title: The EF loop in green proteorhodopsin affects conformation and photocycle dynamics.
abstract: The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced (13)C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.
authors.affiliation: Institute of Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe-University Frankfurt, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Mehler
authors.forename: Frank
authors.initials: F
authors.lastname: Scholz
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Ullrich
authors.forename: Jiafei
authors.initials: J
authors.lastname: Mao
authors.forename: Markus
authors.initials: M
authors.lastname: Braun
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Brown
authors.forename: Richard C D
authors.initials: RC
authors.lastname: Brown
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Fiedler
authors.forename: Johanna
authors.initials: J
authors.lastname: Becker-Baldus
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D055537:Light Signal Transduction; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012176:Retinoids / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025604:Rhodopsins, Microbial
chemical_list: D001426:Bacterial Proteins; D012176:Retinoids; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; C121835:retinylidene chromophore; D012243:Rhodopsin