

(1) “Counterion switch in the photoactivation of G protein-coupled receptor rhodopsin” Yan, E.C.Y.; Kazmi, M.A.; Gamin, Z.; Hou, J. M.; Pan, D.; Chang, B.S.W.; Sakmar, T. P.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9262.
[PDF available here]

“Perspectives on the counterion switch-induced photoactivation of the G protein-coupled receptor rhodopsin” Birge, R.B.; Knox, B.E. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9105.
[PDF available here]

(2) “Function of Extracellular Loop 2 in Rhodopsin: Glutamic Acid 181 Modulates Stability and Absorption Wavelength of Metarhodopsin II” Yan, E.C.Y; Kazmi, M. A.; De, S.; Chang, B.S.W.; Seibert, C.; Marin, E. P.; Mathies, R.A.; Sakmar, T. P. Biochemistry 2002, 41, 3620.
[PDF available here] [DOI link]


Sakmar Lab at Rockefeller University, New York