19F chemical library and 19F-NMR for a weakly bound complex structure.

Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19F-NMR screening method optimizing a 19F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1H-15N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1H and 19F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19F-NMR analysis and the optimized 19F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein.

Sensitivity enhancement by sequential data acquisition for 13C-direct detection NMR.

13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/√2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.

Electrical conductance measurement of HgII-mediated DNA duplex in buffered aqueous solution.

Electrical properties of metal-mediated DNA duplexes (metallo-DNA) have been of particular interest because of their potential applications in DNA-based nanoelectronics. We prepared HgII-mediated DNA duplex with NH2 anchors and measured the electrical conductance of single-molecule metallo-DNA via scanning tunneling microscopy-based break junction method in the buffered solution. Three conductance values were observed that may correspond to different conformations of the metallo-DNA molecule bridged over metallic electrodes.

Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks.

Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.

NMR spectroscopic characterization of a model RNA duplex reflecting the core sequence of hammerhead ribozymes.

Hammerhead ribozymes are a model system for studying molecular mechanism of RNA catalysis. Physicochemical data-driven mechanistic studies are an indispensable step towards understanding the catalysis of hammerhead ribozymes. Here we characterized a model RNA duplex with catalytically important sheared-type G12-A9 base pair and A9-G10.1 metal ion-binding motif in hammerhead ribozymes. By using high magnetic field NMR, all base proton signals, including catalytic residues, were unambiguously assigned. We further characterized structural features of this RNA molecule and found that it reflects the structural features of the A9-G10.1 motif of hammerhead ribozymes. Therefore, this RNA molecule is suitable for extracting an intrinsic physicochemical properties of catalytically important residues.

A metallo-DNA nanowire with uninterrupted one-dimensional silver array.

The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal-DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire-whose structure was solved by high-resolution X-ray crystallography-that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C-Ag-C, as well as G-Ag-G, G-Ag-C and T-Ag-T) and linked to each other through G overhangs involved in interduplex G-Ag-G. The resulting hybrid nanowires are 2 nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix.

Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19F-labeling method was 3.5-fold more sensitive than 15N-labeling, and could be combined with other chemical modification techniques such as lysine 13C-methylation. 13C-dimethylated-19F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

The mechanism of the glycosylase reaction with hOGG1 base-excision repair enzyme: concerted effect of Lys249 and Asp268 during excision of 8-oxoguanine.

The excision of 8-oxoguanine (oxoG) by the human 8-oxoguanine DNA glycosylase 1 (hOGG1) base-excision repair enzyme was studied by using the QM/MM (M06-2X/6-31G(d,p):OPLS2005) calculation method and nuclear magnetic resonance (NMR) spectroscopy. The calculated glycosylase reaction included excision of the oxoG base, formation of Lys249-ribose enzyme-substrate covalent adduct and formation of a Schiff base. The formation of a Schiff base with ΔG# = 17.7 kcal/mol was the rate-limiting step of the reaction. The excision of the oxoG base with ΔG# = 16.1 kcal/mol proceeded via substitution of the C1΄-N9 N-glycosidic bond with an H-N9 bond where the negative charge on the oxoG base and the positive charge on the ribose were compensated in a concerted manner by NH3+(Lys249) and CO2-(Asp268), respectively. The effect of Asp268 on the oxoG excision was demonstrated with 1H NMR for WT hOGG1 and the hOGG1(D268N) mutant: the excision of oxoG was notably suppressed when Asp268 was mutated to Asn. The loss of the base-excision function was rationalized with QM/MM calculations and Asp268 was confirmed as the electrostatic stabilizer of ribose oxocarbenium through the initial base-excision step of DNA repair. The NMR experiments and QM/MM calculations consistently illustrated the base-excision reaction operated by hOGG1.

(1)H, (15)N and (13)C resonance assignments of the conserved region in the middle domain of S. pombe Sin1 protein.

SAPK-interacting protein 1 (Sin1) is an important component of the target of rapamycin (TOR) complex 2 (TORC2). TOR is a serine/threonine-specific protein kinase and forms functionally distinct protein complexes referred to as TORC1 and TORC2. TORC2, conserved from yeast to humans, phosphorylates AGC-family protein kinases and has many cellular functions including the regulation of actin cytoskeleton. The Sin1 subunit of TORC2 is required for the binding of TORC2 to substrates, and the conserved region in the middle (CRIM) domain of Sin1 is important in the substrate recognition of TORC2. Here, we report on the (1)H, (13)C and (15)N resonance assignments of fission yeast Schizosaccharomyces pombe Sin1 (amino acids 247-400) (Sin1CRIM), which possesses the CRIM domain. These data contribute toward the structure determination of Sin1CRIM and an understanding of the interactions of Sin1CRIM with substrates of TORC2.

Utilization of paramagnetic relaxation enhancements for high-resolution NMR structure determination of a soluble loop-rich protein with sparse NOE distance restraints.

NMR structure determination of soluble proteins depends in large part on distance restraints derived from NOE. In this study, we examined the impact of paramagnetic relaxation enhancement (PRE)-derived distance restraints on protein structure determination. A high-resolution structure of the loop-rich soluble protein Sin1 could not be determined by conventional NOE-based procedures due to an insufficient number of NOE restraints. By using the 867 PRE-derived distance restraints obtained from the NOE-based structure determination procedure, a high-resolution structure of Sin1 could be successfully determined. The convergence and accuracy of the determined structure were improved by increasing the number of PRE-derived distance restraints. This study demonstrates that PRE-derived distance restraints are useful in the determination of a high-resolution structure of a soluble protein when the number of NOE constraints is insufficient.

Utilization of lysine ¹³C-methylation NMR for protein-protein interaction studies.

Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive (13)C-methylation of the amino group of the lysine side-chain by (13)C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. (13)C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine (13)C-methylation NMR to monitor protein-protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the (1)H-(13)C HSQC spectra of the (13)C-methylated-lysine methyl groups. The lysine (13)C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.