A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates.
The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30-75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.
Xu
Guoqiang
G
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
Narayan
Mahesh
M
Welker
Ervin
E
Scheraga
Harold A
HA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D004220:Disulfides; D011506:Proteins; D012259:Ribonuclease, Pancreatic