Kinetics of creatine phosphokinase and adenylate kinase. A two-dimensional NMR analysis.

The kinetics of creatine phosphokinase and adenylate kinase catalyzed reactions were studied at equilibrium by two-dimensional Fourier transform phosphorus-31 nuclear magnetic resonance. For the creatine phosphokinase reaction, a pseudo-first-order rate constant of 0.29 s-1 was determined for the transfer of a phosphate group from adenosine triphosphate to creatine phosphate. For the adenylate kinase reaction two slow rate processes were required to describe the experimental results. The conversion of adenosine diphosphate to adenosine monophosphate was found to have a pseudo-first-order rate constant of 1.2 s-1, whereas that for the release of adenosine triphosphate from its enzyme complex occurred at a rate of 14 s-1.

Muscle adenylate kinase catalyzes adenosine 5'-tetraphosphate synthesis from ATP and ADP.

Muscle adenylate kinases from rabbit and porcine sources were found by NMR to catalyze the formation of adenosine tetraphosphate (5'AdoP4) from adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The reaction was completely reversible, with an equilibrium constant of approx. 0.1 at 30 degrees C. The synthesis of 5'AdoP4 from ATP and ADP occurred very slowly, taking over 12 h to reach equilibrium under the conditions used in this study. The sources of micromolar concentrations of 5'AdoP4 found in biological tissues is unknown: potentially, the slow adenylate-kinase-catalyzed breakdown and synthesis of 5'AdoP4 may serve as an important regulator of the steady-state concentration of 5'AdoP4 in muscle tissue.

The three-dimensional structure of the vnd/NK-2 homeodomain-DNA complex by NMR spectroscopy.

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly15N and doubly13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1' resonances. The DNA duplex is nearly straight and its structure is primarily that of B -DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three- dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.

The three-dimensional solution structure of the NK-2 homeodomain from Drosophila.

We describe the NMR determination of the three-dimensional structure of a 77 amino acid residue protein, which consists of the 60 residue NK-2 homeodomain from Drosophila melanogaster and adjacent amino acid residues. The NK-2 homeodomain protein is part of a 723 amino acid residue protein which is expressed early in embryonic development in part of the central nervous system. NK-2 was characterized using both a natural abundance and a uniformly 15N enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square deviation for 30 structures for residues 8 to 53 is 0.40 A for the backbone heavy-atoms and 0.72 A for the backbone and side-chain heavy-atoms. These structures were obtained from 986 NOE-derived upper and lower bound restraints. The three-dimensional structure contains three helices which consist of homeodomain amino acid residues 10 to 22, 28 to 38 and 42 to 52, as well as a turn between helix II and III, characteristic of homeodomains. Residues 53 to 60 of the DNA recognition helix are not fully ordered in the absence of DNA. In the free state this segment adopts a flexible but helix-like structure between residues 53 and 56 and is disordered from residues 57 to 60 although, as shown previously, the helix elongates by eight residues upon binding to DNA. The role of variable residues 52, 54 and 56 in determining the structure and flexibility of the recognition helix, as well as the stability of the NK-2 homeodomain as manifested by its thermal denaturation, are discussed.

Conformational behavior of the linear hexapeptide senktide: a receptor specific tachykinin analog.

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N-methylation of the third amino acid residue in the sequence and the negatively charged N-terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded beta-pleated sheet. The observed senktide-receptor specificity is suggested to result from a selection of this or a closely related conformation.

Nature of the pH-induced conformational changes and exposure of the C-terminal region of chromogranin A.

Chromogranin A is known to undergo pH induced conformational changes, and the difference in conformation is supposed to be responsible for the difference in Ca2+ binding property. To gain insight regarding the overall structure and the nature of pH-induced conformational changes of chromogranin A, limited trypsin digestions were carried out at pH 5.5 and pH 7.5. The resulting fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the amino acid sequences of the tryptic fragments were determined. From these analyses it was shown that the chromogranin A structure consists of an N-terminal compact core region and a rather loosely organized C-terminal region and that the change of pH from 7.5 to 5.5 loosened the overall structure of chromogranin A, exposing the C-terminal region. Since the conserved C-terminal region (residues 407-431) was shown to exist in monomer-dimer and monomer-tetramer equilibria at pH 7.5 and 5.5, respectively, the conformational changes of the region at pH 7.5 and 5.5 were studied by circular dichroism spectroscopy using a synthetic peptide representing the conserved C-terminal region. When the pH was changed from 7.5 to 5.5, the coil structure of the C-terminal peptide decreased with an accompanying increase of alpha-helicity.

Conformational behavior of fragments of adrenocorticotropin and their antisense peptides determined by NMR spectroscopy and CD spectropolarimetry.

An 'antisense' peptide ('HTCA'), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH (1-24) was shown to bind ACTH (1-24) with a Kd of 0.3 nM in a solid-matrix binding assay [( 1986) Biochem. J. 234, 679 683]. Two-dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1-24) and ACTH (1-13), as well as their antisense peptides, HTCA and HTCA(12-24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The Kd values are greater than 1 mM.

Three-dimensional structure and antigenicity of transmembrane-protein peptides of the human immunodeficiency virus type 1. Effects of a neutralization-escape substitution.

A point mutation (Ala-589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV-1 specific antibodies [(1990) J. Virol. 64, 3240-3248]. Here 17-residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV-1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol-water solutions approximately 60% alpha-helix could be induced. The three-dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies.

Two-dimensional J-resolved nuclear magnetic resonance spectral study of two bromobenzene glutathione conjugates.

The application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to determine the structure of two bile metabolites isolated from rats injected interperitoneally with bromobenzene is described. The structures of the two molecules are obtained unambiguously from the proton-proton spin coupling constants. This paper discusses the fundamentals of the technique and demonstrates the resolution of small long-range coupling constants.

15N-edited three-dimensional NOESY-HMQC with water flipback: enhancement of weak labile 1H resonances of protein side chains contacting DNA.

Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein-DNA interface of the vnd/NK-2 homeodomain/DNA complex in an 15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization from the water to the protein by maximizing the z-component of the water magnetization. In one of the pulse sequences, radiation damping during the the indirect 1H and 15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critical for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum of the protein/DNA complex.

Signal enhancement using 45 degrees water flipback for 3D 15N-edited ROESY and NOESY HMQC and HSQC.

A novel implementation of the water flipback technique employing a 45 degrees flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D 15N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45 degrees water flipback are presented using both HMQC and HSQC for the 15N dimension. The 45 degrees flipback pulse is followed by a gradient, a water selective 180 degrees pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t1 and t2 evolution periods is suppressed using gradients. Water resonance planes from NOESY-HMQC and NOESY-HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein-water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.

Conformational analysis of receptor selective tachykinin analogs: senktide and septide.

The conformational behavior in solution of two receptor selective tachykinin agonists, senktide (succinyl-D-F-MeF-G-L-M-NH2) and septide (pQ-F-F-P-L-M-NH2) is described. Two dimensional cross relaxation NMR spectroscopy is used together with coupling constant data to obtain interproton distance constraints. These results are used in conjunction with semi-empirical energy computations to indicate favorable conformations. Senktide is found to have a high degree of conformational order which is attributed to rotational restriction associated with the N-methylation of phenylalanine. The lowest energy conformation in accord with the experimental interproton distances contains a beta-turn. Interproton distances indicate that septide exists as a random coil or in an extended chain conformation. Energy computations suggest that septide is primarily an extended chain with internal reorientation restricted by the proline residue. These results may be related to the selectivity of these peptides for different receptors, in that the analogs, with conformations more stable than tachykinins, are more receptor selective.

Conformational analysis of the tachykinins in solution: substance P and physalaemin.

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.

Stereoselective formation of bromobenzene glutathione conjugates.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.6 +/- 0.1 (mean +/- S.E.). Pretreatment of rats with either phenobarbital or 3-methyl-cholanthrene did not significantly alter the ratio of the two conjugates excreted into bile. When bromobenzene was incubated with rat liver microsomes and glutathione, the same two conjugates were formed in the presence but not in the absence of 100 000 x g supernatant. Furthermore, in the presence of 100 000 x g supernatant from control animals, microsomes from rats treated with phenobarbital formed both conjugates 6 times more rapidly than did microsomes from control rats, whereas microsomes from rats treated with 3-methylcholanthrene formed both conjugates less rapidly than did those from control rats. Thus, the data suggest that both conjugates are formed via bromobenzene 3,4-oxide and that their formation requires in liver cytosol.

Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR.

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayed water cross-relaxation indicated that even at very short mixing times. 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant Asn51 residue, seen in all known homeodomain/DNA structures, is critical for binding affinity and specificity.

Rates of enzyme-catalyzed exchange determined by two-dimensional NMR: a study of glucose 6-phosphate anomerization and isomerization.

The application of two-dimensional (2D) Fourier-transform NMR to the determination of rate constants of complex enzyme-catalyzed reactions in the steady state is described. The yeast phosphoglucose isomerase (EC 5.3.1.9)-catalyzed anomerization of glucose 6-phosphate (Glc-6-P) as well as its isomerization to fructose 6-phosphate (Fru-6-P) was chosen as an example. The 2D technique permitted the simultaneous monitoring of the time course of the anomerization and isomerization steps, from which the various reaction rates were determined. The results obtained in the steady state demonstrate the usefulness of the 2D technique by confirming that the anomerization of Glc-6-P is enzyme catalyzed and that the isomerization of the alpha anomer of Glc-6-P to Fru-6-P is at least 10 times faster than the isomerization of the beta anomer of Glc-6-P. These results are compared with reaction rates obtained by rapid-quench methods and the mechanistic implications are discussed. Extrapolation of these results suggests that the 2D Fourier-transform NMR method should be applicable in intact biological tissues.

In vivo flux between phosphocreatine and adenosine triphosphate determined by two-dimensional phosphorous NMR.

The in vivo unidirectional flux between creatine phosphate and ATP was determined in the leg and head of the anesthetized rat using a two-dimensional NMR technique. The unidirectional flux of creatine phosphate to ATP was 13 mumol/s/g-weight in the leg and 2 mumol/s/g-weight in the head. The unidirectional flux between ATP and inorganic phosphate was too slow to be detected and was estimated to be less than 0.1 mumol/s/g-weight.

Structural basis of an embryonically lethal single Ala --> Thr mutation in the vnd/NK-2 homeodomain.

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to -5 degreesC in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25 degreesC. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

Site-specific deuterium order parameters and membrane-bound behavior of a peptide fragment from the intracellular domain of HIV-1 gp41.

The behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1 in membranes was investigated by solid-state 2H NMR on P828 with the selectively deuterated isoleucines I3, I13, I16, and I20. The quadrupole splittings of the I3 side chain show significant sensitivity to the main phase-transition temperature of the lipid, consistent with partial penetration of the N-terminal peptide region into the hydrophobic core of the membrane. In contrast, the quadrupole splittings of I13, I16, and I20 are in agreement with a location of the C-terminal portion of the peptide near the lipid/water interface. The perturbation of the bilayer by the peptide was studied by 2H NMR on sn-1 chain deuterated 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine membranes. Peptide incorporation results in a significant reduction of lipid chain order toward the bilayer center, but only a modest reduction near the lipid glycerol. These observations suggest a penetration of the partially structured peptide backbone into the membrane/water interface region that reduces lateral packing density and decreases order in the hydrophobic core. In addition, the structure of the peptide was investigated free in water and bound to SDS micelles by high-resolution NMR. P828 is unstructured in water but exists in a flexible partially helical conformation when bound to negatively charged liposomes or micelles. The flexible helix covers the first 14 residues of the peptide, whereas the C-terminus of the peptide, where three of the six positively charged arginine residues are located, appears to be unstructured. The peptide-induced changes in lipid chain order profiles indicate that membrane curvature stress is the driving force for the cytolytic behavior of P828.