Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase.
Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.
Zhang
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Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Gray
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D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002942:Circular Dichroism; D017364:Collagenases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D005978:Glutathione