We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T_m) and unfolding enthalpy (ΔH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than ≈ 1.5 mg·mL⁻¹, or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two‐state transitions, each of which compares well with the two‐state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time‐dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time‐dependent, high‐order aggregation may account for the observed effects. Limited‐proteolysis experiments suggest that in the aggregates the N‐terminal segment 1–63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N‐terminal segment 1–63 has a high propensity for β sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full‐length protein.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N‐terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C‐terminal “tail.” Cleavage within the N‐terminal domain generates an N‐terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Pig‐dependent activator activity was shown to be SK27 (mass 27, 000, residues 147–380), which contains both central and C‐terminal domains, although this activity was reduced approximately 6, 000‐fold relative to that of full‐length SK. The activity of a 36, 000 mass fragment, SK36 (residues 64–380), which differs from SK27 in possessing a portion of the N‐terminal domain, was reduced to 0.1–1.0% of that of SK. Other fragments (masses 7, 000, 11, 000, 16, 000, 17, 000, 25, 000, and 26, 000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1–63), at a 100‐fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50‐ and > 130‐fold, respectively. These findings demonstrate that all of SK's three domains are essential for native‐like SK activity. The central and C‐terminal domains mediate plasminogen‐binding and active site‐generating functions, whereas the N‐terminal domain mediates an activity‐potentiating function.