Two double-cysteine mutants of a small protein judiciously modified so that the cysteines appear at axially opposite sides of the native fold were prepared such that different axes were defined in the two mutants. Upon reduction, the disulfide bonds are broken, and the proteins act as bifunctional ligands toward Ag nanoparticles, encouraging their assembly into nanoparticle dimers and small aggregates such that, when excited with laser light, the proteins are automatically located at electromagnetic hot spots within the aggregates. Because the protein molecules are small (~2.3 nm) and because the electromagnetic energy at a hot spot tends to increase as the size of the interparticle gap decreases, this nanoparticle-protein-nanoparticle geometry significantly enhances the Raman emission at the metallic surface. Exploiting this effect, we have recorded surface-enhanced Raman spectra (SERS) of the proteins at near-single-molecule level. The observed SERS spectra were dominated by the vibrations of molecular groups near the anchor points of the proteins.

First raised some 60 years ago, the question of whether chemically denatured proteins are fully unfolded has, in recent years, seen significantly renewed interest. This increased attention has been spurred, in large part, by new spectroscopic and computational approaches that suggest even the most highly denatured polypeptides contain significant residual structure. In contrast, the most recent scattering results uphold the long-standing view that chemically denatured proteins adopt random coil configurations. Here we review the evidence both for and against residual structure in chemically denatured proteins, and attempt to reconcile these seemingly contradictory observations.

Pure water in a highly 1 H spin-polarized state is proposed as a contrast-agent-free contrast agent to visualize its macroscopic evolution in aqueous media by MRI. Remotely enhanced liquids for image contrast (RELIC) utilizes a 1 H signal of water that is enhanced outside the sample in continuous-flow mode and immediately delivered to the sample to obtain maximum contrast between entering and bulk fluids. Hyperpolarization suggests an ideal contrast mechanism to highlight the ubiquitous and specific function of water in physiology, biology, and materials because the physiological, chemical, and macroscopic function of water is not altered by the degree of magnetization. We present an approach that is capable of instantaneously enhancing the 1 H MRI signal by up to 2 orders of magnitude through the Overhauser effect under ambient conditions at 0.35 tesla by using highly spin-polarized unpaired electrons that are covalently immobilized onto a porous, water-saturated gel matrix. The continuous polarization of radicalfree flowing water allowed us to distinctively visualize vortices in model reactors and dispersion patterns through porous media. A 1 H signal enhancement of water by a factor of ؊10 and ؊100 provides for an observation time of >4 and 7 s, respectively, upon its injection into fluids with a T1 relaxation time of >1.5 s. The implications for chemical engineering or biomedical applications of using hyperpolarized solvents or physiological fluids to visualize mass transport and perfusion with high and authentic MRI contrast originating from water itself, and not from foreign contrast agents, are immediate.Water is the driver of nature.Leonardo da Vinci I n a world where water is so ubiquitous and vital, the exchange and transport characteristics of water are fundamental for the function of an endless range of biological and industrial processes: blood physiology, protein folding, plant metabolism, biomaterial function, and oil recovery from reservoir rocks are only drops in the bucket. However, there exists a paucity of analytical tools capable of directly tracing and quantifying the transport and function of water through these already-watersaturated materials in a chemically selective and noninvasive manner. Although NMR and MRI are the best tools for this purpose, they face two main challenges. One is the lack of sensitivity inherent to all NMR experiments, especially for in vivo NMR studies of transport in biological and biomedical samples. A general approach to this sensitivity issue is the employment of high magnetic fields and cryoprobes, which is not only expensive technology but also is limited to Ͻ1 order of magnitude improvement in sensitivity. The other challenge is the lack of contrast, e.g., between the flowing water molecules being traced and the bulk water or water contained in the specimen. Current perfusion MRI techniques that address this contrast issue are dynamic susceptibility contrast-enhanced imaging (1, 2) and proton electron double-resonance imaging (PEDRI), also known as Overhaus...

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We present a generally applicable approach for monitoring protein aggregation by detecting changes in surface hydration water dynamics and the changes in solvent accessibility of specific protein sites, as protein aggregation proceeds in solution state. This is made possible through the Overhauser dynamic nuclear polarization (DNP) of water interacting with stable nitroxide spin labels tethered to specific proteins sites. This effect is highly localized due to the magnetic dipolar nature of the electron–proton spin interaction, with >80 % of their interaction occurring within 5 Å between the unpaired electron of the spin label and the proton of water. We showcase our tool on the aggregation of tau proteins, whose fibrillization is linked to neurodegenerative disease pathologies known as taupathies. We demonstrate that the DNP approach to monitor local changes in hydration dynamics with residue specificity and local contrast can distinguish specific and neat protein-protein packing leading to fibers from non-specific protein agglomeration or precipitation. The ability to monitor tau assembly with local, residue-specific, resolution, under ambient condition and in solution state will help unravel the mechanism and structural characteristics of the gradual process of tau aggregation into amyloid fibers, which remains unclear to this day.