Backbone dynamics of escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by (15)N NMR relaxation.

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP --> 2ADP. Domains LID and AMPbd execute large-scale movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco were studied comparatively with (15)N NMR relaxation methods. Overall diffusion with correlation times of 15.05 (11.42) ns and anisotropy D(parallel)/D(perp) = 1.25 (1.10), and fast internal motions with correlation times up to 100 ps (50 ps), were determined for AKeco (AKecoAP(5)A). Fast internal motions affect 93% of the AKeco sites, with pronounced preference for domains AMPbd and LID, and 47% of the AKecoAP(5)A sites, with limited variability along the chain. The mean squared generalized order parameters, , of secondary structure elements and loops are affected by ligand binding differentially and in a domain-specific manner. Nanosecond motions predominate within AMPbd. Prominent exchange contributions, associated in particular with residue G10 of the nucleotide-binding P-loop motif, are interpreted to reflect hydrogen-bond dynamics at the inhibitor-binding site. The hypothesis of energetic counter balancing of substrate binding based on crystallographic data is strongly supported by the solution NMR results. Correlations between backbone dynamics and domain displacement are established.

Determination of intramolecular distance distribution during protein folding on the millisecond timescale.

A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).

Design consideration and probes for fluorescence resonance energy transfer studies.

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.

Towards a mechanism of AMP-substrate inhibition in adenylate kinase from Escherichia coli.

Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223-227). The method of time-resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)-AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by an approximately 9 A decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP-substrate inhibition known for E. coli AK.

Domain closure in adenylate kinase.

The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK). A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively. The latter was subsequently labeled with either 5- or 4-acetamidosalicylic acid (the acceptor). From the comparative analysis of AK crystal structures [Schulz, G.E., Müller, C.W., & Diederichs, K. (1990) J. Mol. Biol. 213, 627-630] (apo-AK,AK.AMP complex and AK.AP5A [P1,P5-di(adenosine-5') pentaphosphate] complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure. The domain closure reduces interdomain distances in a two-step manner. Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E. coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex. Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex. The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively. Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A. The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E. coli AK in solution.

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus.

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hydridization response in the microorganisms's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.

[Evidence of the existence of hemolysin II from Bacillus cereus: cloning the genetic determinant of hemolysin II]. / Dokazatel'stvo sushchestovovaniia gemolizina II Bacillus cereus: klonirovanie geneticheskoi determinanty gemolizina II.

The hemolysin genetic determinant distinct from cereolysin AB genetic determinant (lecithinase and sphingomyelinase genes) has been cloned in Escherichia coli and Bacillus subtilis cells as an EcoRI fragment (2.9 kb) of Bacillus cereus VKM-B771 chromosome DNA. The hemolytic product encoded by the cloned DNA fragment possessed all the properties of hemolysin II known to date: it was not inhibited by cholesterol, exhibited the Arrhenius effect, and had a relatively long (in comparison with cereolysin) lag period in erythrocyte lysis. The cloned DNA fragment was concluded to contain the gene of hemolysin II from B. cereus. In contrast to previous suggestions that hemolitic activity ascribed to hemolysin II is due to the combined action of sphingomyelinase and lecithinase, the results obtained present convincing evidence that hemolysin II is an independent B. cereus hemolytic factor different from cereolysin AB.

Refolding kinetics of pig muscle and yeast 3-phosphoglycerate kinases and of their proteolytic fragments.

The time course of refolding of both pig muscle and yeast 3-phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C-terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half-time between 80-120 s, at 20 degrees C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N-terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C-terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.

Reactivation of 3-phosphoglycerate kinase from its unfolded proteolytic fragments.

Limited trypsinolysis of pig muscle 3-phosphoglycerate kinase yielded a nicked enzyme without loss of catalytic activity [Jiang, S. X. & Vas, M. (1988) FEBS Lett. 231, 151-154]. The reactivation rate of the nicked enzyme after denaturation does not differ substantially from the reactivation rate of the denatured intact enzyme: t 1/2 varies between 70-110 s at 25 degrees C, pH 7.0 in both cases. Thus, the absence of a covalent linkage between the two proteolytic fragments of the enzyme molecule apparently does not affect the refolding. The two proteolytic fragments can be separated by FPLC under denaturing conditions. Fluorescence spectra of the isolated fragments may indicate that the tryptic cleavage site is within the N-terminal domain. Thus, the larger fragment (molecular mass about 30 kDa) probably contains the whole nucleotide-binding C-terminal domain plus a small part of the N-terminal domain. The inactive isolated fragments were used in renaturation experiments to study the reassembly of active 3-phosphoglycerate kinase. Kinetic measurements revealed the presence of a bimolecular rate-limiting step of reactivation. Separate preincubation of the fragments under renaturing conditions did not cause substantial acceleration of reactivation. This implies that assembly of the separate structural units (possibly domains) may limit the reactivation of the intact enzyme.

Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase.

Diffuse X-ray-scattering data give evidence for large-scale structural change in pig muscle 3-phosphoglycerate kinase upon substrate binding. Simultaneous binding of 3-phosphoglycerate and MgATP either to the unmodified enzyme or to its active methylated derivative leads to about an 0.1-nm decrease in radius of gyration. These data coincide well with the previous data for yeast 3-phosphoglycerate kinase. When, instead of methylation, the two reactive thiol groups of pig muscle 3-phosphoglycerate kinase are carboxamidomethylated, the enzyme becomes inactive and the radii of gyration of its 'apo' and 'holo' forms do not differ within limits of experimental error. Thus, a correlation exists between the activity of 3-phosphoglycerate kinase and its substrate-induced large-scale conformational change. This correlation is a strong argument in favor of the functional importance of domain locking in the reaction catalyzed by 3-phosphoglycerate kinase.