An infrared spectroscopic study of β-galactosidase structure in aqueous solutions

AbstractFourier-transform infrared spectroscopy has been used to elucidate the secondary structure of E. coli β-galactosidase in aqueous solution. The structure of this enzyme was previously unknown above the level of the amino acid sequence. Spectra have been recorded in both H2O and D2O media; mutually complementing data are obtained, that provide unambiguous structural information. The results show that β-galactosidase contains 40% β-sheet and 35% α-helical structure, with smaller proportions of random coil (12%) and β-turns (13%)

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel ß-sheet a-synuclein aggregates

a-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson 's disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel ß-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that a-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel ß-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside a-synuclein droplets generated by liquid-liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo a-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation