Osmolyte Effects on the Unfolding Pathway ofβ-Lactoglobulin

There are large amounts of osmolytes inside cells,which impact many physiological processes by complicated mechanisms.The osmolyte effects on the stability and folding of proteins have been studied in detail using simple two-state folding proteins.However,many important functional proteins fold in complex pathways involving various intermediates.Little is known about the osmolyte effects on the folding and unfolding of these proteins.It is noted thatβ-lactoglobulin(BLG)is an example of such proteins,whose unfolding involves an obvious intermediate state.Using equilibrium chemical denaturation and stopped-flow kinetics,we investigate the unfolding of BLG in the presence of different osmolytes,e.g.,glycerol,ethylene glycol(EG)and poly(ethylene glycol)400(PEG400).It is found that all these osmolytes can stabilize the unfolding intermediate by modulating the relative unfolding kinetics of the native and the intermediate states.The stabilization effects are similar for EG and PEG400 but distinct for glycerol.Since the unfolding intermediates of many proteins are directly related to protein misfolding diseases,evaluation of the osmolyte effects for the unfolding of these proteins in vitro should be beneficial for the understanding of the occurrence of the related diseases in vivo.