Proteins can be self-assembled into beta-sheets.  This process is thermodynamically driven by protein-protein hydrophobic interactions in an aqueous environment.  An alpha-helix to beta-sheet conformation change has also been shown to be important in self-assembly.  Intra-molecular disulfide bonding between cysteine residues can prevent portions of the polymer chain from opening up and self-assembling.  Here, alpha-lactalbumin, a self-assembling protein, is treated with varying molar amounts of disulfide reducing agents beta-mercaptoethanol (BME) and dithiothreitol (DTT).  Alpha-lactalbumin is 34% alpha-helix.  We have previously shown that it is the hydrophobic regions of the alpha helices that are important in self-assembly.  Fourier transform infrared (FTIR) spectroscopy shows that 125% DTT relative to the molar disulfide amount in the protein results in the greatest conversion of alpha-helix to beta-sheet.  Use of reducing agent yields more conversion of alpha-helix to beta-sheet than in alpha-lactalbumin without reducing agent.  DTT appears to be a more efficient reducing agent than BME.  Atomic force microscopy (AFM) is used to image the beta-sheet structures.  The AFM tip is immersed in the beta-sheets and retracted and the retraction force used as a measure of the level of molecular aggregation.

Role of disulfide bonding in protein aggregation

253rd National Meeting (2017)

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