The L17 ribosomal protein of Bacillus subtilis binds preferentially to curved DNA.

We searched in Bacillus subtilis for proteins that bind preferentially to curved DNA. Two proteins of 9 and 15 kDa displaying this property were purified from exponentially growing cells of B. subtilis strain 168. Sequencing of N-terminal amino acids identified them as the proteins HBsu and L17 respectively. The overproduction of L17 from B. subtilis in Escherichia coli was shown to have a strong effect on nucleoid morphology and segregation.

Conformational dynamics and enzyme activity.

Conformational flexibility and structural fluctuations play an important role in enzyme activity. A great variety of internal motions ranging over different time scales and of different amplitudes are involved in the catalytic cycle. These different types of motions and their functional consequences are considered in the light of experimental data and theoretical analyses. The conformational changes upon substrate binding, and particularly the hinge-bending motion which occurs in enzymes made of two domains, are analyzed from several well documented examples. The conformational events accompanying the different steps of the catalytic cycle are discussed. The last section concerns the motions involved in the allosteric transition which regulates the enzyme activity.

Cleavage points of rabbit skeletal myosin light chains selectively modified in situ by limited proteolysis: structural characteristics of the neoformed isozymes.

The functional significance of myosin light chains in vertebrate striated muscle is an issue of interest any myosin species selectivity modified by papain or trypsin in their LC1 and LC2 light chains are potentially useful for further investigation. We therefore determined the cleavage sites resulting in the (T)-LC1', (P)-LC1' and (T)-LC2'species. Sequence analysis of (T)-LC1' indicated that the cleavage point in LC1 is at Lys7. Under appropriate conditions papain rapidly cleaves a short N-terminal segment from myosin light chain 1 and produces a new isozyme specifically modified in its essential light chain 1. The cleavage occurred at either Ala11, Ala12, or Ala13, the Ala11 cleavage being the most frequent. Trypsin was used to produce a myosin species with a regulatory light chain 2 specifically truncated of a short N-terminal segment. The cleavage was specific at Arg8 with no indication of other significant cleavage sites in this LC2. The effects of trypsin and papain on myosin light chains are different, indicating different proteolytic specificities. None of these modifications, including (CT)-LC2" cleavage at Phe19, changed the K(+)-EDTA- and Ca(2+)-ATPase activities of monomeric myosin significantly, indicating that LC1 and LC2 N-terminal have little or no direct influence on the active site. An electric birefringence study also showed that these modified species retained their average shape and flexibility. These observations are essential in showing that the role of light chain extremities is expressed only in the presence of a minimum of structural organization (filament or acto-myosin complex).

Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70.

The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 11 of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.

Conformational stability of the covalent complex between elastase and alpha 1-proteinase inhibitor.

The equilibrium unfolding-refolding process of the elastase-alpha 1-proteinase inhibitor complex, induced by guanidinium chloride, was followed by spectroscopic methods. A reversible transition with a midpoint at 2.04 +/- 0.04 M guanidinium chloride was observed by fluorescence. This transition was attributed to elastase on the basis of circular dichroism and uv absorption difference data obtained for the covalent complex and for the free proteins. The conformational stability of elastase in the complex was analyzed considering the approximation of a two-state transition. The free energy of denaturation delta GH2O was 4.2 kcal.mol-1 for complexed elastase compared to 10.5 kcal.mol-1 for the free enzyme. Such a decrease in the stability of elastase suggests that, after forming the covalent complex with the inhibitor, the enzyme undergoes not only the expected local modifications of the active site, but also an extensive structural reorganization.

[Structural aspects of the interaction between elastase and macromolecular inhibitors and of the regulation of its activity]. / Aspects structuraux de l'interaction de l'élastase avec des inhibiteurs macromoléculaires et de régulation de son activité.

Conformational states of elastase complexed with macromolecular inhibitors were studied. Chemical reactivities of amino acid side-chains were used as conformational probes providing information on the structure of neighboring regions of the polypeptide chain. Variations in the chemical reactivity of ionizable groups of elastase and anhydroelastase complexed with alpha-1 antiprotease and ovomucoid were evidenced for segments 27-36, 42-53 and 118-136, which are not directly involved in interactions with the inhibitor. Studies of the denaturation of these complexes using differential chemical labeling showed that these conformational modifications were responsible for a decrease in overall stability of the enzyme complexed with the inhibitors. The magnitude of this decrease was estimated at 5 Kcal/mole. This finding suggests that interactions at the active site also induced changes in overall conformation of the molecule.

Conformational changes in intact and papain-modified alpha 1-proteinase inhibitor induced by guanidinium chloride.

Equilibrium unfolding-refolding processes of active and proteolytically modified alpha 1-proteinase inhibitor induced by guanidinium chloride were studied. Spectroscopic methods of ultraviolet absorption, fluorescence emission and circular dichroism were used. The functional inhibitor unfolds following a multistate process: a first transition (midpoint at 0.6 M guanidinium chloride) was observed whatever the method used and was attributed to a limited conformational modification of the region including the two tryptophan residues. At higher denaturant concentrations, two other transitions were observed, one in fluorescence (midpoint at 1.7 M guanidinium chloride), attributed to the unfolding of the polypeptide chain in the same region and the other one, observed in circular dichroism and in ultraviolet absorption (midpoint at 2.3 M guanidinium chloride), leading to the totally unfolded protein. Evidence for several intermediates was also obtained with the proteolytically modified inhibitor. If total unfolding is considered, the modified inhibitor was found to be more stable towards the denaturant than the functional form (obtained at 5.5 M and 3.5 M guanidinium chloride, respectively). The unfolding irreversibility observed was attributed to the C-terminal fragment Ser359-Lys394 associated with the main chain of the cleaved inhibitor.

Transient conformational states in proteins followed by differential labeling.

Refolding of previously denatured and reduced elastase has been followed by titration of chemical reactivities of amino acid side chains to study the topography of the protein in the native state, and the microenvironment variations of protein side chains during the structural transition. Groups accessible to chemical reagents in the denatured form and buried in the "native" form were used as a local conformational probe. Times of labeling, depending on the reagent used, ranged from 100 to 800 ms. The reaction was stopped by isotopic dilution with an excess of unlabeled reagent under denaturing conditions to obtain a chemically homogeneous but heterogeneously labeled material. Peptide fractionation after degradation of the labeled proteins allowed the determination of the amount of radioactive label incorporated by the individual side chains during the refolding. Refolding rates, determined by physicochemical, enzymatic or immunochemical criteria, were compared with the conformational states of protein areas and evaluated by the variation of chemical reactivity at various denaturant concentrations. The importance of the last folding stages is emphasized by the results obtained which indicate that early during the refolding, two domain substructures (H-40 to H-71 and M-180 to H-200)( are stabilized, while the protein remains inactive at the time ranges of the labeling reactions.

[Conformational coupling between structural units. A decisive step in the functional structure formation]. / Couplage conformationnel entre unités structurales. Etape décisive dans la formation de la structure fonctionnelle des protéines.

Proteins built up of several structural domains are compared with oligomeric proteins made up of several subunits. On this basis the functional consequences of a conformational coupling between domains are analyzed. From the data obtained on the elastase molecule it is proposed that the last step in protein folding insures the optimal coupling between domains and the energy of interaction is utilized for catalysis.

Binding of nucleotides ATP and ADP to sarcoplasmic reticulm: study by rate of dialysis.

Binding of the nucleotides ATP and ADP by preparations of sarcoplasmic reticulum was investigated by the method of flow dialysis. For ATP, experimental data could not be analyzed directly in terms of binding since a significant though small amount of hydrolysis could be observed even in presence of EDTA. ADP binding could be analyzed and gave a dissociation constant of 10-20 muM at neutral pH, and a stoichiometry of 0.35 - 0.45 per mole ATPase. The possible significance of this stoichiometry is discussed. Similar experiments were performed after ethoxyformylation of sarcoplasmic reticulum which inhibits the Ca2+ dependnet ATPase activity. The results confirmed the inhibition of ATP hydrolysis and pointed to a considerably reduced affinity for nucleotides. The method based on the measurement of dialysis rates is convenient, and accurate enough to detect the effects of chemical modification on sarcoplasmic reticulum membranes.

The conformational oscillation of delta-chymotrypsin involvement of methionine-192.

In delta-chymotrypsin the reactivity of methionine-192 towards p-nitrophenacyl bromide is strongly reduced when the alpha-amino group of isoleucine-16 has been acetylated. Since acetylation of isoleucine-16 brings delta-chymotrypsin to a conformation similar to its alkaline one this suggests that methionine-192 should present an impaired reactivity in the alkaline conformation of the protein. It is indeed observed that its chemical reactivity as a function of pH depends on the ionization state of the alpha-amino group of isoleucine-16 (pKapp 9 at 15 degrees C) as does the structure of the enzyme. Reciprocally, after chemical reaction of methionine-192 with hydrogen peroxide, isoleucine-16 presents a slower rate of reaction with fluorescamine than when methionine-192 is free. As a result of methionine-192 oxidation the apparent pK of the alkaline transition is shifted from 9 to about 11 at 15 degrees C. This is reflected in the disappearance of the lag phase previously observed for the initial activity of the enzyme when it is incubated at alkaline pH [Eur. J. Biochem. (1973) 39,293-300]. The absence of chemical reactivity of methionine-192 in the alkaline state of the enzyme is confirmed by the appearance of a lag phase in the reaction of the protein with iodoacetate after an incubation at alkaline pH. Such a lag phase does not appear when this incubation is carried out at neutral pH. Since this lag phase is similar to that which shows up in the activity during the isomerization of the enzyme from its alkaline to its neutral state, the present data are interpreted as implying a concerted movement of isoleucine-16 and methionine-192 during this isomerization process. They also indicate that in the alkaline form of the enzyme methionine-192 has moved back into the interior of the protein. Since the spectroscopic properties of the zymogen and of the high-pH form of the enzyme are similar they suggest that methionine-192 occupies in the alkaline conformation of the enzyme a similar position as it does in the zymogen.