Temperature profiling of polypeptides in reversed-phase liquid chromatography. II. Monitoring of folding and stability of two-stranded alpha-helical coiled-coils.
The present study extends the utility of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor folding and stability of de novo designed synthetic two-stranded alpha-helical coiled-coils. Thus, we have compared the effect of temperature on the RP-HPLC retention behaviour of both oxidized (two identical five-heptad alpha-helical peptides linked by a disulfide bridge) and reduced coiled-coil analogues with various amino acids substituted into the hydrophobic core of the coiled-coil. We were able to correlate the RP-HPLC retention behaviour of the oxidized analogues over the temperature range of 10 to 80 degrees C with the stability of the analogues as determined by conventional thermal and chemical denaturation approaches. In addition, the contribution of a disulfide bridge to coiled-coil stability was highlighted by comparing the elution behaviour of the oxidized and reduced analogues. Overall, we demonstrate the excellent potential of "temperature profiling" by RP-HPLC to monitor differences in oligomerization state and protein stability.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D013696:Temperature
D010455:Peptides