Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin-related protein 4. Here, we report a protocol for pilot-scale production in E. coli and the X-ray structure of chimeric avidin. The ligand-binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology.

Plants developed various reversible and non‐reversible acclimation mechanisms to cope with the multifaceted nature of abiotic‐stress combinations. We hypothesized that in order to endure these stress combinations, plants elicit distinctive acclimation strategies through specific trade‐offs between reproduction and defense. To investigate Brachypodium distachyon acclimation strategies to combinations of salinity, drought and heat, we applied a system biology approach, integrating physiological, metabolic, and transcriptional analyses. We analyzed the trade‐offs among functional and performance traits, and their effects on plant fitness. A combination of drought and heat resulted in escape strategy, while under a combination of salinity and heat, plants exhibited an avoidance strategy. On the other hand, under combinations of salinity and drought, with or without heat stress, plant fitness (i.e., germination rate of subsequent generation) was severely impaired. These results indicate that under combined stresses, plants’ life‐history strategies were shaped by the limits of phenotypic and metabolic plasticity and the trade‐offs between traits, thereby giving raise to distinct acclimations. Our findings provide a mechanistic understanding of plant acclimations to combinations of abiotic stresses and shed light on the different life‐history strategies that can contribute to grass fitness and possibly to their dispersion under changing environments.

1 4 1 5 1 6 Running title: Unique acclimations to abiotic-stress combinations 1 7 1 8 2 Abstract 1Plants developed various reversible and non-reversible acclimation mechanisms to cope with the 2 multifaceted nature of abiotic stress combinations. We hypothesized that in order to endure these 3 stress combinations, plants elicit distinctive acclimation strategies through specific trade-offs 4 between reproduction and defense. To investigate Brachypodium distachyon acclimation 5 strategies to combinations of salinity, drought and heat, we applied a system biology approach, 6 integrating physiological, metabolic and transcriptional analyses. We analyzed the trade-offs 7 among functional and performance traits, and their effects on plant fitness. A combination of 8 drought and heat resulted in escape strategy, while under a combination of salinity and heat, 9 plants exhibited avoidance strategy. On the other hand, under combinations of salinity and 1 0 drought, with or without heat stress, plant fitness (i.e. germination rate of subsequent generation) 1 1 was severely impaired. These results indicate that under combined stresses, plants' life-history 1 2 strategies were shaped by the limits of phenotypic and metabolic plasticity and the trade-offs 1 3 between traits, thereby giving raise to distinct acclimations. Our findings provide a mechanistic 1 4 understanding of plant acclimations to combinations of abiotic stresses and shed light on the 1 5 different life-history strategies that can contribute to grass fitness and possibly to their dispersion 1 6 under changing environments. 1 7 1 8 1 9

A structural element termed ‘hydrophobic core’ is a suppressor of spontaneous autophosphorylation in Hog1 and p38s. Practically any mutation in this core of Hog1, but not of p38, evokes spontaneous autophosphorylation. This inherent autophosphorylation suppressor is tighter in mammalian's p38s.

The homotetrameric and biotin-binding properties of avidin and streptavidin have been exploited for a myriad of biotechnological applications and theoretical studies. Among the few differences between the two proteins is the capacity of avidin to hydrolyze biotinyl p-nitrophenyl ester (BNP), as opposed to streptavidin, which fully protects the same pseudosubstrate from hydrolysis. Combined mutagenesis and X-ray analysis have been used to attempt to understand this diametric difference in activities. It was found that a charged residue and one of the loops (L3,4) are together responsible for this difference. Recently, the avidin-related analogue AVR4 was found to have an even more pronounced BNP-hydrolysis activity than avidin. Again, the combination of charged residue(s) (Asp39 and/or Arg112) and the rigid conformation of the L3,4 loop was suggested to be responsible for the observed hydrolysis reaction. However, replacement of the latter charged residues in AVR4 resulted in only a modest reduction in hydrolytic activity at most, whereas replacement of the L3,4 loop of avidin with the rigid loop of AVR4 caused a dramatic increase in the activity of avidin. These results clearly demonstrate that the main feature responsible for the observed differences in rates of hydrolysis among the avidins is the conformational status of the L3,4 loop, which imposes conformational constraints on the pseudosubstrate, thereby rendering it susceptible to nucleophilic attack by solvent. In this context, the hydrolytic properties of the avidins reflect enzyme catalysis, in that subtleties in substrate binding are the determining features of catalytic efficiency.