To elucidate the mechanism of ␣A-crystallin chaperone function, a detailed thermodynamic analysis of its binding to destabilized, site-directed mutants of T4 lysozyme was carried out. The selected mutants form a ladder of stabilities spanning the 5-10 kcal/mol range of free energy of unfolding. The crystal structures of the majority of the mutants have been previously determined and found to be similar to that of the wild type with no evidence of static local unfolding. Complex formation between ␣A-crystallin and T4 lysozyme was observed directly via the changes in the electron paramagnetic resonance lineshape of a nitroxide introduced at a non-destabilizing, solvent exposed site in T4 lysozyme. ␣A-Crystallin differentially interacts with the mutants, binding the more destabilized ones to a larger extent despite the similar structure of their native states. Our results suggest that the states recognized by ␣A-crystallin are non-native excited states distinct from the unfolded state. Stable complexes are formed when the free energy of binding to ␣A-crystallin is on the order of the free energy associated with the transition from the excited state to the native state. Biphasic binding isotherms reveal two modes of interactions with distinct affinities and stoichiometries. Highly destabilized mutants preferentially bind to the high capacity mode, suggesting conformational preference in the use of each mode. Furthermore, binding can be enhanced by increased temperature and pH, which may be reflecting conformational changes in ␣A-crystallin oligomeric structure.In the crowded molecular environment of the cell, protein folding, stability, and solubility are critically dependent on a specialized protein machinery consisting of multiple superfamilies of heat shock proteins, the molecular chaperones. By recognizing and binding non-native protein states, chaperones protect the cell from the toxic and pathogenic consequences of protein aggregation (1, 2). These non-native states are not only a byproduct of the folding process but are also populated through fluctuations in the native structure, the rate of which increases under extreme physicochemical conditions. The diversity of the molecular chaperones has been associated with distinct roles at different stages of the protein life cycle and in response to different cellular conditions (3).The small heat shock protein (sHSP) 1 superfamily consists of oligomeric proteins of 4 -40 subunits with a molecular mass of less than 40 kDa/subunit (4 -6). The exact role of the sHSP in the heat shock response is poorly understood, although they have been associated with a spectrum of functions under permissive temperatures (7-10). In humans, six sHSP have been identified in various tissues (6). The ␣-crystallins are the main protein component in the lens, where they play a critical role in establishing and maintaining its optical properties (11-13). It has been hypothesized that ␣-crystallin chaperone function prevents the early aggregation and precipitation of lens proteins. In non-l...

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Summary 1.Invasion by alien plant species represents a challenge to land managers throughout the world as they attempt to restore frequent fire-adapted ecosystems following decades of fire exclusion. In ponderosa pine Pinus ponderosa forests of western North America, the response of alien species to restoration treatments has not been well documented, particularly for alien species capable of altering environmental conditions (transformers). Understanding alien species dynamics is critical for developing treatments that accomplish restoration goals while minimizing alien invasion. 2. We used a replicated, randomized block experiment to compare the effects of an untreated control and thin-only, burn-only and thin-burn treatments on alien and transformer understorey species at multiple spatial scales (1 m 2 , 100 m 2 and 1000 m 2 ). Data were collected pre-treatment and for multiple post-treatment years. We compared richness and cover of alien species and transformer species among treatments, and identified environmental variables correlated with transformer species cover. Indicator species analysis was used to identify transformer species associated with specific treatments. 3. Alien and transformer species richness and cover were significantly higher in the thin-burn than in all other treatments at all spatial scales. Thin-only and burn-only treatments showed greater alien and transformer species responses than the control at the larger 100-m 2 and 1000-m 2 scales. 4. Increased transformer cover was strongly correlated with increased tree crown scorch height and removal of overstorey trees. 5. The thin-burn treatment had four transformer species as indicators, the thin-only had one, while the burn-only and control had none. 6. Synthesis and applications . The results show that alien species, including transformers, respond to restoration treatments, especially the combined thin-burn treatment. Therefore monitoring for alien species invasion is an essential component of a restoration programme. Abundance of transformer species increased with increasing disturbance intensity, suggesting that less intense single-disturbance treatments (burnonly, thin-only) or incremental treatments may be preferred in some applications. Where more intense treatments are required to meet management objectives, specific strategies, such as seeding of native species, limiting grazing before and after treatment and harvesting over a protective winter snowpack, may be necessary to limit alien invasion.