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

Study Summary

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 MNDSEFHRLADQLWLTIEERLDDWDGDSDIDCEINGGVLTITFENGSKIIINRQEPLHQVWLATKQGGYHFDLKGDEWICDRSGETFWDLLEQAATQQAGETVSFR ; Mutations for sequence MESSTDGQVVPQEVLNLPLEKYHEEADDYLDHLLDSLEELSEAHPDCIPDVELSHGVMTLEIPAFGTYVINKQPPNKQIWLASPLSGPNRFDLLNGEWVSLRNGTKLTDILTEEVEKAISKSQ ; Mutations for sequence GSHMGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1LY7 2002-06-26 The solution structure of the the c-terminal domain of frataxin, the protein responsible for friedreich ataxia
2GA5 2006-03-21 yeast frataxin
1SOY 2004-11-23 Solution structure of the bacterial frataxin orthologue, CyaY
3T3L 2011-08-10 1.15 1.15 A structure of human frataxin variant Q153A
3T3K 2011-08-10 1.24 1.24 A Structure of Friedreich's ataxia frataxin variant Q148R
3S4M 2011-06-29 1.3 Crystal structure of wild-type human frataxin
3S5E 2011-06-29 1.31 Crystal structure of human frataxin variant W155R, one of the Friedreich's ataxia point mutations
3T3T 2011-08-10 1.38 1.38 A structure of human frataxin variant Q148G
1EW4 2000-08-09 1.4 CRYSTAL STRUCTURE OF ESCHERICHIA COLI CYAY PROTEIN REVEALS A NOVEL FOLD FOR THE FRATAXIN FAMILY
2P1X 2007-10-02 1.42 Crystal structure analysis of the complex between CyaY and Eu(III)
3S5D 2011-06-29 1.5 Crystal structure of human frataxin variant W155A
3S5F 2011-06-29 1.5 Crystal structure of human frataxin variant W155F
3T3X 2011-08-10 1.57 1.57 A structure of Friedreich's ataxia frataxin variant R165C
3T3J 2011-08-10 1.7 1.70 A structure of Friedreich's ataxia frataxin variant N146K
2EFF 2007-10-02 1.8 Crystal structure analysis of the complex between CyaY and Co(II)
1EKG 2000-11-08 1.8 MATURE HUMAN FRATAXIN
5KZ5 2016-08-31 14.3 Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Complex Formed by the Iron Donor, the Sulfur Donor, and the Scaffold
5TRE 2017-06-07 15.6 Zinc and the Iron Donor Frataxin Regulate Oligomerization of the Scaffold Protein to Form New Fe-S Cluster Assembly Centers
5T0V 2016-08-31 17.5 Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: the Sub-Complex Formed by the Iron Donor, Yfh1, and the Scaffold, Isu1
3OEQ 2011-08-24 2.96 Crystal structure of trimeric frataxin from the yeast Saccharomyces cerevisiae, with full length n-terminus
4EC2 2013-01-30 3.0 Crystal structure of trimeric frataxin from the yeast Saccharomyces cerevisiae, complexed with ferrous
2FQL 2006-11-07 3.01 Crystal structure of trimeric frataxin from the yeast Saccharomyces cerevisiae
3OER 2011-08-24 3.2 Crystal structure of trimeric frataxin from the yeast saccharomyces cerevisiae, complexed with cobalt
6NZU 2019-05-22 3.2 Structure of the human frataxin-bound iron-sulfur cluster assembly complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Frataxin homolog, mitochondrial Q07540 FRDA_YEAST
90.6 Protein CyaY A7MQK4 CYAY_CROS8
90.6 Protein CyaY A4WG10 CYAY_ENT38
92.5 Protein CyaY B5BIV3 CYAY_SALPK
92.5 Protein CyaY Q5PKM9 CYAY_SALPA
92.5 Protein CyaY C0Q3B5 CYAY_SALPC
92.5 Protein CyaY Q57HR5 CYAY_SALCH
94.3 Protein CyaY B4TNV5 CYAY_SALSV
93.4 Protein CyaY B4SZ48 CYAY_SALNS
94.3 Protein CyaY A9MY64 CYAY_SALPB
94.3 Protein CyaY A9MJ08 CYAY_SALAR
94.3 Protein CyaY P56978 CYAY_SALTY
94.3 Protein CyaY Q8Z3A4 CYAY_SALTI
94.3 Protein CyaY B5RFQ2 CYAY_SALG2
94.3 Protein CyaY B5QVJ3 CYAY_SALEP
94.3 Protein CyaY B5FNU2 CYAY_SALDC
98.1 Protein CyaY Q0SYZ5 CYAY_SHIF8
95.3 Protein CyaY B5EZ64 CYAY_SALA4
96.2 Protein CyaY B4TB34 CYAY_SALHS
94.3 Protein CyaY B5XYK9 CYAY_KLEP3
95.3 Protein CyaY A6TGJ0 CYAY_KLEP7
96.2 Protein CyaY A8ACV5 CYAY_CITK8
99.1 Protein CyaY Q83IX2 CYAY_SHIFL
99.1 Protein CyaY B5YY53 CYAY_ECO5E
99.1 Protein CyaY Q8XAP0 CYAY_ECO57
98.1 Protein CyaY Q8FBN9 CYAY_ECOL6
99.1 Protein CyaY A8A6R1 CYAY_ECOHS
99.1 Protein CyaY B7LU51 CYAY_ESCF3
99.1 Protein CyaY Q31UI1 CYAY_SHIBS
99.1 Protein CyaY B2TUW2 CYAY_SHIB3
99.1 Protein CyaY Q1R4D1 CYAY_ECOUT
99.1 Protein CyaY Q0TAR9 CYAY_ECOL5
99.1 Protein CyaY A1AHX3 CYAY_ECOK1
99.1 Protein CyaY B7N297 CYAY_ECO81
99.1 Protein CyaY B7MH68 CYAY_ECO45
99.1 Protein CyaY B7UNC2 CYAY_ECO27
99.1 Protein CyaY B7NTD8 CYAY_ECO7I
99.1 Protein CyaY B1LLX7 CYAY_ECOSM
100.0 Protein CyaY Q3YVG0 CYAY_SHISS
100.0 Protein CyaY Q329Y2 CYAY_SHIDS
100.0 Protein CyaY B6I4E2 CYAY_ECOSE
100.0 Protein CyaY B7NFA9 CYAY_ECOLU
100.0 Protein CyaY P27838 CYAY_ECOLI
100.0 Protein CyaY B1IW97 CYAY_ECOLC
100.0 Protein CyaY B1XAH3 CYAY_ECODH
100.0 Protein CyaY C4ZZ71 CYAY_ECOBW
100.0 Protein CyaY B7M605 CYAY_ECO8A
100.0 Protein CyaY B7L963 CYAY_ECO55
100.0 Protein CyaY A7ZU09 CYAY_ECO24
96.8 Frataxin, mitochondrial Q8HXX9 FRDA_MACFA
100.0 Frataxin, mitochondrial Q16595 FRDA_HUMAN
95.0 Frataxin, mitochondrial O35943 FRDA_MOUSE
95.1 Frataxin, mitochondrial Q05B87 FRDA_BOVIN