title: Measurement of histidine pKa values and tautomer populations in invisible protein states.
abstract: The histidine imidazole side chain plays a critical role in protein function and stability. Its importance for catalysis is underscored by the fact that histidines are localized to active sites in ∼ 50% of all enzymes. NMR spectroscopy has become an important tool for studies of histidine side chains through the measurement of site-specific pK(a)s and tautomer populations. To date, such studies have been confined to observable protein ground states; however, a complete understanding of the role of histidine electrostatics in protein function and stability requires that similar investigations be extended to rare, transiently formed conformers that populate the energy landscape, yet are often "invisible" in standard NMR spectra. Here we present NMR experiments and a simple strategy for studies of such conformationally excited states based on measurement of histidine (13)Cγ, (13)Cδ2 chemical shifts and (1)Hε-(13)Cε one-bond scalar couplings. The methodology is first validated and then used to obtain pKa values and tautomer distributions for histidine residues of an invisible on-pathway folding intermediate of the colicin E7 immunity protein. Our results imply that the side chains of H40 and H47 are exposed in the intermediate state and undergo significant conformational rearrangements during folding to the native structure. Further, the pKa values explain the pH-dependent stability differences between native and intermediate states over the pH range 5.5-6.5 and they suggest that imidazole deprotonation is not a barrier to the folding of this protein.
authors.affiliation: Departments of Molecular Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Alexandar L
authors.initials: AL
authors.lastname: Hansen
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D030562:Databases, Protein; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D013237:Stereoisomerism
chemical_list: D007093:Imidazoles; D011506:Proteins; D006639:Histidine; C029899:imidazole
keywords: CEST; Im7 protein-folding intermediate; chemical exchange; conformationally excited protein states; pH stability
references.citation: J Am Chem Soc. 2012 Mar 7;134(9):3968-71
references.pmid: 22320129
references.citation: J Biomol NMR. 2011 Aug;50(4):347-55
references.pmid: 21681650
references.citation: Nature. 2011 Aug 21;477(7362):111-4
references.pmid: 21857680
references.citation: J Biomol NMR. 2008 Jul;41(3):113-20
references.pmid: 18574698
references.citation: J Mol Biol. 2014 Feb 6;426(3):763-74
references.pmid: 24211467
references.citation: J Am Chem Soc. 1977 Dec 7;99(25):8149-59
references.pmid: 925263
references.citation: J Biomol NMR. 2011 May;50(1):43-57
references.pmid: 21448735
references.citation: J Mol Biol. 2011 Dec 9;414(4):511-29
references.pmid: 22019474
references.citation: Nature. 2011 Oct 30;480(7376):268-72
references.pmid: 22037310
references.citation: J Biol Chem. 2009 May 15;284(20):13285-9
references.pmid: 19164280
references.citation: J Mol Biol. 2003 Feb 7;326(1):293-305
references.pmid: 12547210
references.citation: Science. 1978 Sep 29;201(4362):1187-91
references.pmid: 694508
references.citation: Biochemistry. 2003 Aug 5;42(30):9227-34
references.pmid: 12885258
references.citation: Proc Natl Acad Sci U S A. 2005 Jan 25;102(4):1006-11
references.pmid: 15657134
references.citation: Chem Rev. 2002 Dec;102(12):4501-24
references.pmid: 12475199
references.citation: Nat Struct Biol. 2000 Sep;7(9):740-3
references.pmid: 10966641
references.citation: J Biol Chem. 1965 Apr;240:1586-92
references.pmid: 14285495
references.citation: Proc Natl Acad Sci U S A. 1981 Dec;78(12):7323-6
references.pmid: 7038675
references.citation: Science. 2009 Apr 10;324(5924):203-7
references.pmid: 19359577
references.citation: Protein Sci. 2009 Jan;18(1):247-51
references.pmid: 19177368
references.citation: J Am Chem Soc. 2011 Feb 9;133(5):1534-44
references.pmid: 21207964
references.citation: Biochem J. 1975 Jun;147(3):605-7
references.pmid: 241331
references.citation: Biochem J. 1998 Jul 1;333 ( Pt 1):183-91
references.pmid: 9639578
references.citation: J Am Chem Soc. 2005 Jun 29;127(25):8904-5
references.pmid: 15969539
references.citation: Nat Chem Biol. 2009 Jan;5(1):15-22
references.pmid: 19088715
references.citation: J Mol Biol. 1995 Dec 15;254(5):980-92
references.pmid: 7500365
references.citation: Protein Sci. 1997 Sep;6(9):1910-9
references.pmid: 9300491
references.citation: Curr Opin Struct Biol. 2007 Feb;17(1):30-7
references.pmid: 17239580
references.citation: Proc Natl Acad Sci U S A. 2001 May 22;98(11):6127-31
references.pmid: 11353859
references.citation: Cell. 1994 May 6;77(3):461-72
references.pmid: 8181064
references.citation: J Chem Phys. 1965 Mar 1;42:1587-99
references.pmid: 14291612
references.citation: Protein Sci. 1993 Apr;2(4):543-58
references.pmid: 8518729
references.citation: J Am Chem Soc. 2009 Oct 7;131(39):13894-5
references.pmid: 19739624
references.citation: Arch Biochem Biophys. 2013 Mar;531(1-2):14-23
references.pmid: 23098780
references.citation: J Biomol NMR. 2013 Jul;56(3):227-41
references.pmid: 23728592
references.citation: J Biomol NMR. 2003 Mar;25(3):243-8
references.pmid: 12652136
references.citation: J Mol Biol. 2001 Sep 28;312(4):849-63
references.pmid: 11575937
references.citation: J Am Chem Soc. 2012 Feb 15;134(6):3178-89
references.pmid: 22300166
references.citation: Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6437-42
references.pmid: 8692833
references.citation: J Am Chem Soc. 2008 Jul 2;130(26):8460-70
references.pmid: 18540585
references.citation: J Biomol NMR. 2008 Sep;42(1):35-47
references.pmid: 18762869
references.citation: Nat Struct Biol. 2001 Jan;8(1):68-72
references.pmid: 11135674
references.citation: J Am Chem Soc. 2005 Sep 14;127(36):12544-54
references.pmid: 16144402
references.citation: Methods Enzymol. 2001;339:204-38
references.pmid: 11462813
references.citation: Proc Natl Acad Sci U S A. 2011 May 31;108(22):9078-83
references.pmid: 21562212
references.citation: J Biol Chem. 2007 Mar 30;282(13):9646-9656
references.pmid: 17202139
references.citation: Adv Protein Chem. 1964;19:223-86
references.pmid: 14268785
references.citation: Biochemistry. 1973 Nov 6;12(23):4732-43
references.pmid: 4204227
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4545-50
references.pmid: 12671071
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13877-82
references.pmid: 18779576
references.citation: Proc Natl Acad Sci U S A. 1967 Oct;58(4):1307-13
references.pmid: 5237865
references.citation: J Mol Biol. 2010 Mar 12;396(5):1329-45
references.pmid: 20053361
references.citation: J Mol Biol. 2004 Mar 12;337(1):183-93
references.pmid: 15001361
references.citation: J Biomol NMR. 2010 Sep;48(1):13-22
references.pmid: 20628786
references.citation: Proc Natl Acad Sci U S A. 1964 Sep;52:736-41
references.pmid: 14212550
references.citation: Philos Trans A Math Phys Eng Sci. 2005 Feb 15;363(1827):433-50; discussion 450-2
references.pmid: 15664892
references.citation: Annu Rev Biochem. 2006;75:333-66
references.pmid: 16756495
references.citation: J Biomol NMR. 2013 Mar;55(3):279-89
references.pmid: 23386228
references.citation: Proc Natl Acad Sci U S A. 2005 May 10;102(19):6679-85
references.pmid: 15870208
references.citation: J Am Chem Soc. 2012 May 16;134(19):8148-61
references.pmid: 22554188
references.citation: J Am Chem Soc. 2005 Nov 2;127(43):14970-1
references.pmid: 16248604
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 25;105(12):4685-90
references.pmid: 18326625
references.citation: Nat Struct Biol. 2002 Mar;9(3):209-16
references.pmid: 11875516
references.citation: Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12867-74
references.pmid: 23868852
references.citation: Biochim Biophys Acta. 2008 Jan;1784(1):100-5
references.pmid: 17728195
references.citation: J Magn Reson. 2000 Mar;143(1):79-87
references.pmid: 10698648
references.citation: Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):105-12
references.pmid: 11136249
references.citation: J Mol Biol. 1990 Oct 5;215(3):403-10
references.pmid: 2231712
references.citation: J Magn Reson. 1998 Apr;131(2):373-8
references.pmid: 9571116
references.citation: Proteins. 2002 Oct 1;49(1):1-6
references.pmid: 12211010
references.citation: Science. 2012 Apr 20;336(6079):362-6
references.pmid: 22517863
references.citation: Q Rev Biophys. 2012 Feb;45(1):57-103
references.pmid: 22085441
references.citation: Proc Natl Acad Sci U S A. 2011 Apr 5;108(14):5602-7
references.pmid: 21422292
references.citation: Protein Sci. 2003 Apr;12(4):794-810
references.pmid: 12649438
references.citation: Science. 2010 Sep 10;329(5997):1312-6
references.pmid: 20829478
references.citation: J Am Chem Soc. 1973 Jan 24;95(2):328-31
references.pmid: 4687673