The energy landscape of a small RNA tetraloop hairpin is explored by temperature jump kinetics and base-substitution. The folding kinetics are single-exponential near the folding transition midpoint T(m). An additional fast phase appears below the midpoint, and an additional slow phase appears above the midpoint. Stem mutation affects the high-temperature phase, while loop mutation affects the low-temperature phase. An adjusted 2-D lattice model reproduces the temperature-dependent phases, although it oversimplifies the structural interpretation. A four-state free energy landscape model is generated based on the lattice model. This model explains the thermodynamics and multiphase kinetics over the full temperature range of the experiments. An analysis of three variants shows that one of the intermediate RNA structures is a stacking-related trap affected by stem but not loop modification, while the other is an early intermediate that forms some stem and loop structure. Even a very fast-folding 8-mer RNA with an ideal tetraloop sequence has a rugged energy landscape, ideal for testing analytical and computational models.

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of ␤-catenin. Activation of PKA is essential for PTH-induced ␤-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of ␤-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity.[Keywords: PTH signaling; LRP6; osteoblasts; ␤-catenin; PKA] Supplemental material is available at http://www.genesdev.org.

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Hydrogels with tunable viscoelasticity hold promise as materials that can recapitulate many dynamic mechanical properties found in native tissues. Here, covalent adaptable boronate bonds are exploited to prepare hydrogels that exhibit fast relaxation, with relaxation time constants on the order of seconds or less, but are stable for long‐term cell culture and are cytocompatible for 3D cell encapsulation. Using human mesenchymal stem cells (hMSC) as a model, the fast relaxation matrix mechanics are found to promote cell–matrix interactions, leading to spreading and an increase in nuclear volume, and induce yes‐associated protein/PDZ binding domain nuclear localization at longer times. All of these effects are exclusively based on the hMSCs' ability to physically remodel their surrounding microenvironment. Given the increasingly recognized importance of viscoelasticity in controlling cell function and fate, it is expected that the synthetic strategies and material platform presented should provide a useful system to study mechanotransduction on and within viscoelastic environments and explore many questions related to matrix biology.

The Y22W͞Q33Y͞G46,48A mutant of the protein 6 -85 folds in a few microseconds at room temperature. We find that its folding kinetics are probe-dependent under a strong bias toward the native state, a new signature for downhill folding. The IR-and fluorescence-detected relaxation time scales converge when the native bias is removed by raising the temperature, recovering activated two-state folding. Langevin dynamics simulations on one-and 2D free energy surfaces tunable from two-state to downhill folding reproduce the difference between the IR and fluorescence experiments, as well as the temperature and viscosity trends. In addition, the 2D surface reproduces the stretched exponential dynamics that we fit to the glucose solution experimental data at short times. Nonexponential dynamics at <10 s is a signature either for local free energy minima along the reaction coordinate (''longitudinal roughness''), or for folding on a higherdimensional free energy surface (''transverse roughness'').fluorescence ͉ infrared ͉ helix bundle ͉ amide band ͉ landscape roughness T he possibility of downhill (type 0) folding is one of the central but least expected predictions of the energy landscape theory. As discussed by Bryngelson et al. (1), folding could proceed downhill in free energy when there is a sufficiently strong bias toward the native state. When the bias is reduced by heat denaturation, activated folding over a barrier (the type 1 scenario) is recovered (1). This switch corresponds to a transition from potentially nonexponential diffusion on a rough free energy surface to activated kinetics with a single rate coefficient, k a . Type 1 folding seems to be the norm among natural proteins, but type 0 folding remains a possibility for proteins with an unusually strong native bias.Significant experimental progress has been made in the search for downhill folding kinetics (2). With the advent of fast laser initiation techniques (3), including laser T-jumps (4-6), the necessary nano-to microsecond time scales have become accessible. In 1999, Sabelko et al. (7) reported downhill formation of a phosphoglycerate kinase (PGK) folding intermediate. The nonexponential dynamics could be tuned toward a single exponential by cold or heat denaturing the protein, decreasing the bias toward the native state. The C-terminal domain of PGK was later shown to be responsible for this behavior (8). Kinetic evidence indicating downhill folding to the native state has also been reported. Activated kinetics of the engineered 6-85 protein were preceded by a fast (k m Ϸ 1 s Ϫ1 ) ''molecular phase'' (9). This new phase was attributed to a substantial population en route between the native and denatured states and accounted for nearly the entire signal in stabilizing glucose solution (10). The experimental downhill folding ''speed limit'' is in agreement with the preexponential factor calculated by Portman et al. (11). Estimates based on diffusion of denatured proteins (12, 13), on generic folding models (14), and on molecular dynamics studies of th...

There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.photodegradable hydrogel | human mesenchymal stem cell | spatial matrix stiffness

We report real-time observations of the folding and melting of DNA by probing two active sites of a hairpin structure, the bases and the stem end, and using an ultrafast T-jump. Studies at different initial temperatures (before, during, and after melting) provide the time scale of water heating (<20 ps), single-strand destacking (700 ps to 2 ns), and hairpin destacking (microseconds and longer) in solutions of various ionic strengths and pH values. The behavior of transient changes gives direct evidence to the existence of intermediate collapsed structures, labile in destacking but compact in nature, and indicates that melting is not a two-state process. We propose a landscape that is defined by these nucleation structures and destacking for efficient folding and melting.biopolymers ͉ macromolecular folding ͉ ultrafast T-jump T he folding of a biomolecule is an inherently complex process involving an energy landscape that describes the possible conformations and nuclear/segment motions (1-7). Different time scales (8-10) are involved and span the slow, largeamplitude motions and fast, local motions. The former is the rate-limiting step in the folding process and involves the passage through basins and barriers of the energy surface, ultimately reaching the final native structure. In contrast, elementary local motions are those of structures that may or may not be transient intermediates; their existence is critical to the description of the pathways and rates of folding and melting. Such elementary motions are often hidden from detection when the time resolution used is relatively long. Much work has been done on protein folding and their energy landscapes. For DNA and RNA, the melting problem (11) is usually identified in textbooks by two states:however, recent reports (12-14) have suggested the existence of intermediates for hairpin nucleic acids from experiments with Ϸ10-ns or longer time resolution. To directly observe the elementary processes of folding and melting, we invoked the methodology of a T-jump with an ultrashort time resolution that reaches the thermal limit of water heating (15). The system is perturbed to a nonequilibrium state, and its relaxation toward a new equilibrium state is monitored in real time by using a probe pulse. We use two active sites for probing: the actual base dynamics through the evolution of the hypochromicity of bases and the stem-end degree of folding through the evolution of a fluorescence marker (Fig. 1). The observations were made in the time window of picoseconds to nanoseconds at different initial temperatures (before, during, and after melting) and for solutions of various pH values and ionic strengths (buffer, water, and sodium hydroxide). The hairpin secondary structure, besides being the building block of tertiary structures, also is important in biological function, e.g., folding initiation, ligand binding, and replication and transcription (16, 17). Results and DiscussionMelting at Steady State. Because UV absorbance increases upon destacking, the so-called hyp...

Electrocatalysts remain vitally important for the rational management of intermediate polysulfides (LiPSs) in the realm of Li−S batteries. In terms of transition-metal-based candidates, in situ evolution of electrocatalysts in the course of an electrochemical process has been acknowledged; nevertheless, consensus has not yet been reached on their real functional states as well as catalytic mechanisms. Herein, we report an all-chemical vapor deposition design of the defective vanadium diselenide (VSe 2 )−vertical graphene (VG) heterostructure on carbon cloth (CC) targeting a high-performance sulfur host. The electrochemistry induces the sulfurization of VSe 2 to VS 2 at Se vacancy sites, which propels the adsorption and conversion of LiPSs. Accordingly, the VSe 2 −VG@CC/S electrode harvests an excellent cycling stability at 5.0 C with a capacity decay of only 0.039% per cycle over 800 cycles, accompanied by a high areal capacity of 4.9 mAh cm −2 under an elevated sulfur loading of 9.6 mg cm −2 . Theoretical simulation combined with operando characterizations reveals the key role played by the Se vacancy with respect to the electrocatalyst evolution and LiPS regulation. This work offers insight into the rational design of heterostructure sulfur hosts throughout defect engineering.