The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability.


Abstract

Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding. Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans. Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps. Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities. Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange. By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins. Thermal stability is also gained by the addition of Fe(2+). This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations. We discuss the implications that our findings have for the role of frataxins as iron-binding proteins. Study holds ProTherm entries: 17079, 17080, 17081, 17082, 17083, 17084, 17085, 17086, 17087, 17088, 17089, 17090, 17091, 17092, 17093, 17094, 17095, 17096, 17097, 17098, 17099, 17100, 17101, 17102, 17103, 17104, 17105, 17106, 17107, 17108, 17109, 17110, 17111, 17112, 17113, 17114, 17115, 17116, 17117, 17118, 17119, 17120, 17121, 17122, 17123, 17124, 17125, 17126, 17127 Extra Details: molecular forces; protein folding; genomic; conformational exchange; iron-binding proteins

Submission Details

ID: jzMtrKBJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Adinolfi S;Nair M;Politou A;Bayer E;Martin S;Temussi P;Pastore A,Biochemistry (2004) The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability. PMID:15157084
Additional Information

Number of data points 49
Proteins Frataxin homolog, mitochondrial ; Frataxin homolog, mitochondrial ; Protein CyaY ; Iron-sulfur cluster assembly protein CyaY ; Frataxin, mitochondrial ; Frataxin, mitochondrial
Unique complexes 3
Assays/Quantities/Protocols Experimental Assay: Tm ionic:NaCl: 150 mM, buffers:Hepes: 10 mM, prot_conc:16 microM ; Experimental Assay: Tm buffers:Hepes: 10 mM, prot_conc:16 microM, ionic:CaCl2: 1 mM ; Experimental Assay: Tm ionic:CaCl2: 150 mM, prot_conc:10 microM, buffers:Hepes: 10 mM ; Experimental Assay: Tm prot_conc:10 microM, buffers:Hepes: 10 mM, ionic:Na2SO4: 150 mM ; Experimental Assay: Tm prot_conc:10 microM, buffers:Hepes: 10 mM, ionic:NaF: 150 mM ; Experimental Assay: Tm ionic:NaCl: 150 mM, prot_conc:10 microM, buffers:Hepes: 10 mM ; Experimental Assay: Tm prot_conc:10 microM, buffers:Hepes: 10 mM, ionic:CaCl2: 1 mM ; Experimental Assay: Tm pH:7.2, prot_conc:10 microM, buffers:Hepes: 10 mM, ionic:NaF: 150 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:Hepes: 10 mM, pH:7.2 ; Experimental Assay: Tm pH:7.5, ionic:: , prot_conc:10 microM, buffers:Hepes: 10 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:phosphate: 200 mM, pH:8.0 ; Experimental Assay: Tm ionic:: , prot_conc:16 microM, buffers:phosphate: 150 mM ; Experimental Assay: Tm pH:6.0, ionic:: , prot_conc:16 microM, buffers:phosphate: 150 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:phosphate: 150 mM, pH:8.0 ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:phosphate: 150 mM ; Experimental Assay: Tm pH:6.0, ionic:: , prot_conc:10 microM, buffers:phosphate: 150 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:phosphate: 20 mM, pH:8.0 ; Experimental Assay: Tm pH:6.0, ionic:: , prot_conc:10 microM, buffers:phosphate: 20 mM ; Experimental Assay: Tm ionic:: , buffers:Hepes: 10 mM, prot_conc:16 microM ; Experimental Assay: Tm ionic:: , buffers:phosphate: 20 mM, prot_conc:16 microM ; Experimental Assay: Tm ionic:: , pH:7.4, prot_conc:10 microM, buffers:Hepes: 10 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:Hepes: 10 mM ; Experimental Assay: Tm ionic:: , prot_conc:10 microM, buffers:phosphate: 20 mM
Libraries Mutations for sequence GSHMGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA ; Mutations for sequence MESSTDGQVVPQEVLNLPLEKYHEEADDYLDHLLDSLEELSEAHPDCIPDVELSHGVMTLEIPAFGTYVINKQPPNKQIWLASPLSGPNRFDLLNGEWVSLRNGTKLTDILTEEVEKAISKSQ ; Mutations for sequence MNDSEFHRLADQLWLTIEERLDDWDGDSDIDCEINGGVLTITFENGSKIIINRQEPLHQVWLATKQGGYHFDLKGDEWICDRSGETFWDLLEQAATQQAGETVSFR
Sequence Assay Result Units