title: Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d.
abstract: Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.
authors: Alabama High Field NMR Laboratory, Laboratory for Structural Biology, University of Alabama in Huntsville, Huntsville, AL 35899, USA.
authors: Andrew T
authors: AT
authors: Clark
authors: Alabama High Field NMR Laboratory, Laboratory for Structural Biology, University of Alabama in Huntsville, Huntsville, AL 35899, USA.
authors: Kelley
authors: K
authors: Smith
authors: Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors: Ranjith
authors: R
authors: Muhandiram
authors: Alabama High Field NMR Laboratory, Laboratory for Structural Biology, University of Alabama in Huntsville, Huntsville, AL 35899, USA. Electronic address: edmonds@uah.edu.
authors: Stephen P
authors: SP
authors: Edmondson
authors: Alabama High Field NMR Laboratory, Laboratory for Structural Biology, University of Alabama in Huntsville, Huntsville, AL 35899, USA. Electronic address: shriverj@uah.edu.
authors: John W
authors: JW
authors: Shriver
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry; D016415:Sequence Alignment; D016944:Sulfolobus / Q000737:chemistry / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D007477:Ions; D012492:Salts; C093228:Sso7d protein, Sulfolobus; C095116:Sac7 protein, Sulfolobus; D004247:DNA
references: J Mol Biol. 2006 Sep 22;362(3):594-604
references: 16934292
references: Cell Mol Life Sci. 2001 Aug;58(9):1216-33
references: 11577980
references: Biochemistry. 2004 Mar 16;43(10):2840-53
references: 15005619
references: Trends Biotechnol. 2001 Apr;19(4):132-5
references: 11250029
references: Proteins. 2007 Jun 1;67(4):991-1001
references: 17357157
references: Biochemistry. 2000 Nov 21;39(46):14292-304
references: 11087378
references: Structure. 1997 May 15;5(5):635-46
references: 9195883
references: Biochemistry. 2002 Apr 30;41(17):5656-67
references: 11969427
references: Proc Natl Acad Sci U S A. 1991 May 15;88(10):4176-80
references: 2034662
references: Curr Opin Struct Biol. 1998 Apr;8(2):211-7
references: 9631295
references: Science. 1978 Sep 29;201(4362):1187-91
references: 694508
references: Nat Struct Biol. 1995 Feb;2(2):122-8
references: 7749916
references: Appl Microbiol Biotechnol. 2007 May;75(2):367-76
references: 17262208
references: Protein Sci. 1994 Dec;3(12):2217-25
references: 7756981
references: Biophys J. 2002 Sep;83(3):1595-612
references: 12202384
references: Biochemistry. 1996 Jun 18;35(24):7819-33
references: 8672483
references: Biochemistry. 1989 Jul 25;28(15):6150-6
references: 2675964
references: Curr Opin Struct Biol. 2006 Apr;16(2):142-51
references: 16540310
references: Proteins. 2004 May 15;55(3):689-704
references: 15103631
references: Biochemistry. 1995 Jul 25;34(29):9424-33
references: 7626612
references: Biochemistry. 2005 Dec 27;44(51):16817-25
references: 16363795
references: J Mol Biol. 1994 May 6;238(3):415-36
references: 8176733
references: Nat Struct Biol. 2000 May;7(5):380-3
references: 10802734
references: J Mol Biol. 1999 Jan 29;285(4):1811-30
references: 9917414
references: J Biochem. 1997 Mar;121(3):448-55
references: 9133613
references: Protein Sci. 1999 Sep;8(9):1843-9
references: 10493585
references: J Mol Biol. 1993 May 20;231(2):459-74
references: 8510157
references: CRC Crit Rev Biochem. 1985;18(2):91-197
references: 3899508
references: Protein Sci. 1998 Jan;7(1):21-38
references: 9514257
references: Proteins. 2001 Sep 1;44(4):400-17
references: 11484218
references: J Mol Biol. 2003 Apr 11;327(5):1135-48
references: 12662936
references: Biochemistry. 1999 Oct 5;38(40):13379-84
references: 10529213
references: Proteins. 2004 Oct 1;57(1):128-41
references: 15326599
references: FEBS J. 2006 Jul;273(13):2851-68
references: 16759231
references: Science. 1988 Jun 17;240(4859):1632-41
references: 2837824
references: Biochemistry. 1995 Apr 11;34(14):4714-23
references: 7718577
references: Biophys J. 2007 Jan 1;92(1):257-66
references: 17040982
references: Adv Protein Chem. 1970;24:1-95
references: 4912353
references: J Mol Biol. 1995 Dec 15;254(5):980-92
references: 7500365
references: Nature. 1975 May 15;255(5505):256-9
references: 1143325
references: Nat Struct Biol. 1994 Nov;1(11):808-19
references: 7634092
references: Protein Sci. 2001 Jun;10(6):1206-15
references: 11369859
references: Protein Sci. 1994 Feb;3(2):211-26
references: 8003958
references: Eur J Biochem. 1987 May 15;165(1):147-55
references: 3569291
references: Biochemistry. 1999 Aug 10;38(32):10247-55
references: 10441118
references: Protein Sci. 2002 May;11(5):1004-16
references: 11967358
references: Biophys J. 2000 Sep;79(3):1637-54
references: 10969024
references: Biochemistry. 1990 May 15;29(19):4659-67
references: 2372549
references: J Biosci Bioeng. 2004;97(1):75-7
references: 16233593
references: Nat Struct Biol. 1998 Sep;5(9):782-6
references: 9731772
references: J Mol Biol. 1983 Aug 25;168(4):867-85
references: 6887253
references: Biochemistry. 1995 Aug 8;34(31):10063-77
references: 7632679
references: J Mol Biol. 1999 Dec 10;294(4):1051-62
references: 10588906
references: Methods Enzymol. 2001;334:129-45
references: 11398456
references: J Mol Biol. 1998 Jul 31;280(5):933-52
references: 9671561
references: J Mol Biol. 1998 Nov 27;284(2):489-502
references: 9813132
references: J Mol Biol. 1999 Jun 4;289(2):357-69
references: 10366510
references: J Biomol NMR. 1991 May;1(1):13-22
references: 1668718
references: Protein Eng. 1994 Jan;7(1):103-8
references: 7908135
references: Biochemistry. 1996 Jan 9;35(1):1-6
references: 8555161
references: Biochemistry. 2003 Feb 18;42(6):1421-9
references: 12578354
references: Biochemistry. 1990 Oct 9;29(40):9343-52
references: 2248951
references: Q Rev Biophys. 1984 Aug;17(3):283-422
references: 6098916
references: J Mol Biol. 1996 Dec 13;264(4):784-805
references: 8980686
references: Protein Sci. 2000 Jul;9(7):1395-8
references: 10933506
references: J Mol Biol. 2003 Jan 31;325(5):1077-92
references: 12527309
references: J Mol Biol. 1999 Jun 25;289(5):1435-44
references: 10373377
references: J Biomol NMR. 1991 May;1(1):99-104
references: 1668719
references: Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12300-5
references: 9770481
references: Biochemistry. 1991 Dec 10;30(49):11521-9
references: 1747370
references: Biochemistry. 1999 Dec 14;38(50):16419-23
references: 10600102
references: J Phys Chem A. 2005 Aug 4;109(30):6634-43
references: 16834015
references: Biochemistry. 2000 Jul 11;39(27):8067-72
references: 10891089
references: Biochemistry. 1995 Oct 17;34(41):13289-304
references: 7577913
references: Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4545-50
references: 12671071
references: Biochemistry. 2000 Feb 8;39(5):872-9
references: 10653630
references: J Biochem. 1995 Nov;118(5):946-52
references: 8749311
references: Chembiochem. 2002 Jul 2;3(7):604-17
references: 12324994
references: J Mol Biol. 2001 Oct 19;313(2):343-57
references: 11800561
references: Biochemistry. 1995 Apr 11;34(14):4724-32
references: 7718578
references: J Comput Chem. 2004 Sep;25(12):1504-14
references: 15224394
references: FEBS Lett. 2006 Jul 24;580(17):4224-30
references: 16831434
references: Biochemistry. 2003 Nov 18;42(45):13122-33
references: 14609322
references: Biochemistry. 1999 Apr 13;38(15):4896-903
references: 10200179
references: Biochemistry. 1990 Nov 6;29(44):10219-25
references: 2271649
references: J Mol Biol. 1998 Feb 13;276(1):203-24
references: 9514720
references: Proteins. 2002 Aug 1;48(2):388-403
references: 12112705
references: Biochemistry. 1990 Mar 6;29(9):2403-8
references: 2337607
references: Biochemistry. 1994 Jul 26;33(29):8826-32
references: 8038174
references: Adv Protein Chem. 1968;23:121-282
references: 4882248
references: J Magn Reson. 2011 Dec;213(2):423-41
references: 22152361
references: Proteins. 2005 Dec 1;61(4):704-21
references: 16231289
references: Science. 1995 May 26;268(5214):1144-9
references: 7761829
references: Methods Enzymol. 1995;259:512-38
references: 8538470
references: Biochemistry. 2001 Aug 28;40(34):10115-39
references: 11513590
references: Appl Bioinformatics. 2004;3(1):21-9
references: 16323963