[Visualization of radiographically occult osteochondrosis dissecans of the talus using MRI]. / Nachweis einer radiographisch okkulten Osteochondrosis dissecans tali mit der MRT.

Posttraumatic disorders of the ankle are a common cause of chronic pain. Magnetic resonance imaging (MRI) has proved to be highly useful in clarifying a wide spectrum of underlying lesions which frequently cannot be detected on radiographs. Even if the assessment of the lateral collateral ligaments of the ankle is not regarded as a primary indication for MRI, it allows the diagnosis of subchondral contusions or cysts, anterolateral impingement syndrome, sinus tarsi syndrome, osteochondral fractures and osteochondrosis dissecans of the talus (ODT),which may not be detected with other imaging modalities. A 42 year old female patient suffered from persisting non-specific pain following an inversion trauma 4 months previously. MRI enabled the detection of an ODT which was not diagnosed on plain radiographs and which was verified upon arthroscopy. A superficial cartilage defect, as shown arthroscopically,was not delineated using MRI. Our observations indicate that MRI may be useful in patients with unclear persisting pain following ankle trauma and that it may contribute to the early detection of lesions which require surgical intervention.

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.

GrpE accelerates peptide binding and release from the high affinity state of DnaK.

The Escherichia coli nucleotide exchange factor GrpE accelerates the rate of ADP dissociation from high affinity ADP-DnaK, thus enabling ATP binding and transition to the low affinity state. We show here that GrpE, in the absence of ATP, accelerates the rates of the forward and reverse reaction ADP-DnaK-P right harpoon over left harpoon ADP-DnaK + P, where P denotes peptide substrate. Specifically, the binding of GrpE to an ADP-DnaK-P (or DnaK-P) complex increases koff and kon by approximately 200-fold and approximately 60-fold, respectively. The results are consistent with a GrpE- induced conformational change in the C-terminal polypeptide binding domain of an ADP-DnaK molecule, which results in a unique low affinity intermediate from which peptide can dissociate. A simulation of peptide dissociation from DnaK as a function of the [ATP] / [ADP] ratio shows that GrpE induced peptide dissociation from ADP-DnaK is important at elevated cellular concentrations of ADP, which typically occur upon stress.

ATP lowers the activation enthalpy barriers to DnaK-peptide complex formation and dissociation.

The role of nucleotide in controlling the pre-steady-state kinetics of peptide binding to the Escherichia coli 70-kDa molecular chaperone DnaK was investigated using stopped-flow fluorescence. The peptide used in this study, fVSV13 (representing amino acids 490-502 of the vesicular stomatitis virus glycoprotein), was dansylated specifically at its N-terminus. We found that (i) between 17 and 35 degrees C in the presence of ATP the second-order rate constant (k(on)) for fVSV13 binding to DnaK exhibited almost no dependence on temperature and did not deviate significantly from 3.8 x 10(5) M(-1) s(-1). In contrast, over the same temperature range in the presence of ADP, k(on) increased by a factor of 32 (7.3 x 10(4) to 2.3 x 10(6) M(-1) s(-1)); and (ii) ATP increased the apparent first-order rate constant for the release of fVSV13 from preformed DnaK-fVSV13 complexes by several orders of magnitude relative to ADP. The activation energy parameters for fVSV13 binding to and dissociation from DnaK are compared to the activation parameters for the binding of an unrelated peptide to DnaK and are also discussed in terms of an open-to-closed equilibrium in the polypeptide-binding domain. On the basis of this comparison, it is suggested that the activation entropy term deltaS++, which is related to the structure of the DnaK-bound peptide or the degree of solvation of the peptide, is a controlling factor in the kinetics of peptide binding to DnaK.

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.

Kinetic evidence for peptide-induced oligomerization of the molecular chaperone DnaK at heat shock temperatures.

The pre-steady-state kinetics of the binding of a fluorescent peptide (dansyl-KLIGVLSSLFRPK, fVSV13) to the Escherichia coli molecular chaperone DnaK were investigated over a range of temperatures (25-42 degrees C). At 42 degrees C, over a wide range of peptide concentrations, the fVSV13 peptide bound to DnaK with biphasic kinetics: a rapid burst in the DnaK-fVSV13 signal in the first 5 s was followed by a gradual reduction in the signal over the next 100 s. The descending portion of each biphasic trace followed the equation F(t) = DeltaF exp(-kdt) + Finfinity, where DeltaF, kd, and Finfinity are the amplitude, the apparent first-order rate constant, and the fluorescence end point, respectively. Both DeltaF and kd increased with increasing concentrations of DnaK, which suggests that the loss of the DnaK-fVSV13 signal is caused by a bimolecular reaction. We propose that (i) the fVSV13 peptide binds to and induces a conformational change in the DnaK monomer [E + P right harpoon over left harpoon (EP)*]; and (ii) the conformational change promotes the formation of oligomeric DnaK-peptide complexes [En + (EP)* right harpoon over left harpoon En-EP]. The term (EP)* denotes a monomeric DnaK-peptide complex in which the bound peptide is fluorescent; En-EP denotes an oligomeric DnaK-peptide complex in which the fluorescence of the bound peptide is quenched. Numerical fitting of the stopped-flow data to reactions (i) and (ii) yielded values for the four rate constants. When the proposed kinetic model was tested by conducting experiments in the presence of excess peptide or excess ATP&sbd;conditions which inhibit oligomerization&sbd;DnaK-fVSV13 complex formation proceeded to stable asymptotes, with no reduction in the DnaK-fVSV13 signal at long times.

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.

Large activation energy barriers to chaperone-peptide complex formation and dissociation.

To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.

Inhibition of class II MHC-peptide complex formation by protease inhibitors.

Studies on the kinetics of antigenic peptide binding to major histocompatibility complex class II molecules have been used extensively to probe major histocompatibility complex (MHC) structure as well as to investigate the molecular mechanism of peptide recognition. Previous experiments have frequently been carried out in the presence of a cocktail of protease inhibitors to inhibit the proteolysis of MHC heterodimers. By using high performance size exclusion chromatography to measure fluorescent peptide binding to MHC protein, we have found that the addition of a commonly used mixture of protease inhibitors leads to a significant reduction in peptide binding to the class II heterodimer.

Formation and dissociation of short-lived class II MHC-peptide complexes.

The incubation of a detergent-solubilized class II MHC protein with excess peptide at 37 degrees C leads to the formation of long-lived protein-peptide complexes (alpha beta P*), which have reported dissociation half-times at 37 degrees C from 30 to 100 h (alpha beta P*-->alpha beta + P*). Here we report an unexpected temperature effect on the reaction between class II MHC and added peptide. When the detergent-solubilized mouse class II MHC protein I-Ad is incubated with excess labeled peptide at 4 degrees C, a large fraction of the resultant complexes are relatively short-lived, with dissociation half-times at 40 degrees C from 2 to 0.2 h. Short-lived complexes formation and dissociation are both characterized by nonexponential kinetics. Short-lived I-A(d)-peptide complexes may contain two peptides, where the second, added fluorescent peptide is prevented from utilizing all the potential intermolecular interactions in the binding site due to the prior partial occupation of the binding site by a prebound peptide.

A first-order reaction controls the binding of antigenic peptides to major histocompatibility complex class II molecules.

Major histocompatibility complex class II molecules have been reported to bind antigenic peptides very slowly in vitro. To investigate the molecular events that govern the slow binding reaction, we have determined the dependence of complex formation and dissociation on peptide concentration. The complex between the purified major histocompatibility complex class II protein I-Ek and a fluoresceinated peptide representing amino acids 89-104 of pigeon cytochrome c (FpCytc) was studied. Two important results emerge from this study. (i) At pH 5.4, the half-time for I-Ek-FpCytc complex formation is equal to approximately 7 hr for peptide concentrations that vary over a range of three orders of magnitude. There is in fact a small but significant decrease in the half-time for complex formation at low peptide concentrations. The small decrease in half-time is related to the release of endogenous peptides. (ii) At large ratios of peptide to protein [( FpCytc]/[I-Ek] greater than 40), the half-times for I-Ek-FpCytc complex formation and dissociation are equal to one another to within a factor of two between pH 7.5 and 4.5. The percent results demonstrate that a slow, first-order reaction precedes complex formation between I-Ek and FpCytc. This first-order reaction may involve a protein conformational change in addition to the release of endogenous peptides.

Room temperature characterization of the dioxygen intermediates of cytochrome c oxidase by resonance Raman spectroscopy.

Resonance Raman spectroscopy was employed to investigate the heme structures of catalytic intermediates of cytochrome c oxidase at room temperature. The high-frequency resonance Raman spectra were obtained for compound C (the two-electron-reduced dioxygen intermediate), ferryl (the three-electron-reduced dioxygen intermediate), and the fully oxidized enzyme. Compound C was formed by photolyzing CO mixed-valence enzyme in the presence of O2. The ferryl intermediate was formed by reoxidation of the fully reduced enzyme by an excess of H2O2. Two forms of the oxidized enzyme were prepared by reoxidizing the fully reduced enzyme with O2. Our data indicate that, in compound C, cyt a3 is either intermediate or low spin and is nonphotolabile and its oxidation state marker band, v4, appears a higher frequency than that of the resting form of the enzyme. The ferryl intermediate also displays a low-spin cyt a3, which is nonphotolabile, and an even higher frequency for the oxidation state marker band, v4. The reoxidized form of cytochrome c oxidase with a Soret absorption maximum at 420 nm has an oxidation state marker band (v4) in a position similar to that of the resting form, while the spin-state region resembles that of compound C. This species subsequently decays to a second oxidized from of the enzyme, which displays a high-frequency resonance Raman spectrum identical with that of the original resting enzyme.

Evidence for a ferryl Fea3 in oxygenated cytochrome c oxidase.

Evidence is reported which shows that a reactive ferryl Fea3/cupric CuB binuclear couple is present at the dioxygen reduction site in "oxygenated" cytochrome c oxidase; when the fully reduced enzyme is reoxidized at low temperatures; and when partially reduced cytochrome c oxidase is reoxidized with dioxygen at room temperature.

Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase.

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.