Biochemistry. 2016 Sep 13;55(36):5095-105. doi: 10.1021/acs.biochem.6b00687.

Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH.

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Rhodopsin is a light-activated protein found in the retina. In the dark it exists in an inactive form. Upon absorption of light it is converted to an active form that interacts with a G-protein called transducin. That interaction triggers an enzyme cascade that results in a highly amplified electrical signal indicating the presence of light.

This paper describes the complex mechanism of bovine rhodopsin activation. By carrying out time-resolved optical absorption measurements of the protein at 15°C and pH 8.7 it was possible to observe the absorption and kinetic characteristics of intermediates which do not accumulate in sufficient concentrations to observe them at higher temperatures and neutral pH even though they participate in the activation mechanism under other conditions.

Activation intermediates were first discovered through low-temperature trapping studies by Wald, Yoshizawa, and coworkers in the middle of the last century. Shining light on rhodopsin at temperatures below -140°C yielded a red-shifted species, later called bathorhodopsin. Warming the sample above this temperature yielded a species slightly blue-shifted from the initial rhodopsin spectrum. This species, called lumirhodopsin, was stable below -40°C. The resulting blue-shifted spectrum was called metarhodopsin I, stable below -15°C, at which point it was converted to metarhodopsin II, which was taken to be the activated state of rhodopsin. This simple mechanism has been used to interpret many structural studies of rhodopsin intermediates and can still be described in textbooks as the true rhodopsin activation mechanism.

The rhodopsin activation mechanism is significantly more complex than the one described above. This has been shown by time-resolved optical absorption spectroscopy carried out at near-physiological temperatures. This paper clearly shows a complex mechanism of the late (microsecond to second) intermediates of the activation process and demonstrates how one must be careful in assigning structural intermediates to specific spectral intermediates of the protein. It provides information about the concentrations of all of the intermediates as a function of time to aid such assignments. The attached figure shows the relationship between the intermediates originally identified by low temperature trapping and those identified in this paper. The intermediates identified using low-temperature trapping methods are shown in blue.



We recently published an interesting follow-up study to this one [C. Funatogawa, I. Szundi, and D.S. Kliger, “A Comparison between the Photoactivation Kinetics of Human and Bovine Rhodopsins,” Biochemistry 55, 7005 – 7013 (2016). DOI:10.1021/acs.biochem.6b00953] in which the activation mechanism of human rhodopsin was studied under the same conditions as used in this study of the bovine protein. We found that the mechanism exhibited by the two proteins is the same, though the kinetic behavior of specific intermediates of the two proteins differ. With this information, and knowing the specific residues that differ in the two proteins, it will now be possible to mutate the two proteins to gain a deeper understanding of the roles of specific residues (and areas of the proteins) in controlling activation kinetics.

In addition to understanding the cause of the differences in the kinetics of the bovine and human rhodopsins, this approach should make it possible to gain new insights into retinal diseases which are caused by mutations in the rhodopsin gene. There are about 40 to 50 such variants associated with autosomal dominant retinitis pigmentosa (ADRP), with most resulting in prevention of proper folding of rhodopsin. However, there are a handful of mutations that result in properly folded proteins that bind retinal, and that interact with transducin (though with reduced activity). Understanding how these mutations affect activation kinetics, combined with knowledge of the effects of specific residues, will lead to better understanding of factors that affect ADRP.