A non-natural variant of human lysozyme (I59T) mimics the in vitro behaviour of the I56T variant that is responsible for a form of familial amyloidosis.
We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein.
Hagan
Christine L
CL
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Johnson
Russell J K
RJ
Dhulesia
Anne
A
Dumoulin
Mireille
M
Dumont
Janice
J
De Genst
Erwin
E
Christodoulou
John
J
Robinson
Carol V
CV
Dobson
Christopher M
CM
Kumita
Janet R
JR
D000682:Amyloid / Q000378:metabolism; D028226:Amyloidosis, Familial / Q000201:enzymology / Q000235:genetics*; D041961:Deuterium Exchange Measurement; D006801:Humans; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D013816:Thermodynamics
D000682:Amyloid; D009113:Muramidase
Nature. 1997 Feb 27;385(6619):787-93
9039909
Protein Sci. 2000 Sep;9(9):1669-84
11045614
Biochemistry. 2008 Oct 21;47(42):11041-54
18816062
J Mol Biol. 1997 Nov 21;274(1):132-42
9398521
J Biomol NMR. 1995 Nov;6(3):277-93
8520220
Protein Eng. 1999 Oct;12(10):841-50
10556244
Hum Mutat. 2006 Jan;27(1):119-20
16329101
Annu Rev Biochem. 2006;75:333-66
16756495
J Biochem. 1991 Dec;110(6):1022-9
1794972
Protein Expr Purif. 1999 Jun;16(1):171-80
10336875
Methods Enzymol. 1986;131:266-80
3773761
J Mol Biol. 1981 Nov 15;152(4):737-62
7334520
J Mol Biol. 2009 Mar 20;387(1):17-27
19361437
Eur J Biochem. 2001 Jan;268(1):155-9
11121116
Nat Struct Biol. 2002 Apr;9(4):308-15
11887182
Biochemistry. 1997 Jul 22;36(29):8733-42
9220960
J Biol Chem. 2009 Feb 13;284(7):4246-54
19074141
Biochemistry. 1999 May 18;38(20):6419-27
10350460
Biochemistry. 1994 May 17;33(19):5867-76
8180215
Methods. 2004 Sep;34(1):51-64
15283915
Kidney Int. 2003 May;63(5):1652-7
12675840
J Mol Graph. 1996 Feb;14(1):51-5, 29-32
8744573
J Biol Chem. 2003 Jul 11;278(28):25910-8
12709420
FEBS J. 2006 Feb;273(4):711-20
16441658
J Struct Biol. 2000 Jun;130(2-3):339-51
10940237
Trends Biochem Sci. 1999 Sep;24(9):329-32
10470028
Gastroenterology. 2002 Oct;123(4):1346-9
12360495
Nature. 2003 Aug 14;424(6950):783-8
12917687
J Mol Biol. 2005 Sep 30;352(4):823-36
16126226
Proteins. 1993 Sep;17(1):75-86
8234246
J Mol Biol. 2005 Feb 25;346(3):773-88
15713462
Hum Mutat. 2000 Aug;16(2):180
10923049
Protein Sci. 2002 Mar;11(3):500-15
11847273
Trends Biochem Sci. 1990 Jan;15(1):14-7
2107612
Kidney Int. 2002 Mar;61(3):907-12
11849445
J Mol Biol. 2007 May 25;369(1):157-67
17407782
Nature. 1993 Apr 8;362(6420):553-7
8464497
Biochemistry. 2001 Apr 17;40(15):4853-8
11294653
Science. 1993 Nov 5;262(5135):896-900
8235611