Vladimír Sklenář

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions.

SummaryA novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H2O at 5 °C is presented.

Spin-echo water suppression for the generation of pure-phase two-dimensional NMR spectra

A new approach is described for water suppression in one- and two-dimensional NMR, generating absorption-mode spectra that are free of baseline distortions. This method involves the use of a 1−1 hard pulse as a read pulse, followed by a 1−1 refocusing pulse which is phase cycled to obtain the greatest possible water suppression. Examples of this approach for water suppression are demonstrated for a 10 mg sample of the decapeptide LH-RH in 90% H2O by recording 2D NOE, 2D HOHAHA, and heteronuclear 1H15N shift correlation spectra.

Gradient-enhanced HSQC experiments for phase-sensitive detection of multiple bond interactions

Observation of remote connectivities and determination of long range coupling constants by 1H-X (13C, 15N and 77Se) gradient-enhanced phase-sensitive HSQC is reported.

Salt-induced conformational transition of poly[d(A-T)]·poly[d(A-T)]

Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.

Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.

Measurement of carbon-13 longitudinal relaxation using 1H detection

13C relaxation rates provide direct information concerning the dynamic behavior at specific sites in a molecule (Z-4). Interpretation of the Ti values can be straightforward because for protonated aliphatic carbons the longitudinal relaxation is dominated by the large ‘H-i3C dipolar interaction. Unfortunately, accurate measurement of 13C Tr’s is ohen very time consuming because 13C has a low magnetogyric ratio and correspondingly low NMR sensitivity. Moreover, i3C relaxation times are often quite long, requiring long delay times between scans. As will be demonstrated here, the sensitivity of the measurement can be increased signifkantly by using the more sensitive proton resonance to monitor changes in 13C magnetization. This indirect 13C T, measurement utilizes the sensitive hydrogen nucleus in a way similar to recently proposed ‘H-detected heteronuclear chemical-shift correlation experiments (57). Unfortunately, for the T1 experiment net magnetization transfer between ‘H and 13C nuclei is required, which makes the experimental schemes more complex than those used for heteronuclear chemical-shift correlation. Two different schemes for ‘H-detected measurement of 13C T,‘s arc depicted in Fig. 1. In the simplest scheme (Fig. la), the amount of 13C z magnetization present is measured by transferring this magnetization to ‘H magnetization by using a reverse DEPT (8,9) transfer. The experiment can be conducted with or without presaturation of the ‘H resonances, i.e., with or without an NOE effect on the 13C. To minimize systematic errors and to make it possible to fit the experimental data by a two parameter fit a difference experiment is performed (IO), with and without inversion of the r3C magnetization at the beginning of the variable relaxation delay. If at the time of this first i3C pulse the 13C magnetization equals aMO, where MO is the equilibrium i3C z magnetization, and the (imperfect) 13C 180” pulse changes this into bA40, then the difference experiment wiIl yield an amount of magnetization transfer proportional to (a b)emBf. For optimal sensitivity, a delay time between scans of about twice the i3C T, value is needed (II). Since the relaxation time of the “C is unknown, the delay time needed may be difficult to estimate. Note, however, that a nonoptimal delay time

Direct identification of relayed nuclear overhauser effects

The measurement of nuclear Overhauser effects (1) for determination of the threedimensional structure of mo lecules in solution is rapidly gaining popularity. The bui ldup rate of the NOE depends on r6, where I is the interproton distance. Measu.rement of the NOE buildup rates therefore provides a means to measure interproton distances. Most commonly, two-dimensional NMR experiments are used for measur ing the NOES in complicated spectra of macromolecules that are in the slow tumbling lim it (UT, > 1). To measure the true bui ldup rate, the 2D experiment has to be repeated fair a series of m ixing times, and the cross-peak intensity is mon itored as a function of this m ixing time (2-5). A complication commonly occurs if two protons, B and C, are close in space and a third proton, A, is close to B but distant from C. In this case, a cross peak can be observed between A and C which could be m istakenly interpreted as a direct NOE connectivity between A and C. Interpretation of the intensity of this cross peak generally will lead to an erroneous AC distance determination, i.e., to an incorrect structure. In principle, the shape of the NOE buildup curve could be used to distinguish direct from relayed NOES. However, to obtain a useful bui ldup curve it is especially important to measure the NOE for very short m ixing times. For these short m ixing times the cross peaks have very low intensities and consequently the sensitivity of this approach is poor. It is demonstrated here that direct and relayed NOES can readily be distinguished by using spin-locked NOE spectroscopy (6, 7). In the spin-locked NOE experiment the NOE effect is always positive and increases with slower mo lecular tumbling. Therefore, cross peaks due to direct NOE are always opposite in sign relative to the diagonal and consequently, cross peaks relayed via an intermediate nucleus will be in phase with the diagonal resonances. O f course, doubly relayed signals would be of a’pposite sign again but these resonances are usually too weak to be detected unless very long m ixing times are used. In 1D NOE difference spectra of small mo lecules (‘UT, < 1) this alternation of the sign of the NOE is commonly observed and is known a.s the “three-spin effect” (I).

Reaction mechanism and stereochemistry of gamma-hexachlorocyclohexane dehydrochlorinase LinA.

γ-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide γ-hexachlorocyclohexane (γ-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of γ-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from γ-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic γ-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic γ-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic γ-PCCH. δ-PCCH was detected as the only product of dehydrochlorination of δ-HCH. LinA requires the presence of a 1,2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1,2-biaxial HCl pairs present on γ-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH. Furthermore, LinA enantiomerically differentiates 1,3(S),4(R),5(R),6(S)-PCCH and 1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of γ-HCH to 1,2,4-TCB by LinA consists of two 1,2-anticonformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.

Refinement of d(GCGAAGC) Hairpin Structure Using One- and Two-Bond Residual Dipolar Couplings

The structure of the 13C,15N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond 1H-13C, 1H-15N, 13C-13C and 13C-15N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions.

Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole

Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by 15N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2-sec-4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature depencence of the motional parameters.