A CLC-ec1 mutant reveals global conformational change and suggests a unifying mechanism for the CLC Cl/H transport cycle.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl for H). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained.

A selective class of inhibitors for the CLC-Ka chloride ion channel.

CLC proteins are a ubiquitously expressed family of chloride-selective ion channels and transporters. A dearth of pharmacological tools for modulating CLC gating and ion conduction limits investigations aimed at understanding CLC structure/function and physiology. Herein, we describe the design, synthesis, and evaluation of a collection of -arylated benzimidazole derivatives (BIMs), one of which (BIM1) shows unparalleled (>20-fold) selectivity for CLC-Ka over CLC-Kb, the two most closely related human CLC homologs.

Plasma membrane regulates Ras signaling networks.

Ras GTPases activate more than 20 signaling pathways, regulating such essential cellular functions as proliferation, survival, and migration. How Ras proteins control their signaling diversity is still a mystery. Several pieces of evidence suggest that the plasma membrane plays a critical role.

Revealing an outward-facing open conformational state in a CLC Cl(-)/H(+) exchange transporter.

CLC secondary active transporters exchange Cl(-) for H(+). Crystal structures have suggested that the conformational change from occluded to outward-facing states is unusually simple, involving only the rotation of a conserved glutamate (Gluex) upon its protonation. Using (19)F NMR, we show that as [H(+)] is increased to protonate Gluex and enrich the outward-facing state, a residue ~20 Å away from Gluex, near the subunit interface, moves from buried to solvent-exposed.

The higher level of complexity of K-Ras4B activation at the membrane.

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction.

GTP-Dependent K-Ras Dimerization.

Ras proteins recruit and activate effectors, including Raf, that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. It has been suspected that dimeric Ras may promote dimerization and activation of Raf.

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function.

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC).

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function.

A novel method for the production of fully modified K-Ras 4B.

Post-translational modifications in proteins play a major functional role. Post-translational modifications affect the way proteins interact with each other, bind nucleotides, and localize in cellular compartments. Given the importance of post-translational modifications in protein biology, development of methods to produce post-translationally modified proteins for biochemical and biophysical studies is timely and significant.

Application of reductive ¹³C-methylation of lysines to enhance the sensitivity of conventional NMR methods.

NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂.