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.
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301, USA.
Guoqiang
G
Xu
Mahesh
M
Narayan
Ervin
E
Welker
Harold A
HA
Scheraga
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