We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Dynamic nuclear polarization (DNP) is apowerful way to overcome the sensitivity limitation of magic-anglespinning (MAS) NMR experiments.However,the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals.H igh-quality DNP-enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800 MHz) and fast MAS (40 kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar-based experiments.T his enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long-range contacts, which are not observed at room temperature,o pening new possibilities for structure determination.Dynamic nuclear polarization (DNP) allows transfer of polarization from the unpaired electrons of ap aramagnetic center to the surrounding nuclei, enhancing the magic-anglespinning (MAS) NMR signals by several orders of magnitude. [1] Thetechnique has been successfully applied in various fields from chemistry and materials to structural and cell biology.H owever,d espite convincing demonstrations on fibrils, [2] membrane-embedded proteins, [3] virus capsids, [4] and whole-cell assemblies, [5] thes ensitivity enhancement provided by DNP on biological systems is always associated with an umber of issues.W hile high-resolution solid-state MAS NMR spectra of proteins can be nowadays obtained at temperatures above 273 K, the resolution of the DNP NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures (at about 100 K) and in the presence of paramagnetic radicals.F urthermore,D NP spectra of protein samples are currently almost exclusively acquired at moderate magnetic fields (usually 9.4 T) for maximum DNP enhancements and using 3.2 mm rotors with MAS rates up to 15 kHz. Under these experimental conditions,t he lines are broadened by both homogeneous broadening,o wing to incomplete averaging of nuclear and hyperfine interactions, and inhomogeneous broadening,a rising from protein conformational disorder. [6] Thelack of resolution associated with the DNP enhancements is therefore amajor obstacle for the detailed structural study of uniformly labeled biomolecular samples.This limitation has been previously addressed by using increased temperatures (180-200 K) [7] and higher magnetic field strengths [8] at the expense of lower DNP enhancements, as well as by exploring improved sample preparation methods. [9] Whilst these initial attempts have shown the potential for improving resolution in DNP spectra, af urther improvement is imperative to take full advantage of the signal amplification potentially provided by DNP.O ne factor that holds promise is the MAS rate.T he availability of prob...

Potent second-generation thrombin aptamers adopt a duplex-quadruplex bimodular folding and recognize thrombin exosite II with very high affinity and specificity. A sound model of these oligonucleotides, either free or in complex with thrombin, is not yet available. Here, a structural study of one of these aptamers, HD22-27mer, is presented. The crystal structure of this aptamer in complex with thrombin displays a novel architecture in which the helical stem is enchained to a pseudo-G-quadruplex. The results also underline the role of the residues that join the duplex and quadruplex motifs and control their recruitment in thrombin binding.

Cononsolvency refers to the experimental finding that poly(N-isopropylacrylamide), PNIPAM, has a coil conformation in both pure water and pure methanol, at 20 °C and 1 atm, but assumes a globule conformation in methanol-water solutions, over the 0.1 ≤ X(MeOH) ≤ 0.4 methanol molar fraction. This strange phenomenon has recently been rationalized by claiming that: (a) MeOH molecules are able to bind two distant monomers in the chain, driving collapse [Nat. Commun., 2014, 5, 4882]; (b) the preferential binding of MeOH stabilizes globule conformations due to a conformational entropy gain of the chain [J. Phys. Chem. B, 2015, 119, 15780]. In the present work a self-consistent application of the approach already used to rationalize the effect of sodium salts, urea and tetramethylurea on PNIPAM collapse [Phys. Chem. Chem. Phys., 2015, 17, 27750; 2016, 18, 14426] leads to a different explanation. The emerging scenario is that cononsolvency is caused by the fact that, on adding methanol, the competition between water and methanol molecules to make attractive interactions with PNIPAM surface causes a decrease in the magnitude of attractive energy with respect to the pure water situation, for basic geometric reasons. Polymer chains collapse to reduce this geometric frustration.

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.

Thrombin plays a pivotal role in the coagulation cascade; therefore, it represents a primary target in the treatment of several blood diseases. The 15-mer DNA oligonucleotide 5′-GGTTGGTGTGGTTGG-3′, known as thrombin binding aptamer (TBA), is a highly potent inhibitor of the enzyme. TBA folds as an antiparallel chair-like G-quadruplex structure, with two G-tetrads surrounded by two TT loops on one side and a TGT loop on the opposite side. Previous crystallographic studies have shown that TBA binds thrombin exosite I by its TT loops, T3T4 and T12T13. In order to get a better understanding of the thrombin-TBA interaction, we have undertaken a crystallographic characterization of the complexes between thrombin and two TBA mutants, TBADT3 and TBADT12, which lack the nucleobase of T3 and T12, respectively. The structural details of the two complexes show that exosite I is actually split into two regions, which contribute differently to TBA recognition. These results provide the basis for a more rational design of new aptamers with improved therapeutic action.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3′–5′ cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3′–5′ cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.