The crystal structure of super ecliptic pHluorin reveals a novel pathway for pH to influence fluorescence
- Authors
- Shin, Sang Chul; Leong, Lee Min; Kim, Heejung; Seong, Jihye; Bake, Bradley J.
- Issue Date
- 2024-02-08
- Publisher
- CELL PRESS
- Citation
- 68th Annual Meeting of the Biophysical-Society, pp.53A
- Abstract
- The fluorescent protein (FP), super ecliptic pHluorin (SEpH), is extremely sensitive to physiological pH with a pKa at 7.2. When fused to a voltage sensing domain, the resulting genetically encoded voltage indicator (GEVI) yields a poor optical response to voltage transients of the plasma membrane. However, a single mutation to SEpH that introduces a negative charge to the external surface of the β-can structure (A227D) improved the optical response by over 15-fold resulting in the GEVI ArcLight. To better understand how A227D contributes to the voltage-dependent optical signal, we solved the crystal structure of SEpH and SEpH A227D in different pH conditions. The structures are remarkably similar showing no discernible pH-induced conformational changes (from pH 4.5-9.5) nor an obvious role for A227D. However, all of the structures regardless of pH show a novel flexibility near the beginning of the 7th β strand of the can structure enabling the sidechain of D147 to form a hydrogen bond with the chromophore while the other oxygen of D147 is exposed to the external surface. The chromophore of SEpH has a hydrogen bond with an aspartic acid that is titratable via the external solvent. A pH-induced conformational change was detected in another mutant of SEpH. A novel GEVI (Ulla) was developed that gets brighter in response to membrane depolarizations and shifts the pKa to 8.2. Structures of the Ulla FP domain below the pKa were nearly identical to the SEpH structures; however, at pH 8.5, the structure of Ulla FP reveals a lengthening of the 7th β sheet causing D147 to rotate completely to the external surface. These results provide new insights into how the surface chemistry of a fluorescent protein can influence its optical properties.
- ISSN
- 0006-3495
- URI
- https://pubs.kist.re.kr/handle/201004/150355
- DOI
- 10.1016/j.bpj.2023.11.388
- Appears in Collections:
- KIST Conference Paper > 2024
- Files in This Item:
There are no files associated with this item.
- Export
- RIS (EndNote)
- XLS (Excel)
- XML
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.