Reversible independent unfolding of the domains of urokinase monitored by sup 1 H NMR

• Published in: Biochemistry (Easton) 1989-08, Vol.28:16
• Main Authors: Bogusky, M.J., Dobson, C.M., Smith, R.A.G.
• Format: Article
• Language: English
• Subjects: 550201 - Biochemistry- Tracer Techniques; ANIMALS; BARYONS; BASIC BIOLOGICAL SCIENCES; CHEMICAL BONDS; CONFORMATIONAL CHANGES; DRUGS; ELEMENTARY PARTICLES; ENZYMES; FERMIONS; FIBRINOLYTIC AGENTS; GROWTH FACTORS; HADRONS; HEMATOLOGIC AGENTS; HYDROLASES; MAGNETIC RESONANCE; MAMMALS; MAN; MITOGENS; MOLECULAR STRUCTURE; NONSPECIFIC PEPTIDASES; NUCLEAR MAGNETIC RESONANCE; NUCLEONS; ORGANIC COMPOUNDS; PEPTIDE HYDROLASES; PH VALUE; PLASMINOGEN; PRIMATES; PROTEINS; PROTONS; RESONANCE; TEMPERATURE EFFECTS; UROKINASE; VERTEBRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00442a028

Human urinary-type plasminogen activator (urokinase) and proteolytic fragments corresponding to the kringle, EGF-kringle, and protease domains have been examined by {sup 1}H NMR spectroscopy. The intact protein shows a very well-resolved spectrum for a molecule of this size (MW 54,000), with resonance line widths not greatly increased from those of the isolated domains. On increasing the temperature, the protein at pH values close to 4 was found to undergo two distinct and reversible conformational transitions. These were identified, by comparison with spectra of the proteolytic fragments, as the unfolding of the kringle (and EGF) domains (at {approximately} 42{degree}C) and of a segment of the protease domain (at {approximately} 60{degree}C). The remaining segment of the protease domain showed persistent structure to at least 85{degree}C at pH 4; only at lower pH values could complete unfolding be achieved. The results indicate that the structures and stabilities of the isolated domains are closely similar to those in the intact protein and suggest that there is a degree of independent motion at least between the kringle and protease domains.