Graduate Student, Case Western Reserve University
Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism
General interest: Humans perceive light with the help of an extremely sensitive rhodopsin visual pigment in the retina. Rhodopsin harbors 11-cis retinal as its chromophore that enables it to sense light. Light is detected when 11-cis retinal absorbs a photon and isomerizes to all–trans retinal causing a conformational change in the protein moiety that propagates the signaling pathway necessary for vision. The process culminates with the release of all-trans retinal that requires multi-step enzymatic reactions to convert back to 11-cis retinal. The complexity of the retinal recycling process increases the potential for retinal defects associated with mutations in any of the retinoid cycle enzymes involved in this conversion. Overall, the release of all-trans retinal from rhodopsin and its enzymatic recycling to 11-cis retinal is the underlying cause for the most devastating blinding retinal defects in humans including retinitis pigmentosa, Stargardt disease and Leber congenital amaurosis.
Key findings of the study: Our work is the first comprehensive structure-function study communicating the ability of vertebrate rhodopsin bound to a synthetic chromophore to function in a photocyclic manner. Bovine rhodopsin bound with a 6-carbon ring retinal chromophore (Rh6mr) does not release its chromophore after activation with light. In fact, the activated Rh6mr photoproduct is re-isomerized back to its inactive 11-cis state thermally. This photocyclic behavior makes rhodopsin self-sustainable, and abrogates the necessity for a complex enzymatic cycle to renew its chromophore. Thereby, potentially alleviating the progression of several retinal degenerative defects associated with chromophore recycling.
This is the first study demonstrating a complete transition from one-way activation of a G protein-coupled receptor (GPCR) into a self-renewable cyclic activation by changing the chromophore. With this photocyclic behavior, Rh6mr can repeatedly activate downstream G protein signaling making it a great candidate for optogenetic approaches that require reversible control of the effector ligands to modulate cellular calcium signaling.
Additionally, our findings address one of the unresolved mysteries regarding the chemistry of vision - why is 11-cis the preferred retinal configuration of our light-sensing chromophore? Our results demonstrate that the rhodopsin binding pocket itself is responsible for this natural selection.
Abstract: Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure–function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis–to–11,13-dicis isomerization.
Pub.: 13 Mar '17, Pinned: 28 Jun '17
Abstract: Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.
Pub.: 08 May '15, Pinned: 28 Jun '17
Abstract: Phototransduction is initiated when the absorption of light converts the 11-cis-retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-cis-retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the visual or retinoid cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-cis-retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-cis-6-membered ring (11-cis-6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-cis-6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro205 in green cone opsin could prevent entry and binding of 11-cis-6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-cis-6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-cis-6mr-retinal. These results and our structural modeling, indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-cis-6mr-retinal.
Pub.: 11 May '17, Pinned: 28 Jun '17