Co-reporter:Dr. Michael Rettig; Markus W. Germann;Shuo Wang ; W. David Wilson
ChemBioChem 2013 Volume 14( Issue 3) pp:323-331
Publication Date(Web):
DOI:10.1002/cbic.201200706
Abstract
With a growing understanding of the microstructural variations of DNA, it has become apparent that subtle conformational features are essential for specific DNA molecular recognition and function. DNA containing an A-tract has a narrow minor groove and a globally bent conformation but the structural features of alternating AT DNA are less well understood. Several studies indicate that alternating AT sequences are polymorphic with different global and local properties from A-tracts. The mobility of alternating AT DNA in gel electrophoresis is extensively reduced upon binding with minor-groove binding agents such as netropsin. Although this suggests that such complexes are bent, similarly to A-tract DNA, direct evidence and structural information on AT DNA and the induced conformational change is lacking. We have used NMR spectroscopy and residual dipolar coupling together with restrained molecular-dynamics simulations to determine the solution structures of an alternating AT DNA segment, with and without netropsin, in order to evaluate the molecular basis of the binding-induced effects. Complex formation causes a significant narrowing of the minor groove and a pronounced change in bending, from a slight bend towards the major groove for the free DNA to a pronounced bend towards the minor groove in the complex. This observation demonstrates that conformational features and the inherent malleability of AT sequences are essential for specific molecular recognition and function. These results take the field of DNA structures into new areas while opening up avenues to target novel DNA sequences.
Co-reporter:Alexander M. Spring, Markus W. Germann
Analytical Biochemistry 2012 Volume 427(Issue 1) pp:79-81
Publication Date(Web):1 August 2012
DOI:10.1016/j.ab.2012.05.003
Low-temperature nuclear magnetic resonance (NMR), especially under supercooled conditions, can give critical insight into biomolecular systems via slowed dynamics and exchange rates. These conditions can also increase correlation times of small molecules, potentially allowing for NMR structural study of small molecules at moderate field strengths. Agarose gels allow for supercooled conditions and are simple to prepare, invisible to NMR, and noninteractive with most biomolecules and organics. Here we demonstrate their use with nucleic acids, small organic molecules, and peptides.
Co-reporter:Michael Rettig, Markus W. Germann, Mohamed A. Ismail, Adalgisa Batista-Parra, Manoj Munde, David W. Boykin, and W. David Wilson
The Journal of Physical Chemistry B 2012 Volume 116(Issue 19) pp:5620-5627
Publication Date(Web):April 24, 2012
DOI:10.1021/jp301143e
Thermodynamic and structural studies are commonly utilized to optimize small molecules for specific DNA interactions, and, thus, a significant amount of binding data is available. However, the dynamic processes that are involved in minor groove complex formation and maintenance are not fully understood. To help define the processes involved, we have conducted 1D and 2D NMR in conjunction with biosensor-SPR experiments with a variety of compounds and symmetric, as well as asymmetric, AT tract DNA sequences. Surprisingly, the NMR data clearly show exchange between equivalent binding sites for strongly binding compounds like netropsin and DB921 (Ka > 108 M–1) that does not involve dissociation off the DNA. A quantitative analysis of the data revealed that these bound exchange rates are indeed much faster than the macroscopic dissociation rates which were independently determined by biosensor-SPR. Additionally, we could show the existence of at least two 1:1 compound DNA complexes at the same site for the interaction of these compounds with an asymmetric DNA sequence. To explain this behavior we introduced a model in which the ligand is rapidly flipping between two orientations while in close association with the DNA. The ligand reorientation will contribute favorably to the binding entropy. As the potential of minor groove binders to form more than a single complex with asymmetric, as well as symmetric, duplexes is widely unknown, the consequences for binding thermodynamics and compound design are discussed.
Co-reporter:Christopher N. Johnson, Alexander M. Spring, Dimitri Sergueev, Barbara R. Shaw, and Markus W. Germann
Biochemistry 2011 Volume 50(Issue 19) pp:
Publication Date(Web):March 28, 2011
DOI:10.1021/bi200083d
Numerous DNA chemistries for improving oligodeoxynucleotide (ODN)-based RNA targeting have been explored. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN’s are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH3 modification on the nucleic acid structure and RNase H1 enzyme activity, we have investigated two DNA/RNA hybrids containing either a RP or SP BH3 modification by nuclear magnetic resonance (NMR) spectroscopy. TM studies show that the stabilities of RP and SP modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to that of an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. To characterize such similar structures, we used a large number of NMR restraints (including dipolar couplings and backbone torsion angles) to determine structural features that were important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total Rx values of <6%) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In the case of the SP modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the SP BH3 group in the major groove while the RP BH3 group points away from the DNA. However, this creates a potential clash of the RP BH3 groups with important RNase H1 residues in a complex, while the SP BH3 groups could be tolerated. We therefore predict that on the basis of our NMR structures a fully RP BH3 DNA/RNA hybrid would not be a substrate for RNase H1.
Co-reporter:Jin Zhang
Biopolymers 2011 Volume 95( Issue 11) pp:755-762
Publication Date(Web):
DOI:10.1002/bip.21642
Abstract
Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH2, Ac-I-G-G-NH2, Ac-I-G-G-N-NH2 and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13–0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4–9 × 10−3 s−1. Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 755-762, 2011.
Co-reporter:Markus W. Voehler, Galen Collier, John K. Young, Michael P. Stone, Markus W. Germann
Journal of Magnetic Resonance 2006 Volume 183(Issue 1) pp:102-109
Publication Date(Web):November 2006
DOI:10.1016/j.jmr.2006.08.002
The pursuit for more sensitive NMR probes culminated with development of the cryogenic cooled NMR probe. A key factor for the sensitivity is the overall resistance of RF circuitry and sample. Lowering the coil temperature to ∼25 K and the use of superconducting coil material has greatly reduced the resistance contribution of the hardware. However, the resistance of a salty sample remains the same and evolves as the major factor determining the signal-to-noise ratio. Several approaches have been proposed to reduce the resistance contribution of the sample. These range from encapsulating proteins in a water cavity formed by reverse micelles in low viscosity fluids to the optimal selection of low mobility, low conductivity buffer ions. Here we demonstrate that changing the sample diameter has a pronounced effect on the sample resistance and this results in dramatic improvements of the signal-to-noise ratio and shorter π/2 pulses. We determined these parameters for common 5 mm NMR tubes under different experimental conditions and compared them to the 2, 3 and 4 mm tubes, in addition, 5 mm Shigemi tubes were included since these are widely used. We demonstrate benefits and applicability of studying NMR samples with up to 4 M salt concentrations in cryogenic probes. Under high salt conditions, best results in terms of short π/2 pulses and high signal-to-noise ratios are obtained using 2 or 3 mm NMR tubes, especially when limited sample is available. The 4 mm tube is preferred when sample amounts are abundant at intermediate salt conditions.
Co-reporter:Subrata H. Mishra;Christopher M. Shelley;Doyle J. Barrow Jr.;Martyn K. Darby
Biopolymers 2006 Volume 83(Issue 4) pp:
Publication Date(Web):6 JUL 2006
DOI:10.1002/bip.20565
The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev–RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C2H2-type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (Kd's) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the α-helical portion of the zinc fingers and target the bulge region of RREIIB-TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements. © 2006 Wiley Periodicals, Inc. Biopoly 83:352–364, 2006
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Co-reporter:Christopher N. Johnson, Alexander M. Spring, Sunil Desai, Richard P. Cunningham, Markus W. Germann
Journal of Molecular Biology (24 February 2012) Volume 416(Issue 3) pp:425-437
Publication Date(Web):24 February 2012
DOI:10.1016/j.jmb.2011.12.051
DNA sequence context has long been known to modulate detection and repair of DNA damage. Recent studies using experimental and computational approaches have sought to provide a basis for this observation. We have previously shown that an α-anomeric adenosine (αA) flanked by cytosines (5′CαAC-3′) resulted in a kinked DNA duplex with an enlarged minor groove. Comparison of different flanking sequences revealed that a DNA duplex containing a 5′CαAG-3′ motif exhibits unique substrate properties. However, this substrate was not distinguished by unusual thermodynamic properties. To understand the structural basis of the altered recognition, we have determined the solution structure of a DNA duplex with a 5′CαAG-3′ core, using an extensive set of restraints including dipolar couplings and backbone torsion angles. The NMR structure exhibits an excellent agreement with the data (total RX < 5.3%). The αA base is intrahelical, in a reverse Watson–Crick orientation, and forms a weak base pair with a thymine of the opposite strand. In comparison to the DNA duplex with a 5′CαAC-3′ core, we observe a significant reduction of the local perturbation (backbone, stacking, tilt, roll, and twist), resulting in a straighter DNA with narrower minor groove. Overall, these features result in a less perturbed DNA helix and obscure the presence of the lesion compared to the 5′CαAC-3′ sequence. The improved stacking of the 5′CαAG-3′ core also affects the energetics of the DNA deformation that is required to form a catalytically competent complex. These traits provide a rationale for the modulation of the recognition by endonuclease IV.Download high-res image (368KB)Download full-size imageHighlights► DNA sequence context modulates recognition of damaged DNA. ► Endonuclease IV recognizes α-anomeric damage with different efficiency depending on flanking sequence. ► Flanking sequences determine kink and minor groove topology, which in turn affects recognition. ► The local thermodynamic stability does not explain the enzyme behavior. ► The C-αA-G sequence environment conceals the lesion; no kink, less opening of the minor groove. This rationalizes the enzyme behavior.
Co-reporter:Subrata H. Mishra, Alexander M. Spring, Markus W. Germann
Journal of Molecular Biology (23 October 2009) Volume 393(Issue 2) pp:369-382
Publication Date(Web):23 October 2009
DOI:10.1016/j.jmb.2009.07.066
The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C2H2 zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf β-sheet. The ΔCp of znf–RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in ΔCp, as well as ΔH and ΔS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.
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