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
7A6A 2020-09-02 1.15 1.15 A structure of human apoferritin obtained from Titan Mono- BCOR microscope
7A4M 2020-10-28 1.22 Cryo-EM structure of mouse heavy-chain apoferritin at 1.22 A
6Z6U 2020-06-24 1.25 1.25 A structure of human apoferritin obtained from Titan Mono-BCOR microscope
6WYF 2020-11-11 1.25 Crystal structure of Human H-chain Ferritin variant 157C Delta C-star Modified with a RAFT Agent Soaked in an Acrylate Solution
7A6B 2020-09-02 1.33 1.33 A structure of human apoferritin obtained from Titan Mono- BCOR microscope
4Y08 2015-09-23 1.34 ONE MINUTE IRON LOADED HUMAN H FERRITIN
7K3V 2020-11-18 1.34 Apoferritin structure at 1.34 angstrom resolution determined from a 300 kV Titan Krios G3i electron microscope with K3 detector
7K3W 2020-11-18 1.36 Apoferritin structure at 1.36 angstrom resolution determined from a 300 kV Titan Krios G3i electron microscope with Falcon4 detector
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
3AJO 2010-08-25 1.52 Crystal structure of wild-type human ferritin H chain
4YKH 2015-09-09 1.52 Thirty minutes iron loaded human H ferritin
6Z9E 2020-06-24 1.55 1.55 A structure of human apoferritin obtained from data subset of Titan Mono-BCOR microscope
4ZJK 2015-09-09 1.56 FIVE MINUTES IRON LOADED HUMAN H FERRITIN
6Z9F 2020-06-24 1.56 1.56 A structure of human apoferritin obtained from data subset of Titan Mono-BCOR microscope
3AJQ 2010-08-25 1.58 Crystal structure of human H ferritin E140Q mutant
6FTV 2018-08-15 1.58 X-ray structure of human heavy chain ferritin in complex with NAMI A
2CHI 2006-12-13 1.6 Recombinant human H ferritin, K86Q and E27D mutant
5JKK 2017-05-10 1.6 Crystal structure of the negatively supercharged variant Ftn(neg) of human heavy chain ferritin
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
6V21 2020-02-12 1.75 Mouse heavy chain apoferritin determined using single-particle cryo-EM at 200 keV
5UP7 2017-06-21 1.79 Crystal Structure of the Ni-bound Human Heavy-Chain Ferritin 122H-delta C-star variant
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)
6KE2 2019-10-23 1.8 ABloop reengineered Ferritin Nanocage
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)
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
4DYX 2013-01-23 1.85 Crystal Structure of the Cu-adduct of Human H-Ferritin variant 4His-delta C-star
3KXU 2010-02-09 1.85 Crystal structure of human ferritin FTL498InsTC pathogenic mutant
2FHA 1997-09-04 1.9 HUMAN H CHAIN FERRITIN
4DYY 2013-01-23 1.9 Crystal Structure of the Cu-adduct of Human H-Ferritin variant MIC1
2FFX 2006-07-11 1.9 Structure of Human Ferritin L. Chain
3AJP 2010-08-25 1.9 Crystal structure of human H ferritin E140A mutant
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)
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)
6WX6 2020-05-20 2.0 Cryo-EM Structure of Human Apoferritin Light Chain Vitrified Using Back-it-up
5N26 2017-08-02 2.05 X-ray structure of human heavy chain ferritin in complex with cisplatin
7JGN 2020-10-14 2.07 Crystal Structure of the Zn-bound Human Heavy-chain variant 122H-delta C-star with meta-benzenedihyrdoxamate collected at 100K
6TSF 2020-02-19 2.09 Crystal structure of human L ferritin (HuLf) Fe(III)-loaded for 60 minutes
2FG4 2006-07-04 2.1 Structure of Human Ferritin L Chain
2CLU 2006-12-13 2.1 Recombinant human H ferritin, K86Q and E107D mutant
6TSA 2020-02-19 2.18 Crystal structure of human L ferritin (HuLf) Fe(III)-loaded for 30 minutes
6TS1 2020-02-19 2.2 Crystal structure of human L ferritin (HuLf) triple variant E60A-E61A-E64A Fe(III)-loaded for 60 minutes
6TS0 2020-02-19 2.2 Crystal structure of human L ferritin (HuLf) triple variant E60A-E61A-E64A Fe(III)-loaded for 30 minutes
2CN6 2006-12-13 2.2 Recombinant human H ferritin, K86Q and E107D mutant, soaked with Zn ions
6WYH 2020-11-11 2.22 Crystal structure of Human H-chain Ferritin variant 157C Delta C-star Modified with a RAFT Agent Soaked in an Acrylate Solution
3WNW 2015-01-28 2.24 Structure of Mouse H-chain modified ferritin
6WYG 2020-11-11 2.27 Crystal structure of Human H-chain Ferritin variant 157C Delta C-star Modified with a RAFT agent
6TSJ 2020-02-19 2.3 Crystal structure of human L ferritin (HuLf) Fe(III)-loaded for 15 minutes
6KE4 2019-10-23 2.3 ABloop reengineered Ferritin Nanocage
4DYZ 2013-01-23 2.3 Crystal Structure of the apo form of Human H-Ferritin variant MIC1
7JGM 2020-10-14 2.31 Crystal Structure of the Ni-bound Human Heavy-chain variant 122H-delta C-star with meta-benzenedihyrdoxamate
7JGL 2020-10-21 2.34 Crystal Structure of the Zn-bound Human Heavy-chain variant 122H-delta C-star with 2,5-furandihyrdoxamate collected at 100K
6M54 2020-05-13 2.4 Human apo ferritin frozen on TEM grid with Amorphous nickel titanium alloy supporting film
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
6TR9 2020-02-19 2.46 Crystal structure of human L ferritin (HuLf) triple variant E60A-E61A-E64A
4DZ0 2013-01-23 2.5 Crystal structure of the Cu-adduct of human H-Ferritin variant MIC1 labeled with a dansyl fluorophore
2FG8 2006-07-04 2.5 Structure of Human Ferritin L Chain
6M52 2020-05-13 2.6 Human apo ferritin frozen on TEM grid with amorphous carbon supporting film
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
7JGK 2020-10-14 2.68 Crystal Structure of the Ni-bound Human Heavy-chain variant 122H-delta C-star with 2,5-furandihyrdoxamate collected at 100K
6IPP 2019-03-13 2.7 Non-native ferritin 8-mer mutant-C90A/C102A/C130A/D144C
7K26 2020-11-11 2.7 Crystal structure of Human H-chain Ferritin variant infused with Sodium Acrylate
2Z6M 2008-07-22 2.72 Crystal structure of Human Ferritin H8 as biotemplate for noble metal nanoparticle synthesis
6SHT 2020-05-13 2.73 Molecular structure of mouse apoferritin resolved at 2.7 Angstroms with the Glacios cryo-microscope
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
6JOB 2020-03-25 2.93 Ferritin variant with 'GMG' motif
5YI5 2018-02-21 3.0 human ferritin mutant - E-helix deletion
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
6IPO 2019-03-13 3.0 Ferritin mutant C90A/C102A/C130A/D144C
7JGQ 2020-10-21 3.01 Crystal Structure of the Ni-bound Human Heavy-chain variant 122H-delta C-star with 2,5-furandihyrdoxamate collected at 278K after one heating/cooling cycle
3ERZ 2008-10-21 3.06 Directing Noble Metal Ion Chemistry within a Designed Ferritin Protein. Mercury Ions on the Three-Fold Channel
7JGO 2020-10-14 3.08 Crystal Structure of the Ni-bound Human Heavy-chain variant 122H-delta C-star with 2,5-furandihyrdoxamate collected at 278K
6IPQ 2019-03-13 3.1 Non-native ferritin 8-mer mutant-C90A/C102A/C130A
6JPS 2019-10-09 3.5 Human H chain ferritin mutant-MBP
5CMR 2015-09-09 3.79 Crystal Structure of Linker-Mediated Zn-bound Human H-Ferritin variant 122H-delta C-star
6J7G 2019-10-16 3.87 Human H-ferritin mutant-C90A/C102A/C130A/D144C
6H5I 2019-03-27 3.9 Single Particle Cryo-EM map of human Transferrin receptor 1 - H-Ferritin complex.
6J4A 2019-09-18 3.99 Human H chain ferritin with an extension peptide
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.
7JGP 2020-10-14 6.42 Crystal Structure of the Ni-bound Human Heavy-chain variant 122H-delta C-star with 2,5-furandihyrdoxamate at 318K

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