Conformational equilibria of bulged sites in duplex DNA studied by EPR spectroscopy.
Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of "dynamic signatures" that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch ("bulge-mismatches") may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulge-mismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the premelting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bistable, temperature dependent, switching between conformations for a particular complex bulged construct.
Smith
Alyssa L
AL
Department of Chemistry, University of Washington, Box 351700 Bagley Hall, Seattle, Washington 98195-1700, USA.
Cekan
Pavol
P
Brewood
Greg P
GP
Okonogi
Tamara M
TM
Alemayehu
Saba
S
Hustedt
Eric J
EJ
Benight
Albert S
AS
Sigurdsson
Snorri Th
ST
Robinson
Bruce H
BH
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning / Q000379:methods; D004247:DNA / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006358:Hot Temperature; D008956:Models, Chemical; D015233:Models, Statistical; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
D004247:DNA
J Mol Biol. 1999 Dec 10;294(4):997-1013
10588902
Biochemistry. 1992 Apr 28;31(16):4004-14
1314654
EMBO J. 1987 May;6(5):1513-22
3608986
Angew Chem Int Ed Engl. 2007;46(15):2655-8
17309085
Biophys J. 1997 Apr;72(4):1861-77
9083690
Biochemistry. 1990 May 1;29(17):4193-204
2361138
Biochemistry. 2000 Jul 4;39(26):7835-46
10869190
Trends Mol Med. 2005 Nov;11(11):488-95
16213790
Annu Rev Biophys Biomol Struct. 2004;33:415-40
15139820
J Mol Biol. 1999 Dec 10;294(4):981-95
10588901
Hum Mol Genet. 1996 Nov;5(11):1841-7
8923014
Biochemistry. 1989 Jan 10;28(1):294-303
2706253
Biopolymers. 1993 Nov;33(11):1725-45
8241430
Biochemistry. 1992 Apr 28;31(16):4015-26
1314655
J Mol Biol. 1966 Jan;15(1):111-23
5912035
Biophys J. 1999 Dec;77(6):3256-76
10585948
Genome Res. 1998 Dec;8(12):1229-31
9872978
J Phys Chem B. 2008 Jul 31;112(30):9219-36
18593148
J Phys Chem A. 2005 May 12;109(18):4049-61
16833727
Science. 1994 Jan 28;263(5146):490-3
8290958
Nucleosides Nucleotides Nucleic Acids. 2007;26(10-12):1601-4
18066835
J Biol Chem. 1989 Mar 5;264(7):3702-12
2917972
Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):7853-8
10393911
Biochemistry. 1995 Apr 25;34(16):5696-704
7727429
Trends Biochem Sci. 2002 Jun;27(6):288-95
12069788
Nucleic Acids Res. 1990 Feb 11;18(3):471-6
2155401
Chem Commun (Camb). 2008 Aug 7;(29):3393-5
18633500
Biochemistry. 1996 Sep 24;35(38):12329-37
8823167
J Magn Reson. 2004 Jul;169(1):129-63
15183364
Methods Enzymol. 1995;259:221-42
8538456
J Mol Biol. 1962 Jun;4:500-17
13885894
Science. 1996 Jun 28;272(5270):1943-6
8658168
Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5
9465037
Nucleic Acids Res. 2007;35(21):7197-208
17947320
Org Biomol Chem. 2006 May 7;4(9):1638-40
16633552
Acta Crystallogr D Biol Crystallogr. 2007 Jun;63(Pt 6):673-81
17505105
Biopolymers. 1997;44(3):283-308
9591480
J Mol Biol. 1984 Mar 25;174(1):221-9
6716478
J Biol Chem. 1990 Jan 15;265(2):636-47
2295611
Biochem Biophys Res Commun. 1983 Aug 30;115(1):100-5
6615518
Chemistry. 2007;13(16):4452-7
17352435
J Mol Biol. 1964 Jul;9:1-9
14200383
Nucleic Acids Res. 2008 Oct;36(18):5946-54
18805908
Biochemistry. 2008 May 6;47(18):5059-67
18399645
Biochemistry. 1999 Aug 24;38(34):11197-208
10460177
J Mol Biol. 1997 May 2;268(2):331-47
9159474
Nature. 2004 Oct 21;431(7011):931-45
15496913
Biochemistry. 2006 Sep 5;45(35):10563-71
16939208
Biochemistry. 2008 Apr 15;47(15):4469-76
18358004
Biochemistry. 1999 Jun 29;38(26):8409-22
10387087
Anal Chim Acta. 2007 Jul 30;597(1):97-102
17658318