Anabaena Sensory Rhodopsin (ASR) is a retinal containing photoactive membrane protein, from the cyanobacterium Anabaena sp. PCC 7120. ASR undergoes an orange light-induced conformational change associated with release of a bound transducer protein ASRT. This protein complex has been proposed to directly control the transcription of the cpc genes involved in chromatic adaption. However, direct binding of DNA with ASR has not been demonstrated. Hindering such binding studies is the lack of a system to stabilize ASR in an accessible native-like phospholipid bilayer. To overcome this barrier we isolated and characterized ASR from engineered E. coli for construction of self-assembled protein lipid nanodiscs. Nanodiscs are soluble bilayers encircled by protein at the hydrophobic lipid tail portion of the bilayer and allow for interaction from both sides of the bilayer. Our isolated ASR nanodiscs were soluble with a UV-visible spectrum possessing a retinal absorbance shift (548-537nm) in response to light exposure. Dynamic light scattering of ASR nanodisc fractions, separated by size exclusion chromatography, displayed two populations of distinct diameters (21.9 ± 6.3 and 31.1 ± 12.5 nm). The two populations may represent different oligomeric states of ASR in nanodiscs. We conclude that ASR is stabilized in the soluble lipid bilayer of the nanodiscs and represents a practical platform to enable further investigation of ASR’s unique role in the transcriptional control of chromatic adaptation. Our next steps will be to examine the lipid environment and investigate DNA binding. Results of these studies may allow for future use of these proteins as photo-active transcriptional regulators.

Nanodiscs stabilize Anabaena sensory rhodopsin for transcriptional regulation studies

251st National Meeting (2016)

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