Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.Allosteric proteins are the basic building blocks in the transmission of biological signals, allowing communication between and within cells and from the extracellular environment to the cytosol (1). Because many allosteric proteins form homo-oligomeric complexes to modulate a ligand-induced biological response (2, 3), intersubunit communication must play a crucial role in the transduction of an allosteric signal (4, 5). Identifying the molecular mechanisms of intersubunit communication has important implications, from understanding how biological systems detect and transduce signals (6, 7) to reengineering of signaling proteins (8 -10) and to developing allosteric therapeutic modulators with enhanced affinities and specificities (11,12). Despite its importance, dissecting the mechanisms of transduction of allosteric signals from one protein subunit to another has proven difficult because it requires monitoring intermediate liganded states that are poorly populated, especially if the protein displays positive ligand binding cooperativity. Therefore, one of the remaining unresolved issues in allostery is dissecting the directionality of pathways of signal transmissions across protein subunits.A widely used s...

In the absence of simple noninvasive measurements, the knowledge of temporal and spatial variations of axons mechanics remains scarce. By extending thermal fluctuation spectroscopy (TFS) to long protrusions, we determine the transverse amplitude thermal fluctuation spectra that allow direct and simultaneous access to three key mechanics parameters: axial tension, bending flexural rigidity and plasma membrane tension. To test our model, we use PC12 cell protrusions-a well-know biophysical model of axons-in order to simplify the biological system under scope. For instance, axial and plasma membrane tension are found in the range of nano Newton and tens of pico Newtons per micron respectively. Furthermore, our results shows that the TFS technique is capable to distinguish quasi-identical protrusions. Another advantage of our approach is the time resolved nature of the measurements. Indeed, in the case of long term experiments on PC12 protrusions, TFS has revealed large temporal, correlated variations of the protrusion mechanics, displaying extraordinary feedback control over the axial tension in order to maintain a constant tension value.

Abstract:We have used data

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRP MTB ) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRP MTB remain unclear. Here, we investigate the binding mechanism between CRP MTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRP MTB forms high-order CRP MTB –DNA oligomers through interactions with nonspecific DNA sequences or preformed CRP MTB –DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRP MTB –DNA oligomers, reduces the affinity of CRP MTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRP MTB –DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRP Ecoli ). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRP MTB –DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRP BCG ). The functional behavior, thermodynamic stability, and dimerization constant of CRP BCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRP Ecoli , illustrating that structural homology does not imply allosteric homology.