Tissue-type plasminogen activator domain-deletion mutant BM 06.022: modular stability, inhibitor binding, and activation cleavage.
Recombinant BM 06.022 (M(r) 39,589) is a domain-deletion mutant of the human tissue-type plasminogen activator (tPA) structured by the kringle 2 and protease modules. Unfolding under various conditions was investigated via 1H-NMR spectroscopy by monitoring the well-resolved high-field methyl resonances at approximately -0.97 ppm (kringle 2) and approximately -0.29 and -0.54 ppm (protease). Reversible acid/base unfolding is manifest under low pH (< 4.8) conditions. It is observed that, relative to the protease, the kringle exhibits higher overall stability at low pH. At pH 4.6, BM 06.022 undergoes two distinct thermal melting transitions, at approximately 334 and approximately 352 K, assigned to an irreversible denaturation of the protease and a reversible unfolding of the kringle 2, respectively. Under the same conditions, the protease reacted with the active site inhibitor 1,5 dansyl-L-glutamylglycyl-L-arginine chloromethyl ketone (EGRck) exhibits a higher (approximately 10 K) thermal stability than the inhibitor-free protease. Upon acidification, the EGRck-modified protease unfolds irreversibly around pH 3.4. As exemplified by BM 06.022, a single-chain protein, as defined by continuity of the polypeptide backbone, can exhibit simultaneous folding reversibility and irreversibility for autonomous segments of the sequence. Conversion of the isolated (single-chain) protease or intact BM 06.022 to their catalytically active two-chain forms via plasminolytic cleavage of the Arg275-Ile276 peptide bond leaves the kringle 2 spectrum unaffected while perturbing the resolved high-field methyl resonances stemming from the protease. The latter also shift when the protease is reacted with EGRck, indicating that these signals are sensitive to events at the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Kohnert
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Wilhelm
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Fischer
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Llinás
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D000143:Acids / Q000009:adverse effects; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D003619:Dansyl Compounds / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D006358:Hot Temperature; D018082:Kringles; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D000143:Acids; D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D011994:Recombinant Proteins; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; C087896:reteplase; D010959:Tissue Plasminogen Activator