Smoothing a rugged protein folding landscape by sequence-based redesign.
The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.|Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, United Kingdom.
Benjamin T
BT
Porebski
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Shani
S
Keleher
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Jeffrey J
JJ
Hollins
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Adrian A
AA
Nickson
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Emilia M
EM
Marijanovic
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Natalie A
NA
Borg
Programa de Computação Científica, Fundação Oswaldo Cruz, 21949900 Rio de Janeiro, Brazil.
Mauricio G S
MG
Costa
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Mary A
MA
Pearce
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Weiwen
W
Dai
Faculty of Information Technology, Monash University, Clayton, Victoria 3800, Australia.
Liguang
L
Zhu
Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, United Kingdom.
James A
JA
Irving
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
David E
DE
Hoke
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Itamar
I
Kass
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.|ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia.
James C
JC
Whisstock
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Stephen P
SP
Bottomley
Faculty of Information Technology, Monash University, Clayton, Victoria 3800, Australia.
Geoffrey I
GI
Webb
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.|Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
Sheena
S
McGowan
Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Ashley M
AM
Buckle
EMBO Rep. 2011 Sep 30;12(10):1011-7
21909074
Proc Natl Acad Sci U S A. 1989 Nov;86(21):8237-41
2682639
J Am Chem Soc. 2007 Aug 22;129(33):10110-2
17665911
Arch Biochem Biophys. 1998 Aug 15;356(2):296-300
9705220
J Mol Biol. 1990 Dec 20;216(4):1031-44
2266554
PLoS One. 2013;8(1):e54766
23382962
Biochemistry. 1996 Dec 24;35(51):16443-8
8987976
Annu Rev Biochem. 1997;66:549-79
9242917
J Biol Chem. 2003 Aug 29;278(35):33060-6
12807889
J Mol Biol. 2009 Apr 24;388(1):109-21
19265707
Biochemistry. 1999 Dec 14;38(50):16419-23
10600102
Nucleic Acids Res. 2016 Jul 8;44(W1):W356-60
27131359
J Biol Chem. 1999 Apr 2;274(14):9482-8
10092631
Biochim Biophys Acta. 1995 Mar 15;1247(2):179-84
7696306
Genome Biol. 2006;7(5):216
16737556
Genome Res. 2000 Dec;10(12):1845-64
11116082
Annu Rev Biochem. 1990;59:1007-39
2197975
Nature. 2000 Oct 19;407(6806):923-6
11057674
Curr Top Microbiol Immunol. 1999;243:55-86
10453638
Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3626-30
7724609
Protein Eng Des Sel. 2015 Mar;28(3):67-78
25691761
Nature. 2012 Nov 8;491(7423):222-7
23135467
J Mol Biol. 1997 Feb 28;266(3):601-9
9067613
Curr Opin Struct Biol. 2006 Dec;16(6):761-8
17079131
Biochemistry. 2000 Jul 25;39(29):8460-9
10913251
Structure. 2003 May;11(5):581-90
12737823
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):453-8
9012804
J Biol Chem. 1999 Apr 2;274(14):9548-55
10092640
J Biol Chem. 2002 Nov 29;277(48):46347-54
12244055
Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14675-80
9843948
J Mol Biol. 2010 Feb 19;396(2):375-83
19944704
Biochemistry. 1998 Nov 17;37(46):16172-82
9819209
Protein Sci. 2008 Dec;17(12):2127-33
18780818
Protein Eng Des Sel. 2007 Feb;20(2):69-79
17277006
Nature. 1988 Jun 23;333(6175):784-6
3386721
Structure. 2012 Mar 7;20(3):504-12
22405009
FEBS Lett. 2002 Jul 17;523(1-3):2-6
12123794
J Biol Chem. 2005 Mar 4;280(9):8435-42
15590653
Q Rev Biophys. 2014 Nov;47(4):285-363
25225856
Protein Eng Des Sel. 2012 Mar;25(3):107-17
22240293
Nature. 1992 Jun 18;357(6379):605-7
1608473
Science. 1990 Aug 24;249(4971):884-91
2392678
Chem Biol. 2012 Apr 20;19(4):449-55
22520751
Proc Natl Acad Sci U S A. 2010 Oct 5;107(40):17146-51
20855577
EMBO Rep. 2007 Jul;8(7):658-63
17557112
Biochem Biophys Res Commun. 1996 Sep 13;226(2):378-84
8806643
J Mol Biol. 1996 Dec 20;264(5):1072-84
9000631
Biochemistry. 2002 Apr 9;41(14):4575-81
11926819
Biophys J. 2012 Jun 20;102(12):2856-65
22735536
Nat Struct Biol. 1997 Jan;4(1):10-9
8989315
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8721-5
1528885
Nat Struct Biol. 2003 Jul;10(7):541-4
12808446
Curr Opin Biotechnol. 2001 Aug;12(4):371-5
11551465
J Synchrotron Radiat. 2015 Jan;22(1):187-90
25537608
J Mol Biol. 2010 Nov 12;403(5):751-62
20691191
Proc Natl Acad Sci U S A. 2013 Oct 29;110(44):17874-9
24128758
Biochem Biophys Res Commun. 2004 Nov 12;324(2):729-35
15474488
Curr Opin Biotechnol. 1995 Aug;6(4):370-4
7579645
Nat Struct Biol. 1996 Jun;3(6):497-500
8646533
J Biol Chem. 2003 Jun 27;278(26):23600-9
12711610
J Mol Biol. 1994 Jul 15;240(3):188-92
8028003
Nature. 2008 Oct 30;455(7217):1255-8
18923394
J Mol Biol. 2003 Sep 12;332(2):449-60
12948494
Protein Sci. 1999 Oct;8(10):2186-93
10548065
Protein Eng Des Sel. 2016 Jul;29(7):245-51
27274091
Annu Rev Biochem. 2013;82:447-70
23746259
PLoS Comput Biol. 2008 Feb 29;4(2):e1000002
18463696
J Biol Chem. 2000 Oct 27;275(43):33663-8
10924508
J Mol Biol. 2009 Apr 24;388(1):11-20
19285087
Protein Sci. 2000 Feb;9(2):417-20
10716194
J Biol Chem. 1994 Apr 1;269(13):9627-31
8144550
J Biochem. 1997 Apr;121(4):787-97
9163532
J Mol Biol. 2011 Nov 4;413(4):888-98
21945526
Nat Rev Genet. 2002 Oct;3(10 ):759-68
12360234
Arch Biochem Biophys. 2001 Jan 15;385(2):322-31
11368013
Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14141-6
25228761
Protein Sci. 2016 Jan;25(1):255-69
26183799
Nat Chem Biol. 2014 Nov;10(11):884-91
25325699
Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4467-72
22392975
J Biol Chem. 1995 Apr 14;270(15):8597-601
7721761
Structure. 1994 Oct 15;2(10):945-51
7866746
Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):67-72
10618372
J Mol Biol. 1991 Apr 5;218(3):595-606
2016749
Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19819-24
18077414
Nat Struct Mol Biol. 2011 Feb;18(2):222-6
21258324
Chem Rev. 2002 Dec;102(12):4751-804
12475206
J Mol Biol. 2009 Apr 10;387(4):857-68
19232354
PLoS One. 2014 Sep 02;9(9):e102617
25181470
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Dec 1;67(Pt 12):1492-7
22139150
J Biol Chem. 2000 Sep 15;275(37):28513-8
10878020
Curr Opin Struct Biol. 1995 Feb;5(1):79-84
7773750
Protein Eng. 2000 Jan;13(1):49-57
10679530
J Biol Chem. 2016 Jul 22;291(30):15674-86
27246852
Biochemistry. 2004 Aug 3;43(30):9834-9
15274637
Protein Eng. 2002 May;15(5):403-11
12034860
Science. 2003 Nov 21;302(5649):1364-8
14631033
J Mol Biol. 2001 Nov 9;313(5):1161-9
11700071
Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2754-9
11880627
Curr Opin Struct Biol. 2008 Feb;18(1):27-34
18243686
Biochemistry. 1990 Oct 9;29(40):9343-52
2248951