Thermal Properties of Rhodopsin

Abstract

Rhodopsin has developed mechanisms to optimize its sensitivity to light by suppressing dark noise and enhancing quantum yield. We propose that an intramolecular hydrogen-bonding network formed by ϳ20 water molecules, the hydrophilic residues, and peptide backbones in the transmembrane region is essential to restrain thermal isomerization, the source of dark noise. We studied the thermal stability of rhodopsin at 55°C with single point mutations (E181Q and S186A) that perturb the hydrogen-bonding network at the active site. We found that the rate of thermal isomerization increased by 1-2 orders of magnitude in the mutants. Our results illustrate the importance of the intact hydrogen-bonding network for dim-light detection, revealing the functional roles of water molecules in rhodopsin. We also show that thermal isomerization of 11-cis-retinal in solution can be catalyzed by wild-type opsin and that this catalytic property is not affected by the mutations. We characterize the catalytic effect and propose that it is due to steric interactions in the retinal-binding site and increases quantum yield by predetermining the trajectory of photoisomerization. Thus, our studies reveal a balancing act between dark noise and quantum yield, which have opposite effects on the thermal isomerization rate. The acquisition of the hydrogen-bonding network and the tuning of the steric interactions at the retinal-binding site are two important factors in the development of dim-light vision.Rhodopsin is a widely studied G protein-coupled receptor responsible for generating visual signals in dim-light environments (1-11). It is expressed in the disc membranes in the outer segment of rod photoreceptor cells (1-3). It has a seven-helical transmembrane structure and incorporates the 11-cis-retinal chromophore via a protonated Schiff base (SB) 6 in the transmembrane region (3,8,9). Upon absorption of a photon, the retinal chromophore isomerizes from the 11-cis-to all-trans-configuration to form the primary photoproduct bathorhodopsin (12, 13), which then evolves into a number of photointermediates on various time scales (14, 15), forming metarhodopsin II (Meta II) in milliseconds. In Meta II, the SB linkage becomes deprotonated, and the absorption changes from the visible to the UV (380 nm) region. Subsequently, Meta II couples to the G protein transducin to induce an exchange of GDP for GTP in the ␣-subunit of transducin (16). The ␣-subunit dissociates from the ␤␥-subunit and activates phosphodiesterase, which catalyzes hydrolysis of cGMP. A decrease in the concentration of cGMP closes the sodium channels of the rod photoreceptor cells and generates hyperpolarization for a visual signal.We asked what molecular properties allow rhodopsin to gain photosensitivity for dim-light vision; rhodopsin has the ability to handle intensities of light spanning orders of magnitude and yet the capacity to detect single photons (16,17). These properties must be related to the optimization of amplification, quantum yield, and dark noise level, three imp...

Chemical Kinetic Analysis of Thermal Decay of Rhodopsin Reveals Unusual Energetics of Thermal Isomerization and Hydrolysis of Schiff Base

“…Crystal structures suggested the presence of an extensive hydrogen-bonding network involving ϳ20 conserved structural water molecules, hydrophilic residues, and the protein backbone in the transmembrane region (7, 24 -28). We hypothesized that this network might stabilize the dark state, preventing thermal decay (22,23). We discovered that at 59°C rhodopsin undergoes thermal decay, isomerization, and hydrolysis of SB two or three times more slowly in D 2 O than in H 2 O, consistent with the fact that stronger hydrogen bonds form in D 2 O (22).…”

“…We discovered that at 59°C rhodopsin undergoes thermal decay, isomerization, and hydrolysis of SB two or three times more slowly in D 2 O than in H 2 O, consistent with the fact that stronger hydrogen bonds form in D 2 O (22). We also introduced the mutations E181Q and S186A to disrupt hydrogen bonds in the binding site of rhodopsin (23). At 55°C, the mutations increase the rates of thermal decay, isomerization, and hydrolysis of SB by almost 2 orders of magnitude compared with WT rhodopsin.…”

“…We perform experiments to measure the rate of SB hydrolysis in Meta II and apply our model to our previously reported data obtained for WT rhodopsin and the E181Q and S186A mutants at 55°C and for WT rhodopsin in H 2 O and D 2 O at 59°C (22,23). Using global fitting, we determine the rate constants for all thermal processes, which allows a quantitative study of the effect of mutations, D 2 O, and temperature on the kinetics of individual chemical reactions involved in the thermal decay process (Fig.…”

“…We designed experiments to study the thermal processes of rhodopsin and showed that thermal decay consists of both thermal isomerization of retinal and hydrolysis of the SB (22,23). What molecular properties control the kinetics of these thermal processes?…”

“…We previously analyzed the data from thermal decay, isomerization, and hydrolysis experiments using single-exponential fitting (22,23). However, this method does not account for overlaps between the thermal processes measured by individual experiments, which precludes the solution of discrete rate constants.…”