Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.


Abstract

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding. Study holds ProTherm entries: 6106, 6107, 6108, 6109, 6110, 6111, 6112, 6113, 6114, 6115, 6116, 6117, 6118, 6119, 6120, 6121, 6122, 6123, 6124, 6125, 6126, 6127, 6128, 6129, 6130, 6131, 6132, 6133, 6134, 6135, 6136, 6137, 6138, 6139, 6140, 6141, 6142, 6143, 6144, 6145, 6146, 6147 Extra Details: measurements were made for the residue V43

Submission Details

ID: PHNTSsG53

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Ragona L;Fogolari F;Romagnoli S;Zetta L;Maubois JL;Molinari H,J. Mol. Biol. (1999) Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements. PMID:10543977
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
1DV9 2000-02-09 STRUCTURAL CHANGES ACCOMPANYING PH-INDUCED DISSOCIATION OF THE B-LACTOGLOBULIN DIMER
1CJ5 2000-04-26 BOVINE BETA-LACTOGLOBULIN A
4CK4 2014-10-08 1.12 Ovine beta-Lactoglobulin at Atomic Resolution
4TLJ 2014-06-18 1.17 Ultra-high resolution crystal structure of caprine Beta-lactoglobulin
4NLJ 2014-03-12 1.4 Crystal structure of sheep beta-lactoglobulin (space group P1)
4GNY 2013-02-13 1.64 Bovine beta-lactoglobulin complex with dodecyl sulfate
5NUK 2018-04-04 1.7 Engineered beta-lactoglobulin: variant I56F-L39A-M107F in complex with chlorpromazine (LG-FAF-CLP)
1BEB 1997-05-15 1.8 BOVINE BETA-LACTOGLOBULIN, LATTICE X
4KII 2013-09-04 1.85 Beta-lactoglobulin in complex with Cp*Rh(III)Cl N,N-di(pyridin-2-yl)dodecanamide
3NQ9 2010-07-14 1.9 Bovine beta-lactoglobulin complex with caprylic acid
4Y0S 2015-07-01 1.9 Goat beta-lactoglobulin complex with pramocaine (GLG-PRM)
3NQ3 2010-07-14 1.9 Bovine beta-lactoglobulin complex with capric acid
4NLI 2014-03-12 1.9 Crystal structure of sheep beta-lactoglobulin (space group P3121)
3UEV 2011-11-09 1.9 Bovine beta-lactoglobulin complex with myristic acid
6GHH 2018-06-20 1.9 Thermodynamic, Crystallographic and Computational Studies of Non Mammalian Fatty Acid Binding to Bovine b-Lactoglobulin
1B8E 2001-05-02 1.95 HIGH RESOLUTION CRYSTAL STRUCTURE OF THE BOVINE BETA-LACTOGLOBULIN (ISOFORMS A AND B) IN ORTHOROMBIC SPACE GROUP
5NUN 2018-04-04 1.95 Engineered beta-lactoglobulin: variant F105L-L39A-M107F in complex with chlorpromazine (LG-LAF-CLP)
5EEE 2016-11-09 1.95 Bovine lactoglobulin complex with decanol
1UZ2 2004-05-20 1.95 The Cys121Ser Mutant of Beta-Lactoglobulin
4Y0Q 2015-07-01 2.0 Bovine beta-lactoglobulin complex with pramocaine crystallized from sodium citrate (BLG-PRM1)
6GFS 2018-06-20 2.0 Thermodynamic, Crystallographic and Computational Studies of Non Mammalian Fatty Acid Binding to Bovine b-Lactoglobulin
5EED 2016-10-26 2.0 Ovine lactoglobulin complex with decanol
6QI6 2019-01-30 2.0 Trigonal form of WT recombinant bovine beta-lactoglobulin
6FXB 2018-05-23 2.0 Bovine beta-lactoglobulin variant A at pH 4.0
3UEW 2011-11-09 2.0 Bovine beta-lactoglobulin complex with palmitic acid
6QPD 2019-02-27 2.0 Engineered beta-lactoglobulin: variant I56F
1QG5 2001-04-21 2.0 HIGH RESOLUTION CRYSTAL STRUCTURE OF THE BOVINE BETA-LACTOGLOBULIN (ISOFORM A)
3KZA 2010-12-22 2.0 Crystal structure of Gyuba, a patched chimera of b-lactglobulin
6RWQ 2019-06-19 2.05 Engineered beta-lactoglobulin: variant F105L in complex with myristic acid
2Q2M 2008-02-26 2.1 Beta-lactoglobulin (native)
3UEU 2011-11-09 2.1 Bovine beta-lactoglobulin complex with lauric acid
4DQ3 2012-02-29 2.1 Bovine beta-lactoglobulin complex with oleic acid
6RWP 2019-06-19 2.1 Engineered beta-lactoglobulin: variant L58F in complex with myristic acid
6RWR 2019-06-19 2.1 Engineered beta-lactoglobulin: variant M107L in complex with myristic acid
6GF9 2018-06-20 2.1 Thermodynamic, Crystallographic and Computational Studies of Non Mammalian Fatty Acid Binding to Bovine b-Lactoglobulin
4DQ4 2012-02-29 2.1 Bovine beta-lactoglobulin complex with linoleic acid
3UEX 2011-11-09 2.1 Bovine beta-lactoglobulin complex with stearic acid
6RYT 2019-06-26 2.1 Engineered beta-lactoglobulin: variant M107L
6RYS 2019-06-26 2.15 Engineered beta-lactoglobulin: variant F105L
6QPE 2019-02-27 2.2 Engineered beta-lactoglobulin: variant L58F
4IB9 2013-06-26 2.2 Bovine beta-lactoglobulin (isoform B) in complex with dodecyltrimethylammonium (DTAC)
4IB6 2013-06-26 2.2 Bovine beta-lactoglobulin (isoform A) in complex with lauric acid (C12)
3NPO 2010-07-14 2.2 Bovine beta lactoglobulin unliganded form
4Y0P 2015-07-01 2.2 Bovine beta-lactoglobulin complex with tetracaine (BLG-TET)
4IB7 2013-06-26 2.2 Bovine beta-lactoglobulin (isoform A) in complex with dodecyltrimethylammonium (DTAC)
1BSQ 1998-09-02 2.22 STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF POINT MUTATIONS OF VARIANTS A AND B OF BOVINE BETA-LACTOGLOBULIN
1BSO 1999-09-02 2.23 12-BROMODODECANOIC ACID BINDS INSIDE THE CALYX OF BOVINE BETA-LACTOGLOBULIN
1BSY 1999-01-27 2.24 STRUCTURAL BASIS OF THE TANFORD TRANSITION OF BOVINE BETA-LACTOGLOBULIN FROM CRYSTAL STRUCTURES AT THREE PH VALUES; PH 7.1
4IB8 2013-06-26 2.3 Bovine beta-lactoglobulin (isoform A) in complex with dodecyl sulphate (SDS)
6NKQ 2019-01-23 2.3 The structure of bovine beta-lactoglobulin in novel crystals grown at pH 3.8
5NUM 2018-04-04 2.3 Engineered beta-lactoglobulin: variant F105L-L39A in complex with chlorpromazine (LG-LA-CLP)
6GE7 2018-06-20 2.3 Thermodynamic, Crystallographic and Computational Studies of Non Mammalian Fatty Acid Binding to Bovine b-Lactoglobulin
4IBA 2013-06-26 2.3 Bovine beta-lactoglobulin (isoform B) in complex with dodecyl sulphate (SDS)
4OMW 2014-11-19 2.3 Crystal structure of goat beta-lactoglobulin (orthorhombic form)
4OMX 2014-11-19 2.3 Crystal structure of goat beta-lactoglobulin (trigonal form)
4Y0R 2015-07-01 2.3 Bovine beta-lactoglobulin complex with pramocaine crystallized from ammonium sulphate (BLG-PRM2)
1GX9 2002-06-13 2.34 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH RETINOIC ACID, TRIGONAL LATTICE Z
1GXA 2002-06-13 2.35 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH RETINOL AND PALMITIC ACID, TRIGONAL LATTICE Z
5HTE 2016-07-20 2.4 Recombinant bovine beta-lactoglobulin variant L1A/I2S (sBlgB#2)
3PH5 2011-09-21 2.4 Bovine beta lactoglobulin crystallized through ligandation of yttrium cations
4LZU 2014-08-06 2.4 Bovine beta-lactoglobulin crystallized in the presence of 2 mM zinc chloride
1GX8 2002-06-13 2.4 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH RETINOL, TRIGONAL LATTICE Z
2GJ5 2007-10-02 2.4 Crystal structure of a secondary vitamin D3 binding site of milk beta-lactoglobulin
4LZV 2014-08-06 2.44 Bovine beta-lactoglobulin crystallized in the presence of 20 mM zinc chloride
2BLG 1999-01-27 2.46 STRUCTURAL BASIS OF THE TANFORD TRANSITION OF BOVINE BETA-LACTOGLOBULIN FROM CRYSTAL STRUCTURES AT THREE PH VALUES; PH 8.2
5K06 2016-07-20 2.5 Recombinant bovine beta-lactoglobulin with uncleaved N-terminal methionine (rBlgB)
5HTD 2016-07-20 2.5 Recombinant bovine beta-lactoglobulin variant L1A/I2S with endogenous ligand (sBlgB#1)
6QI7 2019-01-30 2.5 Engineered beta-lactoglobulin: variant L39Y in complex with endogenous ligand
1B0O 1999-02-02 2.5 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH PALMITATE, LATTICE Z
2Q39 2008-02-26 2.5 Beta-lactoglobulin (low humidity)
5LKF 2016-08-17 2.5 Bovine beta-lactoglobulin complex with myristic acid at high pressure (0.55 GPa)
3PH6 2011-09-21 2.53 Bovine beta lactoglobulin crytsallized through ligandation of yttrium
3BLG 1999-01-27 2.56 STRUCTURAL BASIS OF THE TANFORD TRANSITION OF BOVINE BETA-LACTOGLOBULIN FROM CRYSTAL STRUCTURES AT THREE PH VALUES; PH 6.2
5NUJ 2018-04-04 2.6 Engineered beta-lactoglobulin: variant I56F-L39A in complex with chlorpromazine (LG-FA-CLP)
5Y5C 2018-08-08 2.61 Crystal structure of dha bind to bovine beta-lactoglobulin
5LKE 2016-08-10 2.8 Bovine beta-lactoglobulin complex with myristic acid, ambient pressure
2R56 2007-11-13 2.8 Crystal Structure of a Recombinant IgE Fab Fragment in Complex with Bovine Beta-Lactoglobulin Allergen
5IO5 2016-03-16 2.85 Unliganded form of bovine beta-lactoglobulin, ambient pressure
5IO7 2016-03-16 2.85 Bovine beta-lactoglobulin complex with dodecane at high pressure (0.43 GPa)
2Q2P 2008-02-26 2.96 Beta-lactoglobulin (reverse native)
2AKQ 2005-08-16 3.0 The structure of bovine B-lactoglobulin A in crystals grown at very low ionic strength

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.7 Beta-lactoglobulin P67975 LACB_OVIMU
95.7 Beta-lactoglobulin P67976 LACB_SHEEP
96.3 Beta-lactoglobulin P02756 LACB_CAPHI
98.8 Beta-lactoglobulin P02755 LACB_BUBBU
100.0 Beta-lactoglobulin P02754 LACB_BOVIN