Conformational stability of secretory leucocyte protease inhibitor: a protein with no hydrophobic core and very little secondary structure.
Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymotrypsin. The EMBO J. 7, 345-352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (DeltaG(H20)) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.
Lin
Curtis C-J
CC
Research Center for Protein Chemistry, Institute of Molecular Medicine and the Department of Biochemistry and Molecular Biology, The University of Texas, 2121 W. Holcombe Blvd., Houston, TX 77030, USA.
Lu
Bao-Yuan
BY
Chang
Jui-Yoa
JY
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D017879:Isothiocyanates / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D053481:Secretory Leukocyte Peptidase Inhibitor; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D006146:Guanidines; D017879:Isothiocyanates; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011994:Recombinant Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; C054435:guanidine isothiocyanate; D003553:Cystine; D014508:Urea; D019791:Guanidine; D003545:Cysteine; D004229:Dithiothreitol