We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA(+) proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease.

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Homooligomeric LonA proteases are the key components of the protein quality control system in bacteria and eukaryotes. Domain organization of the common pool of LonA proteases is determined by comparative analysis of primary and secondary structures of a number of bacterial and eukaryotic enzymes. The similarity of individual enzyme domains was estimated, domain-domain linker areas were revealed, regions that are capable to include intercalated peptide fragments were identified. LonA proteases were shown to be unique AAA+ proteins, because in addition to the classic AAA+ module they contain a part of another AAA+ module, namely the alpha-helical domain including a coiled-coil region, which is similar to the alpha-helical domain of the AAA(+)-1 module of the chaperone-disagregases ClpB/Hsp104.

Fibronectin from human plasma and its 180 kDa fragment which retained collagen-binding, cell-attachment and heparin-binding activities, were studied by velocity centrifvgation and 'H-NMR methods. The tibronectin hydrodynamic radius strongly increased at pH 11 while the hydrodynamic properties of the fragment did not change noticeably. 'H-NMR spectroscopy also showed differences in the molecular properties of fibronectin and its 180 kDa fragment. Under physiological conditions the structure of fibronectin differs from that of its 180 kDa fragment. At pH 11 and in 4 M urea no differences in their structures are observed. It is suggested that interdomain and intersubunit interactions play an important role in maintaining the native conformation of intact tibronectin. Fibronectin 180 kDa fibronectin fragment 'H-NMR

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