Conformational impurity of disulfide proteins: detection, quantification, and properties.
The conformations of native proteins are in principle, and in most cases, dictated by the law of thermodynamics. Accordingly, a native protein must always exist in equilibrium with a minor concentration of nonnative (denatured) conformational isomers even at nondenaturing conditions. The presence of an infinitesimal quantity of nonnative conformational isomers at physiological conditions is biologically relevant due to their propensity to aggregate, which is an underlying cause of many neurodegenerative diseases. However, their detection and quantification are inherently difficult. In this article, we describe a simple strategy using the technique of disulfide scrambling to identify and quantify such minute concentrations of nonnative isomers. It is demonstrated that even for small stable proteins such as epidermal growth factor and hirudin, approximately 1% of heterogeneous nonnative isomers coexist with the native proteins under physiological conditions.
Chang
Jui-Yoa
JY
Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas, Houston, TX 77030, USA. rowen.chang@uth.tmc.edu
Lu
Bao-Yuan
BY
Li
Li
L
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002918:Chymotrypsin / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D006629:Hirudins / Q000097:blood / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020033:Protein Isoforms / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D006629:Hirudins; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins; C504439:hirudin (1-49), leech; D004815:Epidermal Growth Factor; D002918:Chymotrypsin; D014357:Trypsin