Protein folding occurs as a set of transitions between structural states within an energy landscape. An oversimplified view of the folding process emerges when transiently populated states are undetected because of limited instrumental resolution. Using force spectroscopy optimized for 1-μs resolution, we reexamined the unfolding of individual bacteriorhodopsin molecules in native lipid bilayers. The experimental data reveal the unfolding pathway in unprecedented detail. Numerous newly detected intermediates—many separated by as few as 2– 3 amino acids—exhibited complex dynamics, including frequent refolding and state occupancies of <10 μs. Equilibrium measurements between such states enabled the folding free-energy landscape to be deduced. These results sharpen the picture of the mechanical unfolding of membrane proteins and, more broadly, enable experimental access to previously obscured protein dynamics.

For the broadest dissemination of solid-state dynamic nuclear polarization (ssDNP) enhanced NMR as a material characterization tool, the ability to employ generic mono-nitroxide radicals as spin probes is critical. A better understanding of the factors contributing to ssDNP efficiency is needed to rationally optimize the experimental condition for the practically accessible spin probes at hand. This study seeks to advance the mechanistic understanding of ssDNP by examining the effect of electron spin dynamics on ssDNP performance at liquid helium temperatures (4-40 K). The key observation is that bi-radicals and mono-radicals can generate comparable nuclear spin polarization at 4 K and 7 T, which is in contrast to the observation for ssDNP at liquid nitrogen temperatures (80-150 K) that finds bi-radicals to clearly outperform mono-radicals. To rationalize this observation, we analyze the change in the DNP-induced nuclear spin polarization (Pn) and the characteristic ssDNP signal buildup time as a function of electron spin relaxation rates that are modulated by the mono- and bi-radical spin concentration. Changes in Pn are consistent with a systematic variation in the product of the electron spin-lattice relaxation time and the electron spin flip-flop rate that constitutes an integral saturation factor of an inhomogeneously broadened EPR spectrum. We show that the comparable Pn achieved with both radical species can be reconciled with a comparable integral EPR saturation factor. Surprisingly, the largest Pn is observed at an intermediate spin concentration for both mono- and bi-radicals. At the highest radical concentration, the stronger inter-electron spin dipolar coupling favors ssDNP, while oversaturation diminishes Pn, as experimentally verified by the observation of a maximum Pn at an intermediate, not the maximum, microwave (μw) power. At the maximum μw power, oversaturation reduces the electron spin population differential that must be upheld between electron spins that span a frequency difference matching the (1)H NMR frequency-characteristic of the cross effect DNP. This new mechanistic insight allows us to rationalize experimental conditions where generic mono-nitroxide probes can offer competitive ssDNP performance to that of custom designed bi-radicals, and thus helps to vastly expand the application scope of ssDNP for the study of functional materials and solids.

The microfossil record suggests that cyanobacteria or cyanobacteria-like prokaryotes were present on the primitive Earth in the Archaean era more than 3.5 billion years ago (28). The exquisite preservation of these microfossils is thought to reflect the intrinsic stability of the extracellular polysaccharide (EPS) and its ability to bind heavy metals as well as resist degradation (13). Extant cyanobacteria dominate the microbial populations of many extreme environments including soda lakes (Spirulina, Cyanospira), the nutrient-poor open ocean (Trichodesmium), thermal springs (Synechococcus and Mastigocladis), and the cold dry polar deserts (Chroococcidiopsis) (35). In these environments the cyanobacteria produce copious amounts of EPSs in the form of sheaths, slimes, and capsules. Very little is known about the diversity, mode of synthesis, structure, or properties of these biopolymers (19). A recent review emphasized the potential role of EPSs in the desiccation tolerance of prokaryotes (23). However, much further research is needed to resolve the specific mechanisms which biopolymers contribute to such a complex process.The terrestrial cyanobacterium Nostoc commune has a marked capacity for desiccation tolerance and can survive storage at Ϫ400 MPa (0% relative humidity) for centuries (23). The cells produce large amounts of an unusual excreted polysaccharide that contributes in at least four ways to the marked stabilization of cells during prolonged storage in the air-dried state, at low or high temperatures. First, the glycan inhibits fusion of membrane vesicles during desiccation and freezedrying (10) and acts as an immobilization matrix for a range of secreted enzymes which remain fully active after long-term air-dried storage (11,27,32). Second, the glycan provides a structural and/or molecular scaffold with rheological properties which can accommodate the rapid biophysical and physiological changes in the community upon rehydration and during recovery from desiccation. The glycan swells from brittle dried crusts to cartilaginous structures within minutes of rehydration. Third, the glycan matrix contains both lipid-and watersoluble UV radiation-absorbing pigments which protect the cell from photodegradation (12). Fourth, although epiphytes colonize the surfaces of Nostoc colonies, there is no penetration of the glycan due in part to a silicon-and calcium-rich pellicle and inherent resistance of the glycan to enzymatic breakdown. Preliminary structural work on one water-soluble UV-absorbing pigment (released from the glycan by acid hydrolysis) indicated the presence of an oligosaccharide (4), raising the possibility that the pigment may be covalently linked to the glycan in the desiccated state.An understanding of the biochemical and biophysical properties of such biopolymers and the isolation of genes and enzymes required for their synthesis and modification can lead to an understanding of the underlying principles of extremophile stability. Furthermore, one can envision the utilization of such materials f...