Thermal stability of rhodopsin and progression of retinitis pigmentosa: comparison of S186W and D190N rhodopsin mutants.

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1-2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor's active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients.

Spectral tuning of ultraviolet cone pigments: an interhelical lock mechanism.

Ultraviolet (UV) cone pigments can provide insights into the molecular evolution of vertebrate vision since they are nearer to ancestral pigments than the dim-light rod photoreceptor rhodopsin. While visible-absorbing pigments contain an 11-cis retinyl chromophore with a protonated Schiff-base (PSB11), UV pigments uniquely contain an unprotonated Schiff-base (USB11). Upon F86Y mutation in model UV pigments, both the USB11 and PSB11 forms of the chromophore are found to coexist at physiological pH. The origin of this intriguing equilibrium remains to be understood at the molecular level. Here, we address this phenomenon and the role of the USB11 environment in spectral tuning by combining mutagenesis studies with spectroscopic (UV-vis) and theoretical [DFT-QM/MM (SORCI+Q//B3LYP/6-31G(d): Amber96)] analysis. We compare structural models of the wild-type (WT), F86Y, S90A and S90C mutants of Siberian hamster ultraviolet (SHUV) cone pigment to explore structural rearrangements that stabilize USB11 over PSB11. We find that the PSB11 forms upon F86Y mutation and is stabilized by an "inter-helical lock" (IHL) established by hydrogen-bonding networks between transmembrane (TM) helices TM6, TM2, and TM3 (including water w2c and amino acid residues Y265, F86Y, G117, S118, A114, and E113). The findings implicate the involvement of the IHL in constraining the displacement of TM6, an essential component of the activation of rhodopsin, in the spectral tuning of UV pigments.

Schiff base protonation changes in Siberian hamster ultraviolet cone pigment photointermediates.

Molecular structure and function studies of vertebrate ultraviolet (UV) cone visual pigments are needed to understand the molecular evolution of these photoreceptors, which uniquely contain unprotonated Schiff base linkages between the 11-cis-retinal chromophore and the opsin proteins. In this study, the Siberian hamster ultraviolet cone pigment (SHUV) was expressed and purified in an n-dodecyl-ß-D-maltoside suspension for optical characterization. Time-resolved absorbance measurements, over a spectral range from 300 to 700 nm, were taken for the purified pigment at time delays from 30 ns to 4.64 s after photoexcitation using 7 ns pulses of 355 nm light. The resulting data were fit globally to a sum of exponential functions after noise reduction using singular-value decomposition. Four exponentials best fit the data with lifetimes of 1.4 µs, 210 µs, 47 ms, and 1 s. The first photointermediate species characterized here is an equilibrated mixture similar to the one formed after rhodopsin's Batho intermediate decays into equilibrium with its successor, BSI. The extremely large red shift of the SHUV Batho component relative to the pigment suggests that SHUV Batho has a protonated Schiff base and that the SHUV cone pigment itself has an unprotonated Schiff base. In contrast to SHUV Batho, the portion of the equilibrated mixture's spectrum corresponding to SHUV BSI is well fit by a model spectrum with an unprotonated Schiff base. The spectra of the next two photointermediate species revealed that they both have unprotonated Schiff bases and suggest they are analogous to rhodopsin's Lumi I and Lumi II species. After decay of SHUV Lumi II, the correspondence with rhodopsin photointermediates breaks down and the next photointermediate, presumably including the G protein-activating species, is a mixture of protonated and unprotonated Schiff base photointermediate species.