Water Flip-back Research Articles

Effects of radiation damping for biomolecular NMR experiments in solution: a hemisphere concept for water suppression.

Abundant solvent nuclear spins, such as water protons in aqueous solution, cause radiation damping in NMR experiments. It is important to know how the effect of radiation damping appears in high-resolution protein NMR because macromolecular studies always require very high magnetic field strengths with a highly sensitive NMR probe that can easily cause radiation damping. Here, we show the behavior of water magnetization after a pulsed-field gradient (PFG) using nutation experiments at 900 MHz with a cryogenic probe: when water magnetization is located in the upper hemisphere (having +Z component, parallel to the external magnetic field), dephasing of the magnetization by a PFG effectively suppresses residual water magnetization in the transverse plane. In contrast, when magnetization is located in the lower hemisphere (having -Z component), the small residual transverse component remaining after a PFG is still sufficient to induce radiation damping. Based on this observation, we designed 1H-15N HSQC experiments in which water magnetization is maintained in the upper hemisphere, but not necessarily along Z, and compared them with the conventional experiments, in which water magnetization is inverted during the t1 period. The result demonstrates moderate gain of signal-to-noise ratio, 0-28%. Designing the experiments such that water magnetization is maintained in the upper hemisphere allows shorter pulses to be used compared to the complete water flip-back and, thereby, is useful as a building block of protein NMR pulse programs in solution.

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3-5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns.

Unambiguous Determination of the Ionization State of a Glycoside Hydrolase Active Site Lysine by 1H−15N Heteronuclear Correlation Spectroscopy

We have investigated the lysine side chain amines in the 34 kDa catalytic domain from Cellulomonas fimi beta-(1,4)-glycosidase Cex (or CfXyn10A) using 1H-detected 15N heteronuclear correlation NMR spectroscopy. Signals from the 1Hzeta ( approximately 8 ppm) and 15Nzeta ( approximately 35 ppm) of Lys302 in the unmodified enzyme and Lys47 in a trapped cellobiosyl-enzyme intermediate were detected in a 1H-15N HMQC spectrum (pH 6.5 and 30 degrees C). The amine of Lys302 forms a buried ion pair, and that of Lys47 is hydrogen bonded to the cellobioside. Both lysines are positively charged, as unambiguously demonstrated by the splitting of their 15Nzeta signals into quartets (|1JNH| approximately 75 Hz) in a 1H-15N HSQC spectrum recorded without 1H decoupling during 15N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of approximately 3:1:1:3 expected for a highly mobile amine. On the basis of the observed ratios of approximately 1:1:1:1 for the quartet of Lys302 and approximately 0.5:1:1:0.5 for Lys47, the amine of the latter active site residue is most rigidly positioned. Signals from at least 8 and 10 additional positively charged, mobile amines in Cex were observed at 10 degrees C and pH 6.5 and 5.6, respectively. By using conditions of reduced temperature, slightly acidic pH, and low general base concentrations, as well as water flipback pulses to minimize the effects of hydrogen exchange, 1H-15N correlation experiments provide a sensitive route to directly investigate the charge states and dynamic properties of the N-terminal and side chain amines in proteins and protein complexes.

The Structure of the Interleukin-15α Receptor and Its Implications for Ligand Binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

Signal Enhancement Using 45° Water Flipback for 3D 15N-Edited ROESY and NOESY HMQC and HSQC

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

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

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly 15N and doubly 13C/ 15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1′ resonances. The DNA duplex is nearly straight and its structure is primarily that of B-DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three-dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.

Gradient and sensitivity enhancement of 2D TROSY with water flip-back, 3D NOESY-TROSY and TOCSY-TROSY experiments.

Previously we demonstrated a sensitivity enhancement of the original TROSY experiment by a factor of [Formula: see text] by the use of the sensitivity enhanced TROSY (en-TROSY) scheme. Here, we develop a gradient and sensitivity enhanced TROSY experiment (gs-TROSY), which is designed to select magnetization transfer pathways that suppress spectral artifacts and reduce the number of required phase cycles while having minimal loss of sensitivity. Both of these experimental methods (en-TROSY and gs- TROSY) have been combined with a water flip-back scheme which provides a further increase in sensitivity for labile NH groups by avoiding water saturation. We also apply these TROSY schemes to 3D NOESY-TROSY and 3D TOCSY-TROSY experiments.

Water-protein NOEs: Optimized scheme for selective water excitation.

An alternative scheme for selective water excitation is proposed. The pulse sequence saturates the resonances from the solute, allowing the observation of water-solute NOEs with low artifact levels. The water resonance is subsequently excited by a relatively non-selective 90° pulse. The scheme is compared to other selective water excitation schemes. 2D NOE-NOESY and ROE-NOESY pulse sequences are proposed which afford high sensitivity by efficient water excitation and flip-back by radiation damping, yet allow the use of short mixing times for the buildup of water-solute NOEs.

Water-macromolecule interactions by NMR: a quadrature-free constant-time approach and its application to CI2.

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a square root 2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-sheet strands were identified, solving previously reported discrepancies between the X-ray and refined NMR structure of CI2. The proposed experiment in particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand-macromolecule complexes.

15N-edited Three-Dimensional NOESY-HMQC with Water Flipback: Enhancement of Weak Labile1H Resonances of Protein Side Chains Contacting DNA

Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein–DNA interface of the vnd/NK-2 homeodomain/DNA complex in an15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization from the water to the protein by maximizing thez-component of the water magnetization. In one of the pulse sequences, radiation damping during the the indirect1H and15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critical for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum of the protein/DNA complex.

Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY.

This paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a [Formula: see text] sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the 'water flip back' technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.

HMQC and HSQC experiments with water flip-back optimized for large proteins.

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N,1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D 15N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.