Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer.
Animals
Bacterial Proteins
/ chemistry
Binding Sites
CHO Cells
Cricetulus
Fluorescence Resonance Energy Transfer
Gene Expression
Genes, Reporter
Green Fluorescent Proteins
/ chemistry
Luminescent Proteins
/ chemistry
Models, Molecular
Molecular Dynamics Simulation
Polyethylene Glycols
/ chemistry
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Isoforms
/ chemistry
Protein Structure, Tertiary
Recombinant Fusion Proteins
/ chemistry
Single Molecule Imaging
Sodium Chloride
/ chemistry
Urea
/ chemistry
Discrete molecular dynamics
FRET
Fluorescent protein
Macromolecular crowding
Supertertiary structure
Journal
Journal of molecular biology
ISSN: 1089-8638
Titre abrégé: J Mol Biol
Pays: Netherlands
ID NLM: 2985088R
Informations de publication
Date de publication:
05 03 2021
05 03 2021
Historique:
received:
07
02
2020
revised:
03
12
2020
accepted:
21
12
2020
pubmed:
4
1
2021
medline:
7
5
2021
entrez:
3
1
2021
Statut:
ppublish
Résumé
Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.
Identifiants
pubmed: 33388290
pii: S0022-2836(20)30718-X
doi: 10.1016/j.jmb.2020.166793
pmc: PMC8059107
mid: NIHMS1676852
pii:
doi:
Substances chimiques
Bacterial Proteins
0
Cyan Fluorescent Protein
0
Luminescent Proteins
0
Protein Isoforms
0
Recombinant Fusion Proteins
0
yellow fluorescent protein, Bacteria
0
Green Fluorescent Proteins
147336-22-9
Polyethylene Glycols
3WJQ0SDW1A
Sodium Chloride
451W47IQ8X
Urea
8W8T17847W
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
166793Subventions
Organisme : NIMH NIH HHS
ID : R01 MH081923
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM119691
Pays : United States
Informations de copyright
Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Références
Cell Mol Life Sci. 2018 Nov;75(21):3907-3929
pubmed: 30066087
Phys Chem Chem Phys. 2017 Jul 19;19(28):18147-18151
pubmed: 28696461
Science. 2018 Aug 31;361(6405):
pubmed: 30166461
Proc Natl Acad Sci U S A. 2015 Nov 24;112(47):14705-10
pubmed: 26553997
Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17863-7
pubmed: 22665776
Methods. 2014 Mar 15;66(2):131-8
pubmed: 23811334
Elife. 2016 Apr 07;5:e13876
pubmed: 27052508
Biol Chem. 2016 Jan;397(1):37-44
pubmed: 26351910
Biophys J. 1979 May;26(2):161-93
pubmed: 262414
Biophys J. 2016 Mar 8;110(5):1013-6
pubmed: 26958875
Biochemistry. 2017 Dec 19;56(50):6565-6574
pubmed: 29168376
Nat Methods. 2019 Sep;16(9):815-829
pubmed: 31471616
J Chem Phys. 2018 Mar 28;148(12):123329
pubmed: 29604882
Chem Rev. 2016 Jun 8;116(11):6391-423
pubmed: 26889708
Biophys J. 2000 Mar;78(3):1606-19
pubmed: 10692345
Science. 1996 Sep 6;273(5280):1392-5
pubmed: 8703075
Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12491-6
pubmed: 15314214
Biophys J. 2010 Aug 4;99(3):961-70
pubmed: 20682275
Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11996-2001
pubmed: 11050231
Proc Natl Acad Sci U S A. 2019 Nov 12;116(46):23124-23131
pubmed: 31659043
Nat Methods. 2005 Dec;2(12):905-9
pubmed: 16299475
Biophys J. 2011 Aug 17;101(4):892-8
pubmed: 21843480
Biophys J. 2006 Dec 15;91(12):L99-L101
pubmed: 17040988
Nat Biotechnol. 1996 Oct;14(10):1246-51
pubmed: 9631087
Nature. 2016 Feb 4;530(7588):45-50
pubmed: 26808899
Proteins. 2003 Nov 1;53(2):220-8
pubmed: 14517973
Proc Natl Acad Sci U S A. 2017 Aug 1;114(31):E6342-E6351
pubmed: 28716919
Nat Methods. 2015 Mar;12(3):227-9, 1 p following 229
pubmed: 25643150
Angew Chem Int Ed Engl. 2015 Feb 16;54(8):2548-51
pubmed: 25557778
Eur J Biochem. 2002 Jan;269(1):2-12
pubmed: 11784292
Protein Sci. 2007 Jul;16(7):1429-38
pubmed: 17586775
Methods Enzymol. 2000;327:472-500
pubmed: 11045004
J Phys Chem Lett. 2020 Dec 3;11(23):10131-10136
pubmed: 33191750
J Phys Chem B. 2019 Jan 17;123(2):379-393
pubmed: 30571116
Proteins. 2004 Dec 1;57(4):829-38
pubmed: 15390267
PLoS Comput Biol. 2010 Jul 01;6:e1000833
pubmed: 20617196
Mol Cell Biol. 2001 Dec;21(24):8318-28
pubmed: 11713268
Sensors (Basel). 2014 Jul 02;14(7):11691-713
pubmed: 24991940
Elife. 2014 Sep 25;3:
pubmed: 25255214
Science. 2002 May 3;296(5569):913-6
pubmed: 11988576
Protein Sci. 2017 Mar;26(3):403-413
pubmed: 27977883
J Biotechnol. 2002 Jan;82(3):181-96
pubmed: 11999689
Chem Biol. 2014 Apr 24;21(4):459-469
pubmed: 24631122
Structure. 2013 Nov 5;21(11):2051-60
pubmed: 24139989
PLoS One. 2012;7(8):e44159
pubmed: 22952912
Biophys J. 2002 Dec;83(6):3652-64
pubmed: 12496132
Sci Rep. 2016 May 10;6:25745
pubmed: 27160511
J Cell Sci. 2001 Mar;114(Pt 5):837-8
pubmed: 11181166
Proc Natl Acad Sci U S A. 2013 Aug 27;110(35):14249-54
pubmed: 23940346
Molecules. 2015 Jan 14;20(1):1377-409
pubmed: 25594347
Structure. 2008 Jul;16(7):1010-8
pubmed: 18611374
Phys Chem Chem Phys. 2012 Mar 28;14(12):4109-17
pubmed: 22331099
J Chem Theory Comput. 2017 Mar 14;13(3):1454-1461
pubmed: 28157327
Curr Opin Chem Biol. 2008 Feb;12(1):60-5
pubmed: 18282482
Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16155-60
pubmed: 22984159
J Phys Chem B. 2012 Jul 26;116(29):8375-82
pubmed: 22280505
J Chem Phys. 2009 Mar 28;130(12):124903
pubmed: 19334885
Annu Rev Biochem. 2011;80:357-73
pubmed: 21529159
PLoS Pathog. 2014 Sep 18;10(9):e1004373
pubmed: 25233449
Nat Chem Biol. 2012 Jun 18;8(7):597-600
pubmed: 22710296
Biochemistry. 2006 Nov 7;45(44):13183-92
pubmed: 17073440
Proc Natl Acad Sci U S A. 2017 Jun 27;114(26):6776-6781
pubmed: 28607089
J Biol Chem. 2001 Apr 6;276(14):10577-80
pubmed: 11279227
Biochem Soc Trans. 2016 Oct 15;44(5):1185-1200
pubmed: 27911701
J Fluoresc. 2005 Mar;15(2):153-60
pubmed: 15883770
Anal Biochem. 2017 Apr 1;522:1-9
pubmed: 28108168
Cell. 1982 Sep;30(2):345-7
pubmed: 6754085
Biophys Chem. 2004 Mar 1;108(1-3):231-43
pubmed: 15043932
BMC Biophys. 2011 May 31;4:13
pubmed: 21627822
Chem Biol. 2015 Jan 22;22(1):98-106
pubmed: 25556943
Biophys J. 2002 Jan;82(1 Pt 1):133-44
pubmed: 11751302
Sci Rep. 2016 Feb 09;6:21613
pubmed: 26857745
Microsc Microanal. 2006 Jun;12(3):238-54
pubmed: 17481360
Proc Natl Acad Sci U S A. 1999 Feb 2;96(3):893-8
pubmed: 9927664
Methods Appl Fluoresc. 2019 Feb 19;7(2):025002
pubmed: 30690439
Structure. 2011 Jun 8;19(6):810-20
pubmed: 21645852
Biophys J. 2017 May 9;112(9):1929-1939
pubmed: 28494963
Proc Natl Acad Sci U S A. 2013 Sep 17;110(38):15259-64
pubmed: 23986495
Nat Commun. 2018 Sep 13;9(1):3724
pubmed: 30214057
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5217-22
pubmed: 23479636
J Biol Chem. 2016 Jun 17;291(25):12951-9
pubmed: 27129240
Trends Biochem Sci. 2008 Jan;33(1):2-8
pubmed: 18054235
Chem Rev. 2014 Jul 9;114(13):6661-714
pubmed: 24901537
Trends Biochem Sci. 2016 Nov;41(11):970-981
pubmed: 27669651
Protein Eng. 2001 Aug;14(8):529-32
pubmed: 11579220
Sensors (Basel). 2015 Oct 16;15(10):26281-314
pubmed: 26501285
Curr Opin Struct Biol. 2000 Apr;10(2):139-45
pubmed: 10753811
ACS Sens. 2018 Aug 24;3(8):1462-1470
pubmed: 29979038
Proc Natl Acad Sci U S A. 2014 Apr 1;111(13):4874-9
pubmed: 24639500
Int J Mol Sci. 2014 Dec 12;15(12):23090-140
pubmed: 25514413
Proc Natl Acad Sci U S A. 2020 Sep 1;117(35):21413-21419
pubmed: 32817491
Annu Rev Phys Chem. 2011;62:257-77
pubmed: 21219136