Disordered Domain Shifts the Conformational Ensemble of the Folded Regulatory Domain of the Multidomain Oncoprotein c-Src.

c-Src kinase is a multidomain non-receptor tyrosine kinase that aberrantly phosphorylates several signaling proteins in cancers. Although the structural properties of the regulatory domains (SH3-SH2) and the catalytic kinase domain have been extensively characterized, there is less knowledge about the N-terminal disordered region (SH4UD) and its interactions with the other c-Src domains. Here, we used domain-selective isotopic labeling combined with the small-angle neutron scattering contrast matching technique to study SH4UD interactions with SH3-SH2. Our results show that in the presence of SH4UD, the radius of gyration (Rg) of SH3-SH2 increases, indicating that it has a more extended conformation. Hamiltonian replica exchange molecular dynamics simulations provide a detailed molecular description of the structural changes in SH4UD-SH3-SH2 and show that the regulatory loops of SH3 undergo significant conformational changes in the presence of SH4UD, while SH2 remains largely unchanged. Overall, this study highlights how a disordered region can drive a folded region of a multidomain protein to become flexible, which may be important for allosteric interactions with binding partners. This may help in the design of therapeutic interventions that target the regulatory domains of this important family of kinases.

Assessment of antimicrobial activity of melittin encapsulated in bicontinuous microemulsions prepared using renewable oils.

The objective of this study is to demonstrate that melittin, a well-studied antimicrobial peptide (AMP), can be solubilized in an active form in bicontinuous microemulsions (BMEs) that employ biocompatible oils. The systems investigated consisted of Winsor-III and -IV BME phases composed of Water/Aerosol-OT (AOT)/Polysorbate 85/isopropyl myristate and a Winsor-IV BME employing Polysorbate 80 and limonene. We found that melittin resided in an α-helix-rich configuration and was in an apolar environment for the AOT/Polysorbate 85 Winsor-III system, suggesting that melittin interacted with the surfactant monolayer and was in an active conformation. An apolar environment was also detected for melittin in the two Winsor-IV systems, but to a lesser extent than the Winsor-III system. Small-angle X-ray scattering analysis indicated that melittin at a concentration of 1.0 g/Laq in the aqueous subphase of the Winsor-IV systems led to the greatest impact on the BME structure (e.g., decrease of quasi-periodic repeat distance and correlation length and induction of interfacial fluidity). The antimicrobial activity of the Polysorbate 80 Winsor-IV system was evaluated against several bacteria prominent in chronic wounds and surgical site infections (SSIs). Melittin-free BMEs inhibited the growth of all tested bacteria due to its oil, limonene, while the inclusion of 1.0 g/Laq of melittin in the BMEs enhanced the activity against several bacteria. A further increase of melittin concentration in the BMEs had no further enhancement. These results demonstrate the potential utility of BMEs as a delivery platform for AMPs and other hydrophilic and lipophilic drugs to inhibit antibiotic-resistant microorganisms in chronic wounds and SSIs.

Generation of the configurational ensemble of an intrinsically disordered protein from unbiased molecular dynamics simulation.

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

Parental age affects somatic mutation rates in the progeny of flowering plants.

In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes.