Wolfgang Bermel

Transformation of homonuclear two-dimensional NMR techniques into one-dimensional techniques using Gaussian pulses

Abstract A general procedure is proposed, which allows the transtormation of most of the homonuclear two-dimensional NMR techniques into one-dimensional sequences by using semiselective Gaussian pulses. These techniques are particularly advantageous in cases where a limited amount of information is required for solving a chemical problem, e.g., a few connectivities, coupling constants, or NOE values. Applications of 1D COSY, 1D NOESY, 1D homonuclear relayed techniques, and 1D TOCSY are demonstrated. Furthermore, a variant of the ID COSY, the refocused ID COSY with z filter is suggested, which in combination with 1 D COSY allows the application of DISCO for the determination of coupling constants from multiplets in crowded regions. The 1 D NOESY sequence allows the measurement of NOES for very short mixing times and thus provides reliable values for buildup rates of transient NOEs. Selective excitation of a certain resonance may be impossible by conventional techniques, because it lies in a crowded region. In such cases by exploiting the J dependence of transfer functions (directed magnetization transfer) magnetization can be transferred selectively from a coupled nucleus to the one of interest. Its NMR parameters (J coupling, NOE) are then accessible via relayed COSY or relayed NOESY.

Proton-detected C,H correlation via long-range couplings with soft pulses; determination of coupling constants

Abstract Selective excitation of carbonyl resonances in C,H-shift correlation (C,H-COSY) considerably improves the resolution. Variations of the basic pulse sequence are discussed with respect to sensitivity and extractability of heteronuclear coupling constants. A procedure based on evaluation of the shift correlation plus a reference 1D or 2D proton spectrum combined with simulations is proposed for the quantitative analysis and demonstrated with the example of a cyclic hexapeptide.

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

SummaryTwo new 3D 1H-15N-13C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the 2JNCα coupling to establish the sequential assignment of backbone NH and 15N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly 15N- and 13C-labelled ribonuclease T1.

Speeding Up 13C Direct Detection Biomolecular NMR Spectroscopy

After the exploitation of (1)H polarization as a starting source for (13)C direct detection experiments, pulse sequences are designed which exploit the accelerated (1)H longitudinal relaxation to expedite (13)C direct detection experiments. We show here that 2D experiments based on (13)C direct detection on a 0.5 mM water sample of ubiquitin can be recorded in a few minutes and 3D experiments in a few hours. We also show that fast methods like nonuniform sampling can be easily implemented. As overall experimental time has always been a counter indication for the use of (13)C direct detection experiments, this research opens new avenues for the application of (13)C NMR to biological molecules.

Speeding up the measurement of one-bond scalar (1J) and residual dipolar couplings (1D) by using non-uniform sampling (NUS)

The accurate and precise measurement of one-bond scalar and residual dipolar coupling (RDC) constants is of prime importance to be able to use RDCs for structure determination. If coupling constants are to be extracted from the indirect dimension of HSQC spectra a significant saving of measurement time can be achieved by non-uniform sampling (NUS). Coupling constants can either be obtained with the same precision as in traditionally acquired spectra in a fraction of the measurement time or the precision can be significantly improved if the same amount of measurement time as for traditionally acquired spectra is invested. The application of NUS for the measurement of (1)J (scalar coupling constants) and (1)T (total couplings constants) from different kinds of ω(1)-coupled spectra (including also J-scaled ones) is examined in detail and the possible gains in time or resolution are discussed. When using the newly proposed compressed sensing (CS) algorithm for processing, the quality of the spectra is comparable to the traditionally sampled ones.

H-start for exclusively heteronuclear NMR spectroscopy: The case of intrinsically disordered proteins

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on 13C direct detection, which exploit the (1)H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.

Anionic charge is prioritized over geometry in aluminum and magnesium fluoride transition state analogs of phosphoryl transfer enzymes.

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.

A Trojan horse transition state analogue generated by MgF3− formation in an enzyme active site

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. β-Phosphoglucomutase catalyses the isomerization of β-glucose-1-phosphate to β-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using 19F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF3− mimics the transferring PO3− moiety. Here we present a detailed characterization of the metal ion–fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of β-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

Speeding up sequence specific assignment of IDPs

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct 13C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment.

One of the stiffest challenges in structural studies of proteins using NMR is the assignment of sidechain resonances. Typically, a panel of lengthy 3D experiments are acquired in order to establish connectivities and resolve ambiguities due to overlap. We demonstrate that these experiments can be replaced by a single 4D experiment that is time-efficient, yields excellent resolution, and captures unique carbon-proton connectivity information. The approach is made practical by the use of non-uniform sampling in the three indirect time dimensions and maximum entropy reconstruction of the corresponding 3D frequency spectrum. This 4D method will facilitate automated resonance assignment procedures and it should be particularly beneficial for increasing throughput in NMR-based structural genomics initiatives.