title: pH-dependent transient conformational states control optical properties in cyan fluorescent protein.
abstract: A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MSλD)) simulations, we have identified a solvated transient state, characterized by a partially open β-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore (namely, local solvation at the deprotonation site and a partial flexibility of the protein β-barrel structure) and provide the first evidence that transient conformational states can control optical properties of fluorescent proteins.
authors: Department of Chemistry and ‡Biophysics Program, University of Michigan , Ann Arbor, Michigan 48109, United States.
authors: Elena N
authors: EN
authors: Laricheva
authors: Garrett B
authors: GB
authors: Goh
authors: Alex
authors: A
authors: Dickson
authors: Charles L
authors: CL
authors: Brooks
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D055620:Optical Phenomena; D017433:Protein Structure, Secondary
chemical_list: C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins
references: J Mol Biol. 2007 Jul 6;370(2):356-71
references: 17512539
references: J Comput Chem. 2007 Nov 15;28(14):2366-77
references: 17600852
references: J Phys Chem B. 2013 Feb 28;117(8):2247-53
references: 23363049
references: J Chem Theory Comput. 2011 Sep 13;7(9):2728-2739
references: 22125476
references: Nature. 1998 Jul 9;394(6689):192-5
references: 9671304
references: Nat Protoc. 2006;1(2):960-7
references: 17406330
references: Physiol Rev. 2010 Jul;90(3):1103-63
references: 20664080
references: Trends Neurosci. 2004 May;27(5):257-61
references: 15111007
references: Ann N Y Acad Sci. 1986;482:1-23
references: 3471099
references: Annu Rev Biophys. 2014;43:303-29
references: 24895855
references: J Comput Chem. 2009 Jul 30;30(10):1545-614
references: 19444816
references: Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):19994-9
references: 23169643
references: J Phys Chem B. 2005 Mar 3;109(8):3606-15
references: 16851399
references: Nat Biotechnol. 2004 Dec;22(12):1567-72
references: 15558047
references: J Mol Biol. 2013 Sep 9;425(17):3091-105
references: 23747975
references: Sci Rep. 2012;2:608
references: 22934131
references: Biochemistry. 2009 Oct 27;48(42):10038-46
references: 19754158
references: J Phys Chem B. 2005 Dec 22;109(50):24121-33
references: 16375404
references: Angew Chem Int Ed Engl. 2014 Jan 27;53(5):1435-8
references: 24339377
references: J Chem Theory Comput. 2006 Mar;2(2):292-9
references: 26626517
references: Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6803-8
references: 9618493
references: J Chem Phys. 2009 Oct 21;131(15):154104
references: 20568844
references: J Mol Graph Model. 2004 May;22(5):377-95
references: 15099834
references: J Phys Chem B. 2012 Aug 30;116(34):10311-6
references: 22861177
references: Nat Neurosci. 2014 Feb;17(2):262-8
references: 24441679
references: J Phys Chem B. 2012 Oct 18;116(41):12426-40
references: 22988813
references: J Comput Chem. 2009 Aug;30(11):1692-700
references: 19421993
references: Proteins. 2014 Jul;82(7):1319-31
references: 24375620
references: J Am Chem Soc. 2014 Jun 18;136(24):8496-9
references: 24842060
references: J Cheminform. 2012 Aug 13;4(1):17
references: 22889332
references: J Chem Theory Comput. 2013 Feb 12;9(2):935-943
references: 23525495
references: Proc Natl Acad Sci U S A. 2014 Jun 17;111(24):8820-5
references: 24889628
references: J Am Chem Soc. 2013 May 8;135(18):6766-9
references: 23506098
references: Proc Natl Acad Sci U S A. 2007 Nov 20;104(47):18473-7
references: 18006656
references: Nat Chem Biol. 2013 Jul;9(7):462-5
references: 23644478
references: J Chem Theory Comput. 2013 Jan 8;9(1):461-469
references: 23316124
references: J Org Chem. 2006 Mar 31;71(7):2829-38
references: 16555839
references: Nat Chem Biol. 2009 Nov;5(11):808-14
references: 19841630
references: Biophys J. 1996 Jan;70(1):97-110
references: 8770190
references: J Phys Chem Lett. 2013 Mar 7;4(5):760-766
references: 23526474
references: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12651-6
references: 12271129
references: Nat Commun. 2012 Mar 20;3:751
references: 22434194
references: Chem Rev. 2005 Aug;105(8):2999-3093
references: 16092826
references: Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12867-74
references: 23868852
references: Science. 2010 Sep 10;329(5997):1312-6
references: 20829478
references: J Chem Theory Comput. 2012 Jan 10;8(1):36-46
references: 22337595
references: J Am Chem Soc. 2011 Jul 6;133(26):10034-7
references: 21631110
references: Proc Natl Acad Sci U S A. 2011 Sep 27;108(39):16247-52
references: 21918110