Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies.


Abstract

Thermodynamic and pH stability of recombinant human L- and H-ferritins were probed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 they were dissociated into subunit monomers and in this pH interval the H-subunit displayed a single calorimetrically-revealed domain with properties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or 169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), a wide transition range (approximately 20 degrees C) and high ANS binding. In contrast, at pH 2 the L-ferritin subunit showed two calorimetric domains with Tm of 35 and 40 degrees C with similar unfolding enthalpies and with moderate extent of interactions, as indicated by the ratio of calorimetric enthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for the thermal transition. A pH increase from 2.0 to 2.8 determined the coupling of the two domains into a single cooperative folding unit and drastic increase of the transition temperature (from 37 to 80 degrees C). The contacts between the two domains in the L-subunit appeared to contribute to about 30% of the total stabilization free energy. The unfolding enthalpies, heat capacity changes and pronounced ANS binding of the L-subunit at pH 2.0-2.8 indicated that part of the structure lacked 'meltable' tertiary interactions. The results indicate that H- and L-subunits are stabilized by largely different intra-chain interactions with a critical contribution to L-subunit stability of embedded salt bridge(s) absent in the H-subunit. Study holds ProTherm entries: 3456, 3457, 3458, 3459, 3460, 3461, 3462 Extra Details: differential scanning calorimetry; ferritin subunits;,protein folding; protein stability

Submission Details

ID: atuxeD5Q4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Martsev SP;Vlasov AP;Arosio P,Protein Eng. (1998) Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies. PMID:9681870
Additional Information

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
6B8F 2018-05-02 1.06 Contracted Human Heavy-Chain Ferritin Crystal-Hydrogel Hybrid
6B8G 2018-05-02 1.13 Twice-Contracted Human Heavy-Chain Ferritin Crystal-Hydrogel Hybrid
4Y08 2015-09-23 1.34 ONE MINUTE IRON LOADED HUMAN H FERRITIN
4OYN 2015-09-09 1.43 Fifteen minutes iron loaded human H ferritin
2CIH 2006-12-13 1.5 Recombinant human H ferritin, K86Q and E27D mutant, soaked with Zn
4YKH 2015-09-09 1.52 Thirty minutes iron loaded human H ferritin
3AJO 2010-08-25 1.52 Crystal structure of wild-type human ferritin H chain
4ZJK 2015-09-09 1.56 FIVE MINUTES IRON LOADED HUMAN H FERRITIN
6FTV 2018-08-15 1.58 X-ray structure of human heavy chain ferritin in complex with NAMI A
3AJQ 2010-08-25 1.58 Crystal structure of human H ferritin E140Q mutant
5JKK 2017-05-10 1.6 Crystal structure of the negatively supercharged variant Ftn(neg) of human heavy chain ferritin
2CHI 2006-12-13 1.6 Recombinant human H ferritin, K86Q and E27D mutant
5N27 2017-08-02 1.74 X-ray structure of human heavy chain ferritin (apo form)
2CN7 2006-12-13 1.75 Recombinant human H ferritin, K86Q, E27D and E107D mutant
5UP7 2017-06-21 1.79 Crystal Structure of the Ni-bound Human Heavy-Chain Ferritin 122H-delta C-star variant
2CEI 2006-12-13 1.8 Recombinant human H ferritin, K86Q mutant, soaked with Zn
5JKM 2016-10-26 1.8 Binary crystal structure of positively and negatively supercharged variants Ftn(pos) and Ftn(neg) from human heavy chain ferritin (Mg acetate condition)
5JKL 2016-10-26 1.8 Binary crystal structure of positively and negatively supercharged variants Ftn(pos) and Ftn(neg) from human heavy chain ferritin (Mg formate condition)
2IU2 2006-12-13 1.8 Recombinant human H ferritin, K86Q, E27D and E107D mutant, soaked with Zn ions
6S61 2019-07-10 1.84 Apoferritin from mouse at 1.84 angstrom resolution
3KXU 2010-02-09 1.85 Crystal structure of human ferritin FTL498InsTC pathogenic mutant
4DYX 2013-01-23 1.85 Crystal Structure of the Cu-adduct of Human H-Ferritin variant 4His-delta C-star
4DYY 2013-01-23 1.9 Crystal Structure of the Cu-adduct of Human H-Ferritin variant MIC1
6H6T 2019-08-14 1.9 Binary crystal structure of positively and negatively supercharged variants Ftn(pos) and Ftn(neg) from human heavy chain ferritin (propandiol condition, coordination number 8)
2FFX 2006-07-11 1.9 Structure of Human Ferritin L. Chain
2FHA 1997-09-04 1.9 HUMAN H CHAIN FERRITIN
3AJP 2010-08-25 1.9 Crystal structure of human H ferritin E140A mutant
5CMQ 2015-09-09 1.93 Crystal Structure of Zn-bound Human H-Ferritin variant 122H-delta C-star
5VTD 2017-06-21 1.95 Crystal Structure of the Co-bound Human Heavy-Chain Ferritin variant 122H-delta C-star
5LG8 2017-02-22 1.98 Human L-type ferritin iron loaded for 60 minutes
6H6U 2019-08-14 2.0 Unitary crystal structure of the positively supercharged variant Ftn(pos) from human heavy chain ferritin (PEG 400 condition)
5N26 2017-08-02 2.05 X-ray structure of human heavy chain ferritin in complex with cisplatin
2CLU 2006-12-13 2.1 Recombinant human H ferritin, K86Q and E107D mutant
2FG4 2006-07-04 2.1 Structure of Human Ferritin L Chain
2CN6 2006-12-13 2.2 Recombinant human H ferritin, K86Q and E107D mutant, soaked with Zn ions
3WNW 2015-01-28 2.24 Structure of Mouse H-chain modified ferritin
4DYZ 2013-01-23 2.3 Crystal Structure of the apo form of Human H-Ferritin variant MIC1
1FHA 1992-07-15 2.4 SOLVING THE STRUCTURE OF HUMAN H FERRITIN BY GENETICALLY ENGINEERING INTERMOLECULAR CRYSTAL CONTACTS
5UP9 2017-09-27 2.45 Crystal Structure of Zn-bound Human Heavy-Chain ferritin variant 122H-delta C-star with para-xylenedihydroxamate
2FG8 2006-07-04 2.5 Structure of Human Ferritin L Chain
4DZ0 2013-01-23 2.5 Crystal structure of the Cu-adduct of human H-Ferritin variant MIC1 labeled with a dansyl fluorophore
5UP8 2017-06-21 2.63 Crystal Structure of the Zn-bound Human Heavy-Chain variant 122H-delta C-star with para-benzenedihydroxamate
5OBB 2018-07-25 2.65 Structure of a modified mouse H chain ferritin with a lanthanide binding motif in complex with Terbium
6IPP 2019-03-13 2.7 Non-native ferritin 8-mer mutant-C90A/C102A/C130A/D144C
2Z6M 2008-07-22 2.72 Crystal structure of Human Ferritin H8 as biotemplate for noble metal nanoparticle synthesis
3ES3 2008-10-21 2.8 Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein. The Complex with Gold ions. Ferritin H8-H9x Mutant
5GN8 2016-12-14 2.81 Structure of a 48-mer protein nanocage fabricated from its 24-mer analogue by subunit interface redesign
5OBA 2018-07-25 2.85 Structure of a modified mouse H-chain ferritin with a lanthanide binding motif
5GOU 2017-02-22 2.91 Structure of a 16-mer protein nanocage fabricated from its 24-mer analogue by subunit interface redesign
6IPO 2019-03-13 3.0 Ferritin mutant C90A/C102A/C130A/D144C
5XB1 2018-02-21 3.0 human ferritin mutant - E-helix deletion
5ZND 2018-11-07 3.0 8-mer nanotube derived from 24-mer rHuHF nanocage
5YI5 2018-02-21 3.0 human ferritin mutant - E-helix deletion
3ERZ 2008-10-21 3.06 Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein. Mercury Ions on the Three-Fold Channel
6IPQ 2019-03-13 3.1 Non-native ferritin 8-mer mutant-C90A/C102A/C130A
5CMR 2015-09-09 3.79 Crystal Structure of Linker-Mediated Zn-bound Human H-Ferritin variant 122H-delta C-star
6H5I 2019-03-27 3.9 Single Particle Cryo-EM map of human Transferrin receptor 1 - H-Ferritin complex.
6IPC 2019-03-13 4.44 Non-native human ferritin 8-mer
6GSR 2019-03-27 5.5 Single Particle Cryo-EM map of human Transferrin receptor 1 - H-Ferritin complex at 5.5 Angstrom resolution.

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
98.9 Ferritin light chain Q5R538 FRIL_PONAB
100.0 Ferritin light chain P02792 FRIL_HUMAN
92.5 Ferritin heavy chain P18685 FRIH_SHEEP
95.7 Ferritin heavy chain P25915 FRIH_RABIT
92.6 Ferritin heavy chain P08267 FRIH_CHICK
90.6 Ferritin heavy chain O46414 FRIH_BOVIN
92.8 Ferritin heavy chain P09528 FRIH_MOUSE
92.2 Ferritin heavy chain Q8MIP0 FRIH_HORSE
94.4 Ferritin heavy chain P19132 FRIH_RAT
93.4 Ferritin heavy chain P29389 FRIH_CRIGR
94.5 Ferritin heavy chain Q2MHN2 FRIH_FELCA
95.6 Ferritin heavy chain Q95MP7 FRIH_CANLF
95.6 Ferritin heavy chain Q9XT73 FRIH_TRIVU
98.9 Ferritin heavy chain Q5R8J7 FRIH_PONAB
99.5 Ferritin heavy chain P02794 FRIH_HUMAN