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
4CK4 2013-12-27T00:00:00+0000 1.12 Ovine beta-Lactoglobulin at Atomic Resolution
4NLI 2013-11-14T00:00:00+0000 1.9 Crystal structure of sheep beta-lactoglobulin (space group P3121)
4NLJ 2013-11-14T00:00:00+0000 1.4 Crystal structure of sheep beta-lactoglobulin (space group P1)
6T44 2019-10-12T00:00:00+0000 2.0 Ovine lactoglobulin complex with decanol
4OMW 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (orthorhombic form)
4OMX 2014-01-27T00:00:00+0000 2.3 Crystal structure of goat beta-lactoglobulin (trigonal form)
4TLJ 2014-05-30T00:00:00+0000 1.17 Ultra-high resolution crystal structure of caprine Beta-lactoglobulin
4Y0S 2015-02-06T00:00:00+0000 1.9 Goat beta-lactoglobulin complex with pramocaine (GLG-PRM)
7LWC 2021-02-28T00:00:00+0000 3.0 Goat beta-lactoglobulin mutant Q59A
1B0O 1998-11-11T00:00:00+0000 2.5 BOVINE BETA-LACTOGLOBULIN COMPLEXED WITH PALMITATE, LATTICE Z

Relevant UniProtKB Entries

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