GTP hydrolysis is modulated by Arg34 in the RASopathy-associated KRASP34R.

RAS proteins are commonly mutated in cancerous tumors, but germline RAS mutations are also found in RASopathy syndromes such as Noonan syndrome (NS) and cardiofaciocutaneous (CFC) syndrome. Activating RAS mutations can be subclassified based on their activating mechanisms. Understanding the structural basis for these mechanisms may provide clues for how to manage associated health conditions. We determined high-resolution X-ray structures of the RASopathy mutant KRASP34R seen in NS and CFCS. GTP and GDP-bound KRASP34R crystallized in multiple forms, with each lattice consisting of multiple protein conformations. In all GTP-bound conformations, the switch regions are not compatible with GAP binding, suggesting a structural mechanism for the GAP insensitivity of this RAS mutant. However, GTP-bound conformations are compatible with intrinsic nucleotide hydrolysis, including one that places R34 in a position analogous to the GAP arginine finger or intrinsic arginine finger found in heterotrimeric G proteins, which may support intrinsic GTP hydrolysis. We also note that the affinity between KRASP34R and RAF-RBD is decreased, suggesting another possible mechanism for dampening of RAS signaling. These results may provide a foothold for development of new mutation-specific strategies to address KRASP34R -driven diseases.

Label-free DNA sensors using ultrasensitive diamond field-effect transistors in solution.

Charge detection biosensors have recently become the focal point of biosensor research, especially field-effect-transistors (FETs) that combine compactness, low cost, high input, and low output impedances, to realize simple and stable in vivo diagnostic systems. However, critical evaluation of the possibility and limitations of charge detection of label-free DNA hybridization using silicon-based ion-sensitive FETs (ISFETs) has been introduced recently. The channel surface of these devices must be covered by relatively thick insulating layers ( SiO2, Si3N4, Al2O3, or Ta2O5) to protect against the invasion of ions from solution. These thick insulating layers are not suitable for charge detection of DNA and miniaturization, as the small capacitance of thick insulating layers restricts translation of the negative DNA charge from the electrolyte to the channel surface. To overcome these difficulties, thin-gate-insulator FET sensors should be developed. Here, we report diamond solution-gate FETs (SGFETs), where the DNA-immobilized channels are exposed directly to the electrolyte solution without gate insulator. These SGFETs operate stably within the large potential window of diamond (>3.0 V). Thus, the channel surface does not need to be covered by thick insulating layers, and DNA is immobilized directly through amine sites, which is a factor of 30 more sensitive than existing Si-ISFET DNA sensors. Diamond SGFETs can rapidly detect complementary, 3-mer mismatched (10 pM) and has a potential for the detection of single-base mismatched oligonucleotide DNA, without biological degradation by cyclically repeated hybridization and denature.