Interactions between a protein and a ligand are essential to all biological processes. Binding and dissociation are the two fundamental steps of ligand-protein interactions, and determine the binding affinity. Intrinsic conformational dynamics of proteins have been suggested to play crucial roles in ligand binding and dissociation. Here, we demonstrate how protein dynamics dictate the binding and dissociation of a ligand through a single-molecule kinetic analysis for a series of maltose-binding protein mutants that have different intrinsic conformational dynamics and dissociation constants for maltose. Our results provide direct evidence that the ligand dissociation is determined by the intrinsic opening rate of the protein.

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 lM through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using fulllength proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PDderived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.

Glucokinase (GK) is a monomeric allosteric enzyme and plays a pivotal role in blood glucose homeostasis. GK is regulated by GK regulatory protein (GKRP), and indirectly by allosteric effectors of GKRP. Despite the critical roles of GK and GKRP, the molecular basis for the allosteric regulation mechanism of GK by GKRP remains unclear. We determined the crystal structure of Xenopus GK and GKRP complex in the presence of fructose-6-phosphate at 2.9 Å. GKRP binds to a super-open conformation of GK mainly through hydrophobic interaction, inhibiting the GK activity by locking a small domain of GK. We demonstrate the molecular mechanism for the modulation of GK activity by allosteric effectors of GKRP. Importantly, GKRP releases GK in a sigmoidal manner in response to glucose concentration by restricting a structural rearrangement of the GK small domain via a single ion pair. We find that GKRP acts as an allosteric switch for GK in blood glucose control by the liver.hexokinase | sigmoidicity I conformational restriction | type 2 diabetes

We performed a precision measurement of the current from a single-parameter electron pump, where the potential-profile for a quantum dot was manipulated by multiple top-metal gates. In an optimally tuned condition, driven with a sinusoidal-waveform microwave at f = 0.95 GHz, B = 11 T, and T = 0.3 K, the relative deviation of the pump current from ef, δI p /ef ≡ (I p − ef)/ef was measured to be (−0.92 ± 1.37) ppm. Our experiment reproduces the current quantisation accuracy of a previous measurement of a single-parameter pump, but in a device fabricated using very different geometry, thereby indicating that accurate single-parameter pumping is insensitive to device details.

According to Bohr's complementarity principle 1 , a particle possesses wave-like properties only when the different paths the particle may take are indistinguishable. In a canonical example of a two-path interferometer with a which-path detector, observation of interference and obtaining which-path information are mutually exclusive 2,3 . Such duality has been demonstrated in optics with a pair of correlated photons 4 and in solid-state devices with phasecoherent electrons 5 . In the latter case, which-path information was provided by a charge detector embedded near one path of a two-path electron interferometer 5 .Note that suppression of interference can always be understood either as obtaining path information or as unavoidable back action by the detector 3 . The present study reports on dephasing of an Aharonov-Bohm (AB) ring interferometer 6 via a coupled charge detector adjacent to the ring. In contrast to the two-path interferometer, charge detection in the ring does not always provide path information. Indeed, we found that the interference was suppressed only when path information could be acquired, even if only in principle. This demonstrates that dephasing does not always take place by coupling the 'environment' to the interfering particle: path information of the particle must be available too. Moreover, this is valid regardlessof the strength of environment-interferometer coupling, which refutes the general notion of the effect of strong interaction with the environment 7 . In other words, it verifies that an acquisition of which-path information is more fundamental than the back-action in understanding quantum mechanical complementarity. Recently, a series of electronic 'which-path' experiments have been performed in mesoscopic solid-state devices. 5 The devices, fabricated in the plane of a highmobility two-dimensional electron gas (2DEG), were based on a double-path interferometer, consisting of an open Aharonov-Bohm (AB) ring, with a source and a drain of electrons weakly coupled to the open ring 5 . In one path of the interferometer a coherent quantum dot (QD) was embedded 5-6,8 , being electrostatically coupled to a quantum-point-contact (QPC) charge detector (in the immediate proximity to the QD).An electron trapped in the QD modified the conductance of the nearby QPC and thus allowed charge detection by the QPC 5,[9][10][11] . Being an open geometry, with multiple grounded drains (bases) along the paths of the electron, assured that only two paths interfered while the backscattered electrons were drained out by the grounded bases.Thus, the detection of a charge inside the QD (by the QPC) provided path information,

We experimentally investigate the charge (isospin) frustration induced by a geometrical symmetry in a triangular triple quantum dot. We observe the ground-state charge configurations of sixfold degeneracy, the manifestation of the frustration. The frustration results in omnidirectional charge transport, and it is accompanied by nearby nontrivial triple degenerate states in the charge stability diagram. The findings agree with a capacitive interaction model. We also observe unusual transport by the frustration, which might be related to elastic cotunneling and the interference of trajectories through the dot. This work demonstrates a unique way of studying geometrical frustration in a controllable way.

Immobilization of proteins in a functionally active form and proper orientation is crucial for effective surface-based analysis of proteins. Here we present a general method for controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid and click chemistry. The utility and potential of this method was demonstrated by applying it to the analysis of interaction between a pathogenic protein DrrA of Legionella pneumophila and its binding partner Rab1 of human. Kinetic analysis of Rab1 binding onto the DrrA-immobilized surfaces using surface plasmon resonance revealed that immobilization of site-specifically biotinylated DrrA results in about 10-fold higher sensitivity in binding assay than the conventional immobilization of DrrA with random orientation. The present method is expected to find wide applications in the fields of the surface-based studies of protein-protein (or ligand) interactions, drug screening, biochip, and single molecule analysis.

The cell division cycle consists of a series of temporally ordered events. Cell cycle kinases and phosphatases provide key regulatory input, but how the correct substrate phosphorylation and dephosphorylation timing is achieved is incompletely understood. Here we identify a PxL substrate recognition motif that instructs dephosphorylation by the budding yeast Cdc14 phosphatase during mitotic exit. The PxL motif was prevalent in Cdc14-binding peptides enriched in a phage display screen of native disordered protein regions. PxL motif removal from the Cdc14 substrate Cbk1 delays its dephosphorylation, whereas addition of the motif advances dephosphorylation of otherwise late Cdc14 substrates. Crystal structures of Cdc14 bound to three PxL motif substrate peptides provide a molecular explanation for PxL motif recognition on the phosphatase surface. Our results illustrate the sophistication of phosphatase-substrate interactions and identify them as an important determinant of ordered cell cycle progression.