Generation of a family of protein fragments for structure-folding studies. 1. Folding complementation of two fragments of chymotrypsin inhibitor-2 formed by cleavage at its unique methionine residue.
The suitability of the barley chymotrypsin inhibitor-2 for study by fragmentation and complementation has been analyzed. The primary residue for binding to proteases, Met-59 (the unique methionine in the sequence), lies in a broad, solvent-exposed loop. The bond between Met-59 and Glu-60 was cleaved by cyanogen bromide. The two fragments thus obtained, i.e., CI-2(20-59) and CI-2(60-83), associate (KD = 42 nM) to yield a complex that has fluorescence and circular dichroism spectra identical to those of uncleaved chymotrypsin inhibitor-2. Recovery of native-like structure is further indicated by the ability of the complex to inhibit chymotrypsin, although the [I]50% is 140-fold higher than for the uncleaved inhibitor. CI-2(60-83) appears to be highly disordered in water, but fragment CI(20-59) forms significative structure, as judged by its circular dichroism spectra and evidence from one-dimensional NMR. The circular dichroism spectra of CI-2(20-59) approach the baseline in 4 M guanidinium chloride but display characteristics of an alpha-helix in the presence of trifluoroethanol. Analytical ultracentrifugation shows no concentration-dependent change in the molecular weight of the monomer of CI-2(20-59). Both one- and two-dimensional NMR of the complex [CI-2(20-59).(60-83)] show unequivocally the presence of a folded structure, which appears to be slightly different from the uncleaved native protein.
de Prat Gay
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Department of Chemistry, Cambridge University, U.K.
Fersht
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AR
D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature
D006146:Guanidines; D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D019791:Guanidine