Accurate Quantitation of Water-amide Proton Exchange Rates Using the Phase-Modulated CLEAN Chemical EXchange (CLEANEX-PM) Approach with a Fast-HSQC (FHSQC) Detection Scheme

Measurement of exchange rates between water and NH protons by magnetization transfer methods is often complicated by artifacts, such as intramolecular NOEs, and/or TOCSY transfer from C protons coincident with the water frequency, or exchange-relayed NOEs from fast exchanging hydroxyl or amine protons. By applying the Phase-Modulated CLEAN chemical EXchange (CLEANEX-PM) spin-locking sequence, 135°(x) 120°(-x) 110°(x) 110°(-x) 120°(x) 135°(-x) during the mixing period, these artifacts can be eliminated, revealing an unambiguous water-NH exchange spectrum. In this paper, the CLEANEX-PM mixing scheme is combined with Fast-HSQC (FHSQC) detection and used to obtain accurate chemical exchange rates from the initial slope analysis for a sample of 15N labeled staphylococcal nuclease. The results are compared to rates obtained using Water EXchange filter (WEX) II-FHSQC, and spin-echo-filtered WEX II-FHSQC measurements, and clearly identify the spurious NOE contributions in the exchange system.     

Measurement of 13Cα-13CO cross-relaxation rates in 15N-/13C-labelled proteins

Summary Internal motions play an important role in the biological function of proteins and NMR relaxation studies may characterize them over a wide range of frequencies. An experimental pulse scheme is proposed for the measurement of the ¹³CO-¹³C cross-relaxation rate. For sensitivity reasons, this measurement is performed in an indirect manner through several coherence transfer steps, which should thus be calibrated independently. Contributions of other relaxation pathways can be eliminated by the determination of the initial slope of the buildup curve. The cross-relaxation rates have been determined on a ¹⁵N-/¹³C-labelled 116-residue protein and the significant variations along the sequence have been interpreted as evidence of an increased amount of fast local motion.     

The influence of anions and inhibitors on the catalytic metal ion in Co(II)-substituted horse liver alcohol dehydrogenase

¹H-NMR and electronic spectroscopic data are reported for the interaction of the effector molecule imidazole and the inhibitor molecule pyrazole with horse liver alcohol dehydrogenase whose catalytic zinc ions were replaced by Co(II). In addition ¹³C-NMR and optical data are given for the binding of acetate to this enzyme species. For the binary complex with imidazole an assignment of the protons of the metal-coordinated imidazole has been made and it was found that the rate of exchange of the effector molecule is slow on the NMR time scale. In the presence of NADH which is bound to the open conformation of the binary complex, the most pronounced change is a shift of the -CH₂ protons of the metal-coordinated cysteine residues which is attributed to hydrogen bonding interactions between the carboxamide group of the nicotinamide moiety with cysteine 46. The ¹H-NMR spectra of the binary complex of Co(II)-HLADH with pyrazole show resonances assigned to the protons in the 3-and 4-positions of the bound inhibitor, the NH proton resonance is not detectable. In the ternary complex with pyrazole and NAD⁺ only the resonances of the -CH₂ protons (beyond 150 ppm) are changed whereas the protons of histidine 67 and the bound inhibitor are unchanged. The data demonstrate that the coordination environment of the catalytic metal ion is changed very little when the protein changes from the open to the closed conformation. The only changes observed are the -CH₂ proton resonances of the metal-coordinating cysteines which are sensitive to local conformational changes within the ternary complex Co(II)-HLADH · Imidazole · NADH in the open conformation or global changes in the ternary complex Co(II)-HLADH · Pyrazole · NAD⁺ in the closed conformation. Acetate which can be regarded as a substrate model was shown to induce a similar change in the optical spectra of the Co(II) enzyme as all other anions observed so far. From the optical changes a dissociation constant of acetate at the catalytic metal site of 200±50 mM was calculated and from the changes of the ¹³C-NMR linewidth of ¹³C acetate direct bonding of the anion to the catalytic Co(II) ion can be demonstrated to occur under the conditions of rapid exchange. The implications of these data for the assessment of tetracoordination around the catalytic metal ion as well as the chemical nature of intermediates occurring along the catalytic pathway are discussed.This work has been performed with contribution of the project Projetto Strategico Biotechnologie CNR and with financial support from the Deutsche Forschungsgemeinschaft, NATO, Bundesminister für Forschung und Technologie, and the Universität des Saarlandes     

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

Summary We present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {¹H,¹H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H₂O to other protons in the oligosaccharide, (b) heteronuclear {¹H,¹³C} NOEs, (c) isotope effects of O¹H/O²H hydroxyl groups on¹³C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-(26)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the (26)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with angles at –60° or 180° and of the 3 staggered rotamers of the angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution. Supplementary material available from the corresponding author: Table containing values for the dihedral angles , , , , and for bond angles , for the six lowest-energy conformations of sialyl-(26)-lactose (1 page).     

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.     

Cross-relaxation bottleneck in water-lysozyme proton magnetization exchange

The proton spin-lattice relaxation parameters in natural and deuterated lysozyme solutions have been measured as a function of temperature (0-50 degrees C). The variation of the apparent magnitudes of the water proton magnetizations in the solutions with temperature indicates that magnetic coupling mixes protein and water proton magnetizations. The results are consistent with an exchange cross-relaxation model (Hills, B. P., Mol Phys 1992, 76, 489-508) in which the cross-relaxation acts between the labile and nonlabile protons, rather than between water and protein protons. Although this cross-relaxation pathway clearly affects the observed magnetization fractions in this protein solution, its influence on the relaxation rates is less apparent.     

AMI and PM3 study of a low molecular weight structural mimic of hydrogen exchange within the catalytic center of aspartic proteases

Based on recent X-ray studies, a low molecular weight model of the active center of aspartic proteases is proposed. The model is small enough to enable unattended geometry optimizations (including search for saddle-points) by molecular orbital methods. It consists of two malonic acid molecules and a water molecule; there is a carboxylic dimer at one end and the water molecule is located between the carboxylate and the carboxyl group at the other. The latter structure reproduces the geometry of the catalytic center of the native enzyme penicillopepsin with a root-mean-square deviation of 0.46 Å for five O--O distances. The AMI and PM3 molecular orbital methods were used to study the H-bond exchange within the model. Both methods lead consistently to the following conclusions: Among 2 pairs of symmetry-equivalent stationary states of the catalytic center there are at least 4 symmetry-independent hydrogen-exchange pathways, and many more when including symmetry of the center. Energetics and geometry of all identified pathways are presented. In summary, they result in juggling all three active center protons (COON and HOH) among all five active center oxygens (COO^–, COOH and H₂O) providing the center with a high delocalisation with respect to the actual position of its anionic site and/or its protonation status. The relevance of the delocalisation of the acidic proton to the mechanism of enzymatic action is briefly discussed. Correspondence to: J. Ciarkowski     

Assessment of protein solution versus crystal structure determination using spin- diffusion-suppressed NOE and heteronuclear relaxation data

A spin-diffusion-suppressed NOE buildup series has been measured for E. coli thioredoxin.The extensive 13C and 15N relaxation data previously reported for this protein allow fordirect interpretation of dynamical contributions to the 1H-1H cross-relaxation rates for a largeproportion of the NOE cross peaks. Estimates of the average accuracy for these derived NOEdistances are bounded by 4% and 10%, based on a comparison to the corresponding X-raydistances. An independent fluctuation model is proposed for prediction of the dynamicalcorrections to 1H-1H cross-relaxation rates, based solely on experimental structural andheteronuclear relaxation data. This analysis is aided by the demonstration that heteronuclearorder parameters greater than 0.6 depend only on the variance of the H-X bond orientation,independent of the motional model in either one- or two-dimensional diffusion (i.e., 1– S2 = 3/4 sin2 2 ). The combination of spin-diffusion-suppressed NOEdata and analysis of dynamical corrections to 1H-1H cross-relaxation rates based onheteronuclear relaxation data has allowed for a detailed interpretation of various discrepanciesbetween the reported solution and crystal structures.     

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly ¹³C/¹⁵N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s^–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide ¹⁵N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H₂O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin.     

1H- and 2H-NMR study of bovine serum albumin solutions

Frozen, native and denatured bovine serum albumin solutions have been studied with a wide-band NMR pulse spectrometer. Both macromolecular and water protons spin-spin and spin-lattice relaxation times--t2m, t1m, t2w, t1w--have been measured between 170 and 360 K. In the native sample, the t2m process is the tumbling rate of the bovine serum albumin molecules. It gives to the spin-lattice relaxation an omega 0(-2) frequency dependence at room temperature in the studied frequency range, 6-90 MHz. An additional process contributes to t1m-1; it arises from internal backbone or segmental motions and provides a lower frequency behaviour. On denaturation, bovine serum albumin molecules lose their tumbling motion and form a rigid network, while internal backbone motions seem unaffected. Calorimetric Cp measurement confirms the occurrence of a phase transition upon denaturation. 1H and 2H spin-lattice relaxation times of water protons depend mainly on bound water mobility. 1H and 2H t2w depend also on the tertiary structure of bovine serum albumin and on its mobility, because of a fast exchange process between water and some protein protons (or deutons), while a cross-relaxation process between protein and water protons contributes to 1H t1w. Denaturation has no influence on bound water motional properties and bound water population.     

Dynamics of β-CH and β-CH2 Groups of Amino Acid Side Chains in Proteins

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 1 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 -CH and -CH2 groups.     

Endor characterization and D2O exchange in the \begin{array}{*{20}c} {\mathop Z\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array} /{\kern 1pt} \begin{array}{*{20}c} {\mathop D\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array} radical in photosystem II

The early suggestion by Lozier and Butler (Photochem. Photobiol. 17, 133–137 (1973)) that EPR Signal II arises from radicals associated with the water-splitting process in PSII has been confirmed and extended over the intervening years. Recent work has identified the Signal II radicals, \(\begin{array}{*{20}c} {\mathop D\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array}\) and \(\begin{array}{*{20}c} {\mathop Z\nolimits^{\begin{array}{*{20}c} + \\ . \\ \end{array} } } \\ \end{array}\) , with plastosemiquinone cation species. In the experiments presented here we have used ENDOR spectroscopy and D₂O/H₂O exchange to characterize these paramagnets in more detail. The ENDOR matrix region, which arises from protons which interact weakly with the unpaired electron spin, is well-resolved at 4 K and at least seven resonances are apparent. A number of hyperfine couplings in the 3–8 MHz range are observed and are suggested to arise from methyl or hydroxyl protons which occur as substituents on the plastosemiquinone cation ring or from amino acid protons hydrogen-bonded to the 1,4-hydroxyl groups. Orientation selection experiments are consistent with these possibilities. D₂O/H₂O exchange shows that the D⁺/Z⁺ site is accessible to solvent. However, the exchange occurs slowly and is not complete even after 72 hours which suggests that the free radicals are functionally isolated from solvent water.     

Bisintercalation of ditercalinium into a d[CpGpCpG]2 minihelix: structure and dynamics aspects--a 400-MHz 1H-NMR study

The structure of the complex formed in aqueous solution at pH 5.5 between ditercalinium, a potent antitumoral 7H-pyrido[4,3-c]carbazole rigid dimer, and the self-complementary tetranucleotide d[CpGpCpG], was investigated by 400-MHz ¹H-nmr. For a 1:2.5 drug-to-helix ratio, the dimer was only found in bound form, whereas free and complexed tetranucleotide were in slow exchange. This allowed unambiguous assignment of the protons in the complex through exchange polarization transfer measurements. The tetranucleotide existed as a right-handed double helix in the complex. The strong upfield shifts measured on most aromatic protons on both drug and nucleobases as well as on DNA imino protons were consistent with bisintercalation of the dimer. According to the negative nuclear Overhauser effects generated to protons on the convex edge of the bound drug rings by saturation of sugar protons, it was concluded that ditercalinium was intercalated with its rigid bis-ethyl bispiperidine spacer fitting the major groove of the helix. Difference in antitumor activity of various pyridocarbazole dimers is discussed in relation to the binding kinetics and the complex geometry determined in this study.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides

An isotopomer-selected NOE (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the partner hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the partner hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the OO distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 Å , the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.     

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

Summary A method for measuring three-bond ¹³C-¹H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings ³J_C1H2 and ³J_C2H1 in a natural-abundance ¹³C-₁-half-filtered {¹H,¹H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy     

Water–Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T₂^′ -selective ¹H–¹³C–¹³C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T₂^′ selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on ¹³C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

High-resolution solid-state 13C NMR of the LH1 from Rhodospirillum rubrum

High-resolution solid-state 13C NMR spectra of the light-harvesting antenna complex (LH1) from Rhodospirillum rubrum were observed for the first time by cross-polarization (CP), magic angle spinning (MAS) methods with a total elimination of spinning side band technique (TOSS). Chemical shift analysis of the CP/MAS/TOSS 13C NMR spectra confirmed that the LH1 consists mainly of -helices in the solid state. Time constants of cross polarization (TCH) and relaxation time T1 in a rotating frame (T1H) were determined from the experiments at various contact times. Smaller values of TCH were obtained for the carbons attached directly with protons in a rigid state. Relaxation times T1H revealed the dynamic structure of the complex and showed that bacteriochlorophyll a in the LH1 has high internal mobility even in the solid state. The proton spin-lattice relaxation time in a laboratory frame (T1H) determined by the 13C NMR signal amplitude changes suggested that protons in the LH1 proteins have such strong interaction among them that the spins of all protons in the protein can diffuse through spin-lattice-relaxation.