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The signal-to-noise ratio is one of the limiting factors in neutron macromolecular crystallography. Protein perdeuteration, which replaces all H atoms with deuterium, is a method of improving the signal-to-noise ratio of neutron crystallography experiments by reducing the incoherent scattering of the hydrogen isotope. Detailed analyses of perdeuterated and hydrogenated structures are necessary in order to evaluate the utility of perdeuterated crystals for neutron diffraction studies. The room-temperature X-ray structure of perdeuterated diisopropyl fluorophosphatase (DFPase) is reported at 2.1 A resolution. Comparison with an independently refined hydrogenated room-temperature structure of DFPase revealed no major systematic differences, although the crystals of perdeuterated DFPase did not diffract neutrons. The lack of diffraction is examined with respect to data-collection and crystallo­graphic parameters. The diffraction characteristics of successful neutron structure determinations are presented as a guideline for future neutron diffraction studies of macromolecules. X-ray diffraction to beyond 2.0 A resolution appears to be a strong predictor of successful neutron structures.

Hydrogen atoms constitute about half of all atoms in proteins and play a critical role in enzyme mechanisms and macromolecular and solvent structure. Hydrogen atom positions can readily be determined by neutron diffraction, and as such, neutron diffraction is an invaluable tool for elucidating molecular mechanisms. Joint refinement of neutron and X-ray diffraction data can lead to improved models compared with the use of neutron data alone and has now been incorporated into modern, maximum-likelihood based crystallographic refinement programs like CNS. Joint refinement has been applied to neutron and X-ray diffraction data collected on crystals of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of detoxifying organophosphorus nerve agents. Neutron omit maps reveal a number of important features pertaining to the mechanism of DFPase. Solvent molecule W33, coordinating the catalytic calcium, is a water molecule in a strained coordination environment, and not a hydroxide. The smallest Ca–O–H angle is 53°, well beyond the smallest angles previously observed. Residue Asp-229, is deprotonated, supporting a mechanism involving nucleophilic attack by Asp-229, and excluding water activation by the catalytic calcium. The extended network of hydrogen bonding interactions in the central water filled tunnel of DFPase is revealed, showing that internal solvent molecules form an important, integrated part of the overall structure.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.

Hydrogen bonding plays a key role in the tight binding of the FMN cofactor and the regulation of its redox properties in flavodoxins. Hydrogen bonding interactions can be directly observed in solution by multidimensional heteronuclear NMR spectroscopy through the scalar couplings between donor and acceptor nuclei. Here we report on the detection of intermolecular trans-hydrogen-bond couplings ((h)J) between the flavin ring system and the backbone of Desulfovibrio vulgaris flavodoxin in the oxidized and the two-electron reduced states. For this purpose, experiments are adapted from pulse sequences previously applied to determining (h)J coupling constants in nucleic acid-base pairs and proteins. The resulting (h2)J(N,N), (h4)J(N,N), (h3)J(C,N), and (h1)J(H,N) couplings involve the (15)N(1), (13)C(2), and (15)N(3) nuclei of the pyrimidine moiety of FMN, whereas no such interactions are detectable for (13)C(4) and (15)N(5). Several long-range (15)N-(15)N, (13)C-(15)N, and (1)H-(15)N J-coupling constants within the flavin are obtained as "by-products". The magnitudes of both (h)J and regular J couplings are found to be dependent on the redox state. In general, good correlations between (h)J coupling constants and donor-group (1)H chemical shifts and also crystallographic donor-acceptor distances are observed.

We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 μm cross-sectional layer of a 100 μm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts. © 2004 Wiley Periodicals, Inc.

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.

Side-chain proton and carbon-13 resonance assignments of [13C;15N]-enriched proteins usually rely on combinations of several multi-dimensional experiments. Here, we describe a four-dimensional pulse sequence, H(C)C–COSY–TOCSY–(CACO)NH, which provides the information required to assign completely aliphatic side-chain resonance frequencies. As in widely used HCC(CO)NH–TOCSY experiments, problems due to spectral crowding are alleviated by exploiting the dispersion of backbone amide 1H and 15N signals. The modification introduced here allows signals from different side-chains to be distinguished even in the case of overlap in the 1HN–15N plane of the spectra. For illustration, the new method is applied to two proteins with molecular masses of 11 and 23 kDa. Copyright © 2004 John Wiley & Sons, Ltd.

We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed alpha-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.

Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the excisionase protein (Xis) from the λ-like bacteriophage HK022 and to study its sequence-specific DNA interaction. As wild-type Xis was previously characterized as a generally unstable protein, a biologically active HK022 Xis mutant with a single amino acid substitution Cys28Ser was used in this work. This substitution has been shown to diminish the irreversibility of Xis denaturation and subsequent degradation, but does not affect the structural or thermodynamic properties of the protein, as evidenced by NMR and differential scanning calorimetry. The solution structure of HK022 Xis forms a compact, highly ordered protein core with two well-defined α-helices (residues 5–11 and 18–27) and five β-strands (residues 2–4, 30–31, 35–36, 41–44 and 48–49). These data correlate well with 1H2O-2H2O exchange experiments and imply a different organization of the HK022 Xis secondary structure elements in comparison with the previously determined structure of the bacteriophage λ excisionase. Superposition of both Xis structures indicates a better correspondence of the full-length HK022 Xis to the typical ‘winged-helix’ DNA-binding motif, as found, for example, in the DNA-binding domain of the Mu-phage repressor. Residues 51–72, which were not resolved in the λ Xis, do not show any regular structure in HK022 Xis and thus appear to be completely disordered in solution. The resonance assignments have shown, however, that an unusual connectivity exists between residues Asn66 and Gly67 owing to asparagine-isoaspartyl isomerization. Such an isomerization has been previously observed and characterized only in eukaryotic proteins.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid 1H and 13C transverse relaxation. However, 1H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D2O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H2O. As a side-effect partial Cα protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary 1HN- and 1Hα-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D2O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of Cα protonation of individual amino acids is determined quantitatively and transverse relaxation properties of 1HN and 15N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone (1)J(NC'), (1)J(NCalpha), and (2)J(NCalpha) coupling constants are obtained for the two [(15)N;(13)C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [(2)H;(15)N;(13)C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J(NCalpha) values on protein backbone psi torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated (15)N-(13)C residual dipolar couplings.

We have solved the solution structure of the peptidyl-prolyl cis – trans isomerase (PPIase) domain of the trigger factor from Mycoplasma genitalium by homo- and heteronuclear NMR spectroscopy. Our results lead to a well-defined structure with a backbone rmsd of 0.23 A. As predicted, the PPIase domain of the trigger factor adopts the FK506 binding protein (FKBP) fold. Furthermore, our NMR relaxation data indicate that the dynamic behavior of the trigger factor PPIase domain and of FKBP are similar. Structural variations when compared to FKBP exist in the flap region and within the bulges of strand 5 of the β sheet. Although the active-site crevice is similar to that of FKBP, subtle steric variations in this region can explain why FK506 does not bind to the trigger factor. Sequence variability (27% identity) between trigger factor and FKBP results in significant differences in surface charge distribution and the absence of the first strand of the central β sheet. Our data indicate, however, that this strand may be partially structured as “nascent” β strand. This makes the trigger factor PPIase domain the most minimal representative of the FKBP like protein family of PPIases.

Side-chain carbon resonance assignments are difficult to obtain for larger proteins. While standard methods require protons for excitation and detection of magnetization, their presence is often unacceptable and often leads to unacceptable relaxation losses at the directly bound carbon sites. In this paper, pulse sequences are presented which provide connectivities between aliphatic side-chain (13)C and amide (1)H and (15)N chemical shifts in fully deuterated, (13)C/(15)N-enriched proteins. Magnetization either starts off from carbons or from both nitrogens and protons and is passed along the side-chain via (13)C-(13)C isotropic mixing. Direct rather than (13)CO-relayed (15)N--(13)C(alpha) or (13)C(alpha)--(15)N transfer steps allow the detection of intraresidual as well as sequential correlations. To avoid ambiguities between these two types in the three-dimensional version of the experiments, a fourth dimension can be introduced to achieve their separation along a (13)C(alpha) frequency axis. The novel methods are demonstrated with the uniformly (2)H/(13)C/(15)N labeled 35-kDa protein diisopropylfluorophosphatase from Loligo vulgaris.

Methods are described to correlate aromatic 1Hδ 2/13Cδ 2 or 1He 1/15Ne 1 with aliphatic 13Cβ chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with β-carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine δ-CH and tryptophan e-NH groups.

Collective internal motions are known to be important for the function of biological macromolecules. It has been discussed in the past whether the application of superimposing algorithms to remove the overall motion from a structural ensemble introduces artificial correlations between distant atoms. Here we present a new method to eliminate residual rotation and translation from cartesian modes derived from a normal mode analysis or from a principal component analysis. Bias-free separation is based on the idea that the addition of modes of pure rotation/translation can compensate the residual overall motion. Removal of overall motion must reduce the “total amount of motion” (TAM) in the mode. Our algorithm allows to back-calculate revised covariance matrices. The approach was applied to two model systems that show residual overall motion, when analyzed using all atoms as reference for the superimposing algorithm. In both cases, our algorithm was capable of eliminating residual covariances caused by the overall motion, while retaining internal covariances even for very distant atoms. A structural ensemble obtained for a 13-ns molecular dynamics simulation of the protein Ribonuclease T1 showed a covariance matrix of the corrected modes with significantly sharper contours after applying the bias-free separation. Proteins 2001;45:207–218. © 2001 Wiley-Liss, Inc.

pK a values of amino acid side chains of ribonuclease T1 have been determined from the pH dependence of 13C and 15N resonances. It was possible to derive pK a values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pK a values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pK a and a decrease of the E82 pK a. The pK a of E28 at the C-terminal end of the α-helix was increased because of the dipolar character of the α-helix. D76 did not titrate in the investigated pH range of about 2–9. From the chemical shift value this buried side chain seems to be protonated. The pK a values of side chains in the active site deviate from a normal behaviour. The lower pK a value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional 1H(13Cδ)13Cγ correlation experiment was developed to observe the pH dependence of the chemical shifts of the Cγ resonances of histidine residues. From the inspection of the Cγ chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values.

The experimental NMR data for the individual titratable groups in ribonuclease T1 presented in the preceding paper were analysed by means of a continuum dielectric model. The role of two factors, the alteration of hydrogen loci on the ionizable groups and the conformational flexibility, were analysed. It was suggested that the position of the titratable hydrogen is essential mainly for strongly interacting groups. For groups which are accessible to the solvent and whose ionization is not coupled with the ionization of neighbouring groups, this factor can be neglected. The influence of the conformational flexibility on the electrostatic interactions becomes apparent for the environment of K25. For some strongly interacting groups, non-sigmoidal ionization curves were calculated. On this basis the pH dependence of the NMR chemical shift of the 13Cepsilon2 resonance of H27, whose ionization is coupled with E82, was reproduced.

Background: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin. Results: Crystals were grown of both the native and the selenomethionine-labeled enzyme. The X-ray crystal structure of the DFPase from Loligo vulgaris has been solved by MAD phasing and refined to a crystallographic R value of 17.6% at a final resolution of 1.8 A. Using site-directed mutagenesis, we have structurally and functionally characterized essential residues in the active site of the enzyme. Conclusions: The crystal structure of the DFPase from Loligo vulgaris is the first example of a structural characterization of a squid-type DFPase and the second crystal structure of a PTE determined to date. Therefore, it may serve as a structural model for squid-type DFPases in general. The overall structure of this protein represents a six-fold β propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this β propeller has not yet been observed in other β propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed.

The thermoinactivation of native diisopropylfluorophosphatase (DFPase, EC 3.8.2.1) is highly calcium dependent, first-order kinetic. Deactivation is coupled with a simultaneous reduction in beta-sheet content. We report herein our attempts to enhance the thermostability of DFPase by irreversibly incorporating the enzyme into polyurethane polymers. Immobilized DFPase has biphasic deactivation kinetics. Our data demonstrate that the initial rapid deactivationof immobilized DFPase leads to the formation of a hyperstable and still active form of enzyme. Like native DFPase, DFPase-containing polyurethanes exhibit a calcium-dependent thermostability. Since bioplastics cannot be analyzed by spectroscopy, the structural mechanisms involved in thermoinactivation of immobilized DFPase were determined using PEG-modified DFPase. The thermoinactivation profile of highly modified DFPase mirrors the stepwise deactivation pattern of bioplastics. Spectroscopic studies enable a structural analysis of the hyperstable intermediate.

Kinetic measurements, chemical modification and site-directed mutagenesis have been employed to gain deeper insights into the reaction mechanism of the diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. Analysis of the kinetics of diisopropyl fluorophosphate hydrolysis reveals optimal enzyme activity at pH/=8, 35 degrees C and an ionic strength of 500 mM NaCl, where k(cat) reaches a limiting value of 526 s(-1). The pH rate profile shows that full catalytic activity requires the deprotonation of an ionizable group with an apparent pK(a) of 6.82, DeltaH(ion) of 42 kJ/mol and DeltaS(ion) of 9.8 J/mol K at 25 degrees C. Chemical modification of aspartate, glutamate, cysteine, arginine, lysine and tyrosine residues indicates that these amino acids are not critical for catalysis. None of the six histidine residues present in DFPase reacts with diethyl pyrocarbonate (DEPC), suggesting that DEPC has no accessibility to the histidines. Therefore, all six histidine residues have been individually replaced by asparagine in order to identify residues participating in catalysis. Only substitution of H287 renders the enzyme catalytically almost inactive with a residual activity of approx. 4% compared to wild-type DFPase. The other histidine residues do not significantly influence the enzymatic activity, but H181 and H274 seem to have a stabilizing function. These results are indicative of a catalytic mechanism in which H287 acts as a general base catalyst activating a nucleophilic water molecule by the abstraction of a proton.

Recent advances in the characterization of fatty acid-binding proteins (FABPs) by NMR have enabled various research groups to investigate the function of these proteins in aqueous solution. The binding of fatty acid molecules to FABPs, which proceeds through a portal region on the protein surface, is of particular interest. In the present study we have determined the three-dimensional solution structure of human heart-type FABP by multi-dimensional heteronuclear NMR spectroscopy. Subsequently, in combination with data collected on a F57S mutant we have been able to show that different fatty acids induce distinct conformational states of the protein backbone in this portal region, depending on the chain length of the fatty acid ligand. This indicates that during the binding process the protein accommodates the ligand molecule by a ‘selected-fit’ mechanism. In fact, this behaviour appears to be especially pronounced in the heart-type FABP, possibly due to a more rigid backbone structure compared with other FABPs, as suggested by recent NMR relaxation studies. Thus differences in the dynamic behaviours of these proteins may be the key to understanding the variations in ligand affinity and specificity within the FABP family.

Di-isopropylfluorophosphatase (DFPase) is shown to contain two high-affinity Ca2+-binding sites, which are required for catalytic activity and stability. Incubation with chelating agents results in the irreversible inactivation of DFPase. From titrations with Quin 2 [2-({2-[bis(carboxymethyl)amino]-5-methylphenoxy}-methyl)-6-methoxy-8-[bis(carboxymethyl)-amino]quinoline], a lower-affinity site with dissociation constants of 21 and 840nM in the absence and the presence of 150mM KCl respectively was calculated. The higher-affinity site was not accessible, indicating a dissociation constant of less than 5.3nM. Stopped-flow experiments have shown that the dissociation of bound Ca2+ occurs in two phases, with rates of approx. 1.1 and 0.026s-1 corresponding to the dissociation from the low-affinity and high-affinity sites respectively. Dissociation rates depend strongly on temperature but not on ionic strength, indicating that Ca2+ dissociation is connected with conformational changes. Limited proteolysis, CD spectroscopy, dynamic light scattering and the binding of 8-anilino-1-naphthalenesulphonic acid have been combined to give a detailed picture of the conformational changes induced on the removal of Ca2+ from DFPase. The Ca2+ dissociation is shown to result in a primary, at least partly reversible, step characterized by a large decrease in DFPase activity and some changes in enzyme structure and shape. This step is followed by an irreversible denaturation and aggregation of the apo-enzyme. From the temperature dependence of Ca2+ dissociation and the denaturation results we conclude that the higher-affinity Ca2+ site is required for stabilizing DFPase's structure, whereas the lower-affinity site is likely to fulfil a catalytic function.

An NMR pulse sequence is proposed for the simultaneous determination of side chain χ1 torsion-angle related 3 J N,Cγ and 3 J C′,Cγ couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated 15 N and 1 H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly 13 C/ 15 N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.

Hydrogen bonding plays a major role in the tight binding of the FMN cofactor in flavodoxins. The present NMR investigation provides direct experimental evidence for hydrogen bonds involving the phosphate moiety of FMN in Desulfovibrio vulgaris flavodoxin. Several trans-hydrogen bond J couplings between the phosphorus nucleus and backbone amide as well as side chain hydroxyl protons of the apoprotein have been detected. It is shown that relaxation interference between 1H chemical shift anisotropy and 1H−31P dipolar interactions can also lead to correlations of these nuclei in HMBC spectra. The size of the 2hJPH coupling constants was determined using a simple 31P-detected quantitative J correlation experiment. For at least one amide group a scalar three-bond coupling between the phosphorus and nitrogen has been observed in a [15N,1H]-TROSY-type 15N−{31P} spin−echo difference experiment. With approximately 1.7 Hz its magnitude is larger than that of the 31P−1H couplings, which ranged from 0.5 to 1.6 Hz.

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 ± 0.10 A and 1.09 ± 0.09 A for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.

Constant-time 3D heteronuclear relayed E.COSY [Schmidt et al. (1996) J. Biomol. NMR, 7, 142–152], as based on generic 2D small-flip-angle HMQC-COSY [Schmidt et al. (1995) J. Biomol. NMR, 6, 95–105], has been modified to allow for quantitative determination of heteronuclear three-bond 3 J(Hα,Cγ) couplings. The method is applicable to amino acid spin topologies with carbons in the γ position which lack attached protons, i.e. to asparagine, aspartate, and aromatic residues in uniformly 13C-enriched proteins. The pulse sequence critically exploits heteronuclear triple-quantum coherence (HTQC) of CH2 moieties involving geminal Hβ proton pairs, taking advantage of improved multiple-quantum relaxation properties, at the same time avoiding scalar couplings between those spins involved in multiple-quantum coherence, thus yielding E.COSY-type multiplets with a splitting structure that is simpler than with the original scheme. Numerical least-squares 2D line-shape simulation is used to extract 3 J(Hα,Cγ) coupling constants which are of relevance to side-chain χ1 dihedral-angle conformations in polypeptides. Methods are demonstrated with recombinant 15N,13C-enriched ribonuclease T1 and Desulfovibrio vulgaris flavodoxin with bound oxidized FMN.

A TROSY-based triple-resonance pulse scheme is described which correlates backbone 1H and 15N chemical shifts of an amino acid residue with the 15N chemical shifts of both the sequentially preceding and following residues. The sequence employs 1 J NCα and 2 J NCα couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly 2H/13C/15N-labelled proteins in the 30-kDa range.

Major urinary proteins (MUPs) form an ensemble of protein isoforms which are expressed and secreted by sexually mature male mice only. They belong to the lipocalin superfamily and share with other members of this family the capacity to bind hydrophobic molecules, some of which are odorants. MUPs, either associated with or free of their natural ligands, play an important role in the reproductive cycle of these rodents by acting as pheromones. In fact, they are able to interact with receptors in the vomeronasal organ of the female mice, inducing hormonal and physiological responses by an as yet unknown mechanism. In order to investigate the structural and dynamical features of these proteins in solution, one of the various wild-type isoforms (rMUP: 162 residues) was cloned and subsequently isotopically labeled. The complete 1H, 13C and 15N resonance assignment of that isoform, achieved by using a variety of multidimensional heteronuclear NMR experiments, has been reported recently. Here, we describe the refined high-resolution three-dimensional solution structure of rMUP in the native state, obtained by a combination of distance geometry and energy minimization calculations based on 2362 NOE-derived distance restraints. A comparison with the crystal structure of the wild-type MUPs reveals, aside from minor differences, a close resemblance in both secondary structure and overall topology. The secondary structure of the protein consists of eight antiparallel β-strands forming a single β-sheet and an α-helix in the C-terminal region. In addition, there are several helical and hairpin turns distributed throughout the protein sequence, mostly connecting the β-strands. The tertiary fold of the β-sheet creates a β-barrel, common to all members of the lipocalin superfamily. The shape of the β-barrel resembles a calyx, lined inside by mostly hydrophobic residues that are instrumental for the binding and transport of small nonpolar ligand molecules.

A triple-resonance NMR pulse sequence termed HA[HB,HN](CACO)NH is proposed that provides two types of 3JHH coupling constants sharing the Cα proton in amino acid spin systems. The experiment is of the quantitative J correlation-type and takes advantage of the favorable relaxation properties of 1Ha−13Cα multiple-quantum coherence as compared to schemes relying on antiphase magnetization. The problem of signal overlap is considerably reduced by calculating J values from cross-peak−cross-peak intensity ratios rather than referencing diagonal peaks. Application to flavodoxin from Desulfovibrio vulgaris yields a total of 282 3JHα,Hβ and 3JHN,Hα coupling constants, normally determined in two separate experiments, to be used in conjunction with heteronuclear couplings to derive angular constraints for the protein's backbone and side chains. While 3JHα,Hβ coupling constants indicate some extent of conformational averaging not in accord with fixed side chain torsion, excellent correlation of the experimental 3JHN,...

A combination of gradient-purged isotope-filtered NMR experiments is presented, which allows for the detection of long-lived bound water molecules in proteins. The discrimination of direct water–protein exchange from NOE effects between bound water and protein protons is achieved by NOE/ROE cancellation during the mixing time in one of the otherwise identical experiments. The method was applied successfully to 13 C / 15 N -labelled serine protease PB92, and allowed for the identification of 22 protein–water NOEs in this 269-residue enzyme.

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the ‘model-free’ approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the μsec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.

Experiments are proposed for the measurement of the vicinal coupling constants between β-carbons and either amide protons of the same or carbonyl carbons of the preceding amino acid residue in 13C/15N-labeled proteins. Both couplings depend on the backbone torsional angle φ. The three-dimensional pulse sequences give rise to E.COSY-like multiplet patterns in which heteronuclear one-bond couplings separate the doublet components corresponding to the two spin states of the respective passive nuclei. Thus, in contrast to previously published pulse schemes which employed the homonuclear 1J(Cα,Cβ) interaction, difficulties due to overlap of spectral regions of active and passive spins are avoided. A major drawback of the novel sequences is their limited sensitivity. Nevertheless, application to Desulfovibrio vulgaris flavodoxin yielded coupling constants for more than 85% of all non-glycine and non-proline residues.

The concept of self-consistent J coupling evaluation exploits redundant structure information inherent in large sets of 3J coupling constants. Application to the protein Desulfovibrio vulgaris flavodoxin demonstrates the simultaneous refinement of torsion-angle values and related Karplus coefficients. The experimental basis includes quantitative coupling constants related to the polypeptide backbone φ torsion originating from a variety of heteronuclear 2D and 3D NMR correlation experiments, totalling 124 3J(HN,Hα), 129 3J(HN,C′), 121 3J(HN,Cβ), 128 3J(C′i−1,Hαi), 121 3J(C′i−1,C′i), and 122 3J(C′i−1,Cβi). Without prior knowledge from either X-ray crystallography or NMR data, such as NOE distance constraints, accurate φ dihedral angles are specified for 122 non-glycine and non-proline residues out of a total of 147 amino acids. Different models of molecular internal mobility are considered. The Karplus coefficients obtained are applicable to the conformational analysis of φ torsions in other polypeptides.

Methods are described which allow the measurement of geminal and vicinal15N–15N coupling constants in isotopically labeled proteins. The experiments rely on a quantitativeJ-correlation strategy and include either N,N-COSY or N,N-TOCSY magnetization transfer steps. The pulse sequences are demonstrated with a 16-kDa protein. Although the3JNNcouplings are almost vanishingly small, they give rise to visible cross peaks for 56% of the amino acid residues. A dependence of the vicinal coupling constants on the backbone torsion angle ψ is observed. However, only in favorable cases can the3Jvalues be translated into local structural information. In arginine side chains, correlations between ϵ- and η-nitrogens via two-bond scalar couplings are obtained that can be exploited for the resonance assignment of the guanidino groups.

Two- and three-dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty-acid-binding protein (heart FABP). NOE and rotating-frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two-dimensional H2O-ROE/NOE-1H,15N-heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N-enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel beta-strands (betaA-betaJ), forming two nearly orthogonal beta-sheets, and a short helix-turn-helix motif connecting beta-strands A and B. The spatial folding resembles a beta-barrel. Most of the water molecules are localized in the gap between beta-strands D and E, and near the two alpha-helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three-dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N-labeled sample of apo-heart FABP.

The hydration of uncomplexed RNase T1 was investigated by NMR spectroscopy at pH 5.5 and 313 K. Two-dimensional heteronuclear NOE and ROE difference experiments were employed to determine the spatial proximity and the residence times of water molecules at distinct sites of the protein. Backbone carbonyl oxygens involved in intermolecular hydrogen bonds to water molecules were identified based on1 J coupling constants. These coupling constants were determined from 2D-H(CA)CO and 15N-HSQC experiments with selective decoupling of the 13CCα nuclei during the t1 evolution time. Our results support the existence of a chain of water molecules with increased residence times in the interior of the protein which is observed in several crystal structures with different inhibitor molecules and serves as a space filler between the α-helix and the central β-sheet. The analysis of1 JNC' coupling constants demonstrates that some of the water molecules seen in crystal structures are not involved in hydrogen bonds to backbone carbonyls as suggested by crystal structures. This is especially true for a water molecule, which is probably hydrogen bonded by the protonated carboxylate group of D76 and the hydroxyl group of T93 in solution, and for a water molecule, which was reported to connect four different amino acid residues in the core of the protein by intermolecular hydrogen bonds.

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the χ1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 β-CH and β-CH2 groups.

Limits of NMR structure determination using multidimensional NMR spectroscopy, variable target function calculations and relaxation matrix analysis were explored using the model protein ribonuclease T1(RNase T1). The enzyme consists of 104 amino acid residues and has a molecular mass of approximately 11 kDa. Primary experimental data comprise 1856 assigned NOE intensities, 493 3J coupling constants and 62 values of amid proton exchange rates. From these data, 2580 distance bounds, 168 allowed ranges for torsional angles and stereospecific assignments for 75% of β-methylene protons as well as for 80% of diastereotopic methyl groups were derived. Whenever possible, the distance restraints were refined in a relaxation matrix analysis including amid proton exchange data for improvement of lower distance limits. Description of side-chain conformations were based on various models of motional averaging of 3J coupling constants. The final structure ensemble was selected from the starting ensemble comparing the global precision of structures with order parameters derived from 15N relaxation time measurements. Significant differences between the structure of RNase T1 in solution and in the crystal became apparent from a comparison of the two highly resolved structures.

Heteronuclear editing has found widespread use in the detection ofproton–proton dipolar interactions in isotopically labelled proteins.However, in cases where both the resonances of protons and directly bound13C or 15N spins of two or more sites aredegenerate, unambiguous assignments are difficult to obtain by conventionalmethods. Here, we present simple extensions of well-known triple-resonancepulse sequences which improve the dispersion of NOESY spectra. In order torecord the chemical shifts of backbone nuclei which allow a resolution ofoverlapping cross peaks, the magnetization is relayed via the scalarcoupling network either before or after the NOE mixing period. The novelpulse sequences are applied to flavodoxin from the sulfate-reducing organismDesulfovibrio vulgaris. A number of previously unassigned NOE interactionsinvolving α-, β- and amide protons can be unequivocallyidentified, suggesting that the accuracy of protein structure determinationcan be improved.

The backbone dynamics of uniformly 13C/15N-enriched ribonuclease T1 have beeninvestigated using carbonyl carbon relaxation times recorded at three different spectrometerfrequencies. Pulse sequences for the determination of the longitudinal (T1) and transverse (T2)relaxation times are presented. The relaxation behaviour was analysed in terms of a multispinsystem. Although the chemical shift anisotropy relaxation mechanism dominates at highmagnetic field strength, the contributions of the dipole-dipole interactions and thecross-correlation between these two relaxation mechanisms have also been considered.Information about internal motions has been extracted from the relaxation data using themodel-free approach of Lipari and Szabo in order to determine order parameters (S2) andeffective internal correlation times (taui). Using a relatively simple relation between themeasured relaxation rates and the spectral density function, an analytical expression for themicrodynamical parameters in dependence of T1 and T2 has been derived. The spectraldensity mapping technique has been applied in order to study the behaviour of the carbonylcarbon resonances in more detail.

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly13C/15N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the3J(C′i−1,Hiα), 3J(C′i−1,Ciβ) and3J(C′i−1,C′i)coupling constants in the indirectly detected 13C′dimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone φ-angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for3J(C′i−1,Hiα),3J(C′i−1,Ciβ) and3J(C′i−1,C′i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.

Recombinant Desulfovibrio vulgaris flavodoxin was produced inEscherichia coli. A complete backbone NMR assignment for the two-electronreduced protein revealed significant changes of chemical shift valuescompared to the oxidized protein, in particular for the flavinemononucleotide (FMN)-binding site. A comparison of homo- and heteronuclearNOESY spectra for the two redox states led to the assumption that reductionis not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgarisflavodoxin were investigated using two-dimensional15N-1H correlation NMR spectroscopy.T1, T2 and NOE data are obtained for 95%of the backbone amide groups in both redox states. These values wereanalysed in terms of the ’model-free‘ approach introduced by Lipari andSzabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. Acomparison of the two redox states indicates that in the reduced speciessignificantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H groupof FMN bound to the reduced protein compared to the oxidized state.