Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules.
Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix. Whereas, deletion or substitution of the C-terminal proline could noticeably change the bioluminescence activity, stability or the yield of an active photoprotein complex. Therefore, modifications of the first α-helix Arg has a clear destructive effect on the main photoprotein properties. A C-terminal hydrogen-bond network is proposed to be important for the stability of photoprotein molecules towards external disturbances, when taking part in the formation of locked protein conformations and isolation of coelenterazine-binding cavities.
Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia. l_eremeeva@mail.ru.
Elena V
EV
Eremeeva
Ludmila P
LP
Burakova
Vasilisa V
VV
Krasitskaya
Alexander N
AN
Kudryavtsev
Osamu
O
Shimomura
Ludmila A
LA
Frank
D000331:Aequorin / Q000737:chemistry* / Q000235:genetics; D001120:Arginine / Q000737:chemistry*; D003460:Crystallization; D004926:Escherichia coli; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011392:Proline / Q000737:chemistry*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011719:Pyrazines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D007093:Imidazoles; D008164:Luminescent Proteins; D011719:Pyrazines; D011994:Recombinant Proteins; C010497:obelin; C017144:coelenterazine; D000331:Aequorin; D001120:Arginine; D011392:Proline