Effect of temperature on the secondary structure of β-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis

Abstract

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Effect of Heparin on Protein Aggregation: Inhibition versus Promotion

“…Although there are minor discrepancies in the mechanisms of thermal BLG aggregation proposed in the literature, there is wide agreement about the initial steps in thermal aggregation, that is, that BLG loses on the order of 90% of its native structure within 12 to 15 min at T > T m . 74−77 Although the nature of ultimate or intermediate species may be sensitive to conditions, it is apparent that the aggregation of denatured protein is initiated by the rate-determining dissociation of dimer to monomer, 76,78 the species considered most suscep- tible to rapid and irreversible unfolding above T m . Conversion of dimer to monomer, in principle an equilibrium, is kinetically controlled by the slow conversion of unfolded monomer to aggregate.…”

New insight into the pH‐dependent conformational changes in bovine β‐lactoglobulin from Raman optical activity

“…As well as changes in oligomerization, b-lactoglobulin also undergoes interesting conformational changes with changes in pH, temperature and concentration which have attracted attention due to their possible relevance to general problems in protein folding, function, and design~Molinari et al., 1996;Hoffmann et al, 1997;Ikeguchi et al, 1997;Qi et al, 1997;Ragona et al, 1997!. The most important of the pH-induced conformational changes shown by b-lactoglobulin is a reversible transition, first detected by Tanford et al~1959!…”

β-Lactoglobulin Binds Palmitate within Its Central Cavity

“…Thus, many studies on the pure protein are being directed toward unraveling the molecular mechanisms that are responsible for its thermal denaturation because this denaturation is thought to initiate the wider aggregation of milk proteins during thermal processing (9 -12). The initial stage of the thermal denaturation process also involves dimer dissociation (12,13).…”