title: Role of a helix B lysine residue in the photoactive site in channelrhodopsins.
abstract: In most studied microbial rhodopsins two conserved carboxylic acid residues (the homologs of Asp-85 and Asp-212 in bacteriorhodopsin) and an arginine residue (the homolog of Arg-82) form a complex counterion to the protonated retinylidene Schiff base, and neutralization of the negatively charged carboxylates causes red shifts of the absorption maximum. In contrast, the corresponding neutralizing mutations in some relatively low-efficiency channelrhodopsins (ChRs) result in blue shifts. These ChRs do not contain a lysine residue in the second helix, conserved in higher efficiency ChRs (Lys-132 in the crystallized ChR chimera). By action spectroscopy of photoinduced channel currents in HEK293 cells and absorption spectroscopy of detergent-purified pigments, we found that in tested ChRs the Lys-132 homolog controls the direction of spectral shifts in the mutants of the photoactive site carboxylic acid residues. Analysis of double mutants shows that red spectral shifts occur when this Lys is present, whether naturally or by mutagenesis, and blue shifts occur when it is replaced with a neutral residue. A neutralizing mutation of the Lys-132 homolog alone caused a red spectral shift in high-efficiency ChRs, whereas its introduction into low-efficiency ChR1 from Chlamydomonas augustae (CaChR1) caused a blue shift. Taking into account that the effective charge of the carboxylic acid residues is a key factor in microbial rhodopsin spectral tuning, these findings suggest that the Lys-132 homolog modulates their pKa values. On the other hand, mutation of the Arg-82 homolog that fulfills this role in bacteriorhodopsin caused minimal spectral changes in the tested ChRs. Titration revealed that the pKa of the Asp-85 homolog in CaChR1 lies in the alkaline region unlike in most studied microbial rhodopsins, but is substantially decreased by introduction of a Lys-132 homolog or neutralizing mutation of the Asp-212 homolog. In the three ChRs tested the Lys-132 homolog also alters channel current kinetics.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas.
authors.forename: Hai
authors.initials: H
authors.lastname: Li
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Govorunova
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas.
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Sineshchekov
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas. Electronic address: john.l.spudich@uth.tmc.edu.
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
mesh_terms: D000595:Amino Acid Sequence; D002696:Chlamydomonas / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007700:Kinetics; D008027:Light; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000528:radiation effects*; D012545:Schiff Bases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
chemical_list: D050505:Mutant Proteins; D011522:Protons; D012545:Schiff Bases; D012243:Rhodopsin; D008239:Lysine
references.citation: J Am Chem Soc. 2006 Aug 23;128(33):10808-18
references.pmid: 16910676
references.citation: Biophys J. 1991 Dec;60(6):1477-89
references.pmid: 19431816
references.citation: J Biol Chem. 2012 Feb 10;287(7):4818-25
references.pmid: 22139833
references.citation: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10131-6
references.pmid: 11504917
references.citation: J Biol Chem. 2003 Sep 5;278(36):33985-91
references.pmid: 12821661
references.citation: Phys Chem Chem Phys. 2013 May 28;15(20):7567-76
references.pmid: 23588588
references.citation: J Biol Chem. 2013 Oct 11;288(41):29911-22
references.pmid: 23995841
references.citation: Nat Methods. 2011 Jan;8(1):26-9
references.pmid: 21191368
references.citation: Cell. 2011 Dec 23;147(7):1446-57
references.pmid: 22196724
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8689-94
references.pmid: 12060707
references.citation: J Phys Chem B. 2011 Dec 22;115(50):15119-28
references.pmid: 22077286
references.citation: Plant Cell. 2008 Jun;20(6):1665-77
references.pmid: 18552201
references.citation: J Chem Theory Comput. 2011 Jul 12;7(7):2284-95
references.pmid: 26606496
references.citation: J Chem Theory Comput. 2011 Feb 8;7(2):525-37
references.pmid: 26596171
references.citation: FEBS Lett. 2009 Nov 19;583(22):3676-80
references.pmid: 19854176
references.citation: J Biol Chem. 2009 May 15;284(20):13285-9
references.pmid: 19164280
references.citation: J Biol Chem. 1991 Oct 5;266(28):18674-83
references.pmid: 1917991
references.citation: Sci Transl Med. 2013 Mar 20;5(177):177ps5
references.pmid: 23515075
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13940-5
references.pmid: 14615590
references.citation: J Biol Chem. 2012 Mar 2;287(10):7456-66
references.pmid: 22241469
references.citation: J Biol Chem. 2013 Apr 12;288(15):10451-8
references.pmid: 23439646
references.citation: Biophys J. 2004 May;86(5):3112-20
references.pmid: 15111424
references.citation: Proc Natl Acad Sci U S A. 1988 Jun;85(12):4148-52
references.pmid: 3288985
references.citation: Biophys J. 1998 Aug;75(2):999-1009
references.pmid: 9675200
references.citation: J Mol Biol. 1999 Aug 27;291(4):899-911
references.pmid: 10452895
references.citation: J Biol Chem. 2005 Apr 15;280(15):14663-8
references.pmid: 15710603
references.citation: Biophys J. 2013 Feb 19;104(4):807-17
references.pmid: 23442959
references.citation: Photochem Photobiol. 1997 Dec;66(6):788-91
references.pmid: 9421965
references.citation: J Biol Chem. 1989 Aug 25;264(24):14202-8
references.pmid: 2547788
references.citation: Dev Growth Differ. 2013 May;55(4):474-90
references.pmid: 23550617
references.citation: Biochemistry. 2002 Apr 30;41(17):5348-58
references.pmid: 11969395
references.citation: Biochemistry. 1993 Oct 5;32(39):10331-43
references.pmid: 8399176
references.citation: Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):E1273-81
references.pmid: 23509282
references.citation: Biochem Biophys Res Commun. 1991 Aug 15;178(3):1273-9
references.pmid: 1872847
references.citation: mBio. 2011 Jun 21;2(3):e00115-11
references.pmid: 21693637
references.citation: Biophys J. 2004 Apr;86(4):2342-9
references.pmid: 15041672
references.citation: Science. 2001 Aug 24;293(5534):1499-503
references.pmid: 11452084
references.citation: Photochem Photobiol. 2009 Mar-Apr;85(2):564-9
references.pmid: 19192197
references.citation: Structure. 1999 Aug 15;7(8):909-17
references.pmid: 10467143
references.citation: Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3185-9
references.pmid: 7724537
references.citation: Photochem Photobiol. 2012 Jan-Feb;88(1):119-28
references.pmid: 22044280
references.citation: Angew Chem Int Ed Engl. 2007;46(1-2):269-71
references.pmid: 17120281
references.citation: Photochem Photobiol. 2003 Jan;77(1):96-100
references.pmid: 12856889
references.citation: Nature. 2012 Jan 22;482(7385):369-74
references.pmid: 22266941
references.citation: Biochemistry. 1977 May 3;16(9):1955-9
references.pmid: 870032
references.citation: Proc Natl Acad Sci U S A. 1990 Feb;87(3):1018-22
references.pmid: 2153966
references.citation: Science. 2002 Jun 28;296(5577):2395-8
references.pmid: 12089443
references.citation: Nat Neurosci. 2010 Mar;13(3):387-92
references.pmid: 20081849
references.citation: Biophys J. 1991 Dec;60(6):1490-8
references.pmid: 1777569
references.citation: Biophys J. 1993 Jul;65(1):124-30
references.pmid: 8369421