Protein Environment and DNA Orientation Affect Protein-Induced Cy3 Fluorescence Enhancement.
Journal
Biophysical journal
ISSN: 1542-0086
Titre abrégé: Biophys J
Pays: United States
ID NLM: 0370626
Informations de publication
Date de publication:
09 07 2019
09 07 2019
Historique:
received:
23
04
2019
revised:
23
05
2019
accepted:
29
05
2019
pubmed:
27
6
2019
medline:
1
8
2020
entrez:
26
6
2019
Statut:
ppublish
Résumé
The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.
Identifiants
pubmed: 31235181
pii: S0006-3495(19)30448-5
doi: 10.1016/j.bpj.2019.05.026
pmc: PMC6626848
pii:
doi:
Substances chimiques
Carbocyanines
0
DNA-Binding Proteins
0
Escherichia coli Proteins
0
Fluorescent Dyes
0
Replication Protein A
0
SSB protein, E coli
0
cyanine dye 3
0
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
66-73Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM030498
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM045948
Pays : United States
Informations de copyright
Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Références
Nat Struct Biol. 2000 Aug;7(8):648-52
pubmed: 10932248
J Biol Chem. 2001 Sep 28;276(39):36446-53
pubmed: 11479296
Biochemistry. 2001 Oct 2;40(39):11794-810
pubmed: 11570880
Biochemistry. 2002 May 14;41(19):6032-44
pubmed: 11993998
Biochemistry. 2002 Oct 1;41(39):11611-27
pubmed: 12269804
Biophys J. 2004 Apr;86(4):2530-7
pubmed: 15041689
J Mol Biol. 2004 Jun 11;339(4):731-50
pubmed: 15165847
Methods Enzymol. 1992;212:424-58
pubmed: 1518458
J Mol Biol. 2004 Dec 10;344(5):1287-309
pubmed: 15561144
Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10076-81
pubmed: 16009938
Nature. 2005 Oct 27;437(7063):1321-5
pubmed: 16251956
Nucleic Acids Res. 2006;34(15):4126-37
pubmed: 16935876
Mol Cell. 2007 May 11;26(3):335-47
pubmed: 17499041
J Biol Chem. 2007 Sep 14;282(37):27076-85
pubmed: 17631491
Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12610-5
pubmed: 17640918
J Phys Chem B. 2007 Sep 20;111(37):11064-74
pubmed: 17718469
Methods Enzymol. 1991;208:258-90
pubmed: 1779838
Crit Rev Biochem Mol Biol. 2008 Sep-Oct;43(5):289-318
pubmed: 18937104
Science. 2009 Feb 20;323(5917):1070-4
pubmed: 19119185
Mol Cell. 2009 Jul 10;35(1):105-15
pubmed: 19595720
Methods. 2010 Jul;51(3):269-76
pubmed: 20371288
Cell. 2010 Aug 20;142(4):544-55
pubmed: 20723756
Nat Struct Mol Biol. 2010 Oct;17(10):1210-7
pubmed: 20852646
Microbiol Rev. 1990 Dec;54(4):342-80
pubmed: 2087220
Q Rev Biophys. 2011 Feb;44(1):123-51
pubmed: 21108866
Proc Natl Acad Sci U S A. 2011 May 3;108(18):7414-8
pubmed: 21502529
J Mol Biol. 2012 Apr 20;418(1-2):32-46
pubmed: 22342931
Methods Mol Biol. 2012;875:85-104
pubmed: 22573437
J Mol Biol. 2012 Oct 26;423(3):303-14
pubmed: 22820092
Methods Mol Biol. 2012;922:55-83
pubmed: 22976177
Genes Dev. 2012 Oct 15;26(20):2337-47
pubmed: 23070815
Nat Commun. 2013;4:2281
pubmed: 23939144
Chem Soc Rev. 2014 Feb 21;43(4):1221-9
pubmed: 24056732
J Mol Biol. 2014 Sep 23;426(19):3246-3261
pubmed: 25058683
Bioessays. 2014 Dec;36(12):1156-61
pubmed: 25171654
J Phys Chem Lett. 2015 May 21;6(10):1819-23
pubmed: 26263254
J Mol Biol. 1989 May 5;207(1):269-88
pubmed: 2661833
Biochemistry. 2016 Apr 12;55(14):2174-86
pubmed: 26998673
J Phys Chem B. 2016 Jul 7;120(26):6401-10
pubmed: 27184889
Proc Natl Acad Sci U S A. 2016 May 31;113(22):6194-9
pubmed: 27185951
Biophys Chem. 2017 Mar;222:7-24
pubmed: 28092802
Biophys Chem. 2017 Mar;222:25-40
pubmed: 28095332
J Phys Chem B. 2017 Mar 9;121(9):2039-2048
pubmed: 28177636
Biochemistry. 1986 Jan 14;25(1):21-5
pubmed: 3006753
Biochemistry. 1988 Jan 12;27(1):456-71
pubmed: 3280021
Biochemistry. 1987 Jun 2;26(11):3099-106
pubmed: 3300771
Biochemistry. 1986 Dec 2;25(24):7799-802
pubmed: 3542037
J Biol Chem. 1985 Mar 25;260(6):3594-603
pubmed: 3882711
Annu Rev Biochem. 1994;63:527-70
pubmed: 7979247
J Mol Biol. 1994 Feb 11;236(1):106-23
pubmed: 8107097
EMBO J. 1993 Mar;12(3):861-7
pubmed: 8458342
Biochemistry. 1996 Feb 20;35(7):2117-28
pubmed: 8652554
Biochemistry. 1996 Feb 20;35(7):2268-82
pubmed: 8652567
Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6264-8
pubmed: 8692803
Annu Rev Biochem. 1997;66:61-92
pubmed: 9242902