Temperature profiling of polypeptides in reversed-phase liquid chromatography: II. Monitoring of folding and stability of two-stranded α-helical coiled-coils

• Published in: Journal of Chromatography A Vol. 1009; no. 1; pp. 45 - 59
• Main Authors: Mant, Colin T., Tripet, Brian, Hodges, Robert S.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 15.08.2003; Elsevier
• Subjects: Amino Acid Sequence; Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, High Pressure Liquid; Fundamental and applied biological sciences. Psychology; Molecular Sequence Data; Peptides; Peptides - chemistry; Polypeptides; Protein Conformation; Protein Folding; Proteins; Temperature; Coiled-coils; Polypeptides; Peptides; Temperature effects; Amphipathic compound; Structure stability; Coiled structure; Chromatographic retention; HPLC chromatography; Temperature effect; Conformational changes; Separation method; Reversed phase chromatography; Synthetic product; Disulfide bond; Polypeptide; Helical structure; Folding
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(03)00919-1

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 α-helical coiled-coils. Thus, we have compared the effect of temperature on the RP-HPLC retention behaviour of both oxidized (two identical five-heptad α-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 °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.