Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR.

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C-13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011 Å and 1.060 ± 0.011 Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010 Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041 Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083 Å and 1.087 Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041 Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance.

Multidrug resistance is a major challenge to cancer chemotherapy. The multidrug resistance phenotype is associated with the overexpression of the adenosine triphosphate (ATP)-driven transmembrane efflux pumps in cancer cells. Here, we report a lipid membrane-coated silica-carbon (LSC) hybrid nanoparticle that targets mitochondria through pyruvate, to specifically produce reactive oxygen species (ROS) in mitochondria under near-infrared (NIR) laser irradiation. The ROS can oxidize the NADH into NAD+ to reduce the amount of ATP available for the efflux pumps. The treatment with LSC nanoparticles and NIR laser irradiation also reduces the expression and increases the intracellular distribution of the efflux pumps. Consequently, multidrug-resistant cancer cells lose their multidrug resistance capability for at least 5 days, creating a therapeutic window for chemotherapy. Our in vivo data show that the drug-laden LSC nanoparticles in combination with NIR laser treatment can effectively inhibit the growth of multidrug-resistant tumors with no evident systemic toxicity.