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.

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.

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.