Entropic Control of an Excited Folded-Like Conformation in a Disordered Protein Ensemble

Many intrinsically disordered proteins switch between unfolded and folded-like forms in the presence of their binding partner. The possibility of a pre-equilibrium between the two macrostates is challenging to discern given the complex conformational landscape. Here, we show that CytR, a disordered DNA-binding domain, samples a folded-like excited state in its native ensemble through equilibrium multi-probe spectroscopy, kinetics and an Ising-like statistical mechanical model. The population of the excited state increases upon stabilization of the native ensemble with an osmolyte, while decreasing with increasing temperatures. A conserved proline residue, the mutation of which weakens the binding affinity to the target promoter, is found to uniquely control the population of the minor excited state. Semi-quantitative statistical mechanical modeling reveals that the conformational diffusion coefficient of disordered CytR is an order of magnitude slower than the estimates from folded domains. The osmolyte and proline mutation smoothen and roughen up the landscape, respectively, apart from modulation of populations. Our work uncovers general strategies to probe for excited structured states in disordered ensembles, and to measure and modulate the roughness of the disordered landscapes, inter-conversion rates of species and their populations.     

Lettuce production and antioxidant capacity are differentially modified by salt stress and light intensity under ambient and elevated CO2

As a consequence of the increasing importance of vegetables in the human diet, there is an interest in enhancing both the productivity and quality of vegetables. A number of factors, including plant genotype and environmental growing conditions, can impact the production and quality of vegetables. The objective of this study was to determine whether elevated CO₂, salinity, or high light treatments assayed individually, or salinity or high light in combination with elevated CO₂, increased biomass production and antioxidant capacity in two lettuce cultivars. Elevated CO₂ and its combination with salinity or high light increased biomass production in both cultivars, while high light treatment alone increased production in green-leaf lettuce but not in red-leaf lettuce. On the other hand, elevated CO₂ and its combination with salinity or high light increased the antioxidant capacity of both cultivars, while high light treatment alone increased the antioxidant capacity of red-leaf lettuce, but not of green-leaf lettuce.     

Sensory rhodopsin II/transducer complex formation in detergent and in lipid bilayers studied with FRET

The photophobic receptor from Natronomonas pharaonis (NpSRII) forms a photo-signalling complex with its cognate transducer (NpHtrII). In order to elucidate the complex formation in more detail, we have studied the intermolecular binding of both constituents (NpSRII and NpHtrII₁₅₇; truncated at residue 157) in detergent buffers, and in lipid bilayers using FRET. The data for hetero-dimer formation of NpSRII/NpHtrII in detergent agrees well with K_D values (∼ 200 nM) described in the literature. In lipid bilayers, the binding affinity between proteins in the NpSRII/NpHtrII complex is at least one order of magnitude stronger. In detergent the strength of binding is similar for both homo-dimers (NpSRII/NpSRII and NpHtrII/NpHtrII) but significantly weaker (K_D  ∼ 16 μM) when compared to the hetero-dimer. The intermolecular binding is again considerably stronger in lipid bilayers; however, it is not as strong as that observed for the hetero-dimer. At a molar transducer/lipid ratio of 1:2000, which is still well above physiological concentrations, only 40% homo-dimers are formed. Apparently, in cell membranes the formation of the assumed functionally active oligomeric 2:2 complex depends on the full-length transducer including the helical cytoplasmic part, which is thought to tighten the transducer-dimer association.     

The 1.7 A crystal structure of Dronpa: a photoswitchable green fluorescent protein

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.