Characterization of a lidless form of the molecular chaperone DnaK: deletion of the lid increases peptide on- and off-rate constants.

The C-terminal, polypeptide binding domain of the 70-kDa molecular chaperone DnaK is composed of a unique lidlike subdomain that appears to hinder steric access to the peptide binding site. We have expressed, purified, and characterized a lidless form of DnaK to test the influence of the lid on the ATPase activity, on interdomain communication, and on the kinetics of peptide binding. The principal findings are that loss of the lid creates an activated form of DnaK which is not equivalent to ATP-bound DnaK. For example, at 25 degrees C the NR peptide (NRLLLTG) dissociates from the ADP and ATP states of DnaK with observed off-rate constants of 0.001 and 4.8 s(-1), respectively. In contrast, for DnaK that lacks most of the helical lid, residues 518-638, the NR peptide dissociates with observed off-rate constants of 0.1 and 188 s(-1). These results show that the loss of the lid does not interfere with interdomain communication, that the beta-sandwich peptide binding domain can exist in two discrete conformations, and that the lid functions to increase the lifetime of a DnaK.peptide complex. We discuss several mechanisms to explain how the lid affects the lifetime of a DnaK.peptide complex.

Peptide-induced conformational changes in the molecular chaperone DnaK.

DnaK, the 70 kDa molecular chaperone of Escherichia coli, adopts a high-affinity state in the presence of ADP that tightly binds its target peptide, whereas replacement of ADP by ATP induces a structural switch to a low-affinity chaperone state that weakly binds its target. An approximately 15% decrease in tryptophan fluorescence of DnaK occurs in concert with this switch from the high- to low-affinity state. The reversibility of this structural transition in DnaK was investigated using rapid mixing and equilibrium fluorescence methods. The Cro peptide (MQERITLKDYAM) was used to mimic an unfolded substrate. When the Cro peptide is rapidly mixed with preformed low-affinity DnaK complexes (DnaK-ATP), a rapid increase (kobs = 3-30 s-1) in the tryptophan fluorescence of DnaK occurs. We suggest that the Cro peptide induces the transition of the low-affinity state of DnaK back to the high-affinity state, without ATP hydrolysis. The combined results in this report are consistent with the minimal mechanism ATP + EP if ATP-EP if ATP-E + P, where ATP binding (K1) induces a conformational change and concerted peptide release (koff), and peptide binding (kon) to the low-affinity state (ATP-E) induces the transition back to ATP-EP, a high-affinity state. At 25 degreesC, in the presence of the Cro peptide, values for K1, koff, and kon are 22 microM, 3.3 s-1, and 2. 4 x 10(4) M-1 s-1, respectively. Evidence for an equilibrium between closed and open forms of DnaK in the absence of ATP and peptide is also presented.

Visualization of a slow, ATP-induced structural transition in the bacterial molecular chaperone DnaK.

Recent reports have shown that the binding of ATP to a 70-kDa molecular chaperone induces a rapid global conformational transition from a "high affinity" state to a "low affinity" state, where these states are defined by tight and weak binding to (poly)peptides, respectively. To complete the activity cycle, a chaperone molecule must ultimately return to the high affinity state. In this report, this return to the high affinity state was studied using a chemical cross-linking assay in conjunction with SDS-polyacrylamide gel electrophoresis. The basis for this assay is that in the absence of nucleotide or in the presence of ADP, conditions that stabilize the high affinity state, cross-linking of the Escherichia coli molecular chaperone DnaK yielded two monomeric forms, with apparent molecular masses of 70 kDa (77%) and 90 kDa (23%), whereas cross-linking yielded only the 70-kDa monomeric form in the presence of ATP. This ATP-dependent difference in cross-linking was used to follow the kinetics of the low affinity to high affinity transition under single turnover conditions. The rate of this transition (kobs = 3.4 (+/-0.6) x 10(-4) s-1 at 25 degrees C) is almost identical to the reported rate of ATP hydrolysis (khy = 2.7 (+/-0.7) x 10(-4) s-1 at 22 degrees C). These results are consistent with a two-step sequential reaction where rate-limiting ATP hydrolysis precedes the conformational change. Models for the formation of two cross-linked DnaK monomers in the absence of ATP are discussed.

Kinetics of the reactions of the Escherichia coli molecular chaperone DnaK with ATP: evidence that a three-step reaction precedes ATP hydrolysis.

The mechanism of the ATPase cycle of the 70-kDa Escherichia coli molecular chaperone DnaK was investigated by following ATP-induced changes in the tryptophan fluorescence of DnaK. Three steps in the cycle were investigated. (i) Stopped-flow experiments revealed that ATP induces a biphasic reduction in the tryptophan fluorescence of DnaK. The rate of the fast fluorescence transition exhibited a hyperbolic dependence on the ATP concentration, with a maximum rate equal to 56 (+/- 10) s-1 at 35 degrees C, whereas the rate of the slow fluorescence transition was nearly independent of the ATP concentration (4.2 +/- 0.2 s-1). These results are consistent with the three-step sequential reaction E + ATP<-->E-ATP<-->E*-ATP<-->E**-ATP prior to DnaK-catalyzed ATP hydrolysis, where the formation of a collisional complex (E-ATP) causes no change in fluorescence but is followed by two first-order transitions that reduce the fluorescence. (ii) The kinetics of ADP replacement from preformed DnaK-ADP complexes by ATP followed simple exponential kinetics, kADP = 0.038 (+/- 0.002) s-1 at 35 degrees C. The ADP off rate was reduced approximately 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 microM) revealed a slow, first-order increase in tryptophan fluorescence [k(obs) = 0.0015 (+/- 0.0001) s-1, 37 degrees C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k(hy) = 0.0014 (+/- 0.0001) s-1, 37 degrees C]. This slow increase in fluorescence is consistent with a E**-->E conformational transition. A model for the ATPase cycle of DnaK is proposed in which ATP has two distinct functions: ATP binding to the ATPase domain triggers two conformational transitions in a chaperone molecule, and ATP hydrolysis--the slow step in the reaction cycle--reverses the transitions.

Reactions of protamine with the molecular chaperone DnaK.

Molecular chaperones of the 70 kDa family mediate protein-protein interactions by selectively binding to partially unfolded segments of other proteins in an ATP-dependent activity cycle. Previous investigations of chaperone substrate selectivity have shown that chaperones have a propensity to bind to partially unfolded segments of polypeptides that contain bulky hydrophobic residues. However, recent investigations have shown that 70 kDa chaperones such as DnaK, which is expressed by Escherichia coli, also bind short basic peptides and even polycations. We report here that DnaK specifically binds to the polycation protamine when [protamine]/[DnaK] is near unity, whereas protamine induces the aggregation of DnaK when [protamine]/[DnaK] > or = 20. Complexes between DnaK and protamine were detected using fluorescently labeled protamine (protamine*) in conjunction with high performance size exclusion chromatography. We found that: (i) an unlabeled peptide of known affinity for DnaK partially inhibited the formation of DnaK-protamine* complexes; (ii) Mg-ATP (and Mg-gamma-S-ATP) significantly reduced the affinity of protamine* for DnaK; and (iii) the rate of DnaK-protamine* complex dissociation is highly temperature-sensitive, with apparent activation enthalpies (delta H*) equal to 32 +/- 4 and 28 +/- 1 kcal mol-1 in the absence of added nucleotide and in the presence of ADP, respectively. The results are consistent with the specific binding of protamine* at the (poly)peptide binding site of DnaK. A model is proposed to account for the protamine-induced aggregation of DnaK.

[Degree of structural homology of lactoferrins from milk and neutrophilic granulocytes]. / O stepeni strukturnoi gomologii laktoferrinov moloka i neitrofil'nykh granulotsitov.

Some physico-chemical properties of human and pig lactoferrins from milk and neutrophilic granulocytes were compared. It was shown that the lactoferrins from different cell and tissue sources of the same species (humans or pigs) are identical in terms of electrophoretic mobility, molecular weight, iron-binding capacity, absorbance spectra, amino acid and sugar compositions and peptide maps. Human and pig lactoferrins show a high degree of structural homology (approximately 50%), but are immunochemically different.

[Isolation and physico-chemical properties of lactoferrin from swine neutrophils]. / Poluchenie i nekotorye fiziko-khimicheskie svoistva laktoferrina i neitrofilov svin'i.

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.

[Comparison of various physico-chemical properties of pig and cattle myeloperoxidases]. / Sravnitel'noe issledovanie nekotorykh fiziko-khimicheskikh svoistv mieloperoksidaz svin'i i korovy.

The myeloperoxidases possessing the specific activity of 500 000-700 000 of o-dianisidine units per 1 mg of protein were isolated and purified from the lysosome-like granules of pig and cattle neutrophylic leukocytes. The absorbance spectra for the enzymes in the visible region are identical; their molecular weight is 140 000-160 000. The enzymes are made up of two subunits, each containing a heavy (m. w. 68 000) and a light (m. w. 10 000) polypeptide chains. The pH optimum for myeloperoxidase oxidation of o-dianisidine lies around 5.8-6.2 for both enzymes. The dependence of the reaction rate on H2O2 concentration does not obey the Michaelis--Menten kinetics. The optimal concentrations of the reaction substrates are 0.34 mM for o-dianisidine and 0.075 mM for H2O2. The amino acid composition and the peptide maps for pig and cattle myeloperoxidases are in many ways similar, showing no coincidence of enzymes, however, in terms of antigenic determinants. The similarities of some physico-chemical properties of pig and cattle enzymes and their immunological differences are discussed.