Visualization 1.mp4

Manipulation of a microbubble between two horizontally opposed optical fibers due to the switching of the temperature gradient

Carbohydrate Affinity for the Glucose–Galactose Binding Protein Is Regulated by Allosteric Domain Motions

Protein function, structure, and dynamics are intricately correlated, but studies on structure–activity relationships are still only rarely complemented by a detailed analysis of dynamics related to function (functional dynamics). Here, we have applied NMR to investigate the functional dynamics in two homologous periplasmic sugar binding proteins with bidomain composition: <i>Escherichia coli</i> glucose/galactose (GGBP) and ribose (RBP) binding proteins. In contrast to their structural and functional similarity, we observe a remarkable difference in functional dynamics: For RBP, the absence of segmental motions allows only for isolated structural adaptations upon carbohydrate binding in line with an <i>induced fit</i> mechanism; on the other hand, GGBP shows extensive segmental mobility in both <i>apo</i> and <i>holo</i> states, enabling selection of the most favorable conformation upon carbohydrate binding in line with a <i>population shift</i> mechanism. Collective segmental motions are controlled by the hinge composition: by swapping two identified key residues between RBP and GGBP we also interchange their segmental hinge mobility, and the doubly mutated GGBP* no longer experiences changes in conformational entropy upon ligand binding while the complementary RBP* shows the segmental dynamics observed in wild-type GGBP. Most importantly, the segmental interdomain dynamics always increase the apparent substrate affinity and thus, are functional, underscoring the allosteric control that the hinge region exerts on ligand binding

Calibration-Free Electrochemical Biosensors Supporting Accurate Molecular Measurements Directly in Undiluted Whole Blood

The need to calibrate to correct for sensor-to-sensor fabrication variation and sensor drift has proven a significant hurdle in the widespread use of biosensors. To maintain clinically relevant (±20% for this application) accuracy, for example, commercial continuous glucose monitors require recalibration several times a day, decreasing convenience and increasing the chance of user errors. Here, however, we demonstrate a “dual-frequency” approach for achieving the calibration-free operation of electrochemical biosensors that generate an output by using square-wave voltammetry to monitor binding-induced changes in electron transfer kinetics. Specifically, we use the square-wave frequency dependence of their response to produce a ratiometric signal, the ratio of peak currents collected at responsive and non- (or low) responsive square-wave frequencies, which is largely insensitive to drift and sensor-to-sensor fabrication variations. Using electrochemical aptamer-based (E-AB) biosensors as our test bed, we demonstrate the accurate and precise operation of sensors against multiple drugs, achieving accuracy in the measurement of their targets of within better than 20% across dynamic ranges of up to 2 orders of magnitude without the need to calibrate each individual sensor

Simulation-Based Approach to Determining Electron Transfer Rates Using Square-Wave Voltammetry

The efficiency with which square-wave voltammetry differentiates faradic and charging currents makes it a particularly sensitive electroanalytical approach, as evidenced by its ability to measure nanomolar or even picomolar concentrations of electroactive analytes. Because of the relative complexity of the potential sweep it uses, however, the extraction of detailed kinetic and mechanistic information from square-wave data remains challenging. In response, we demonstrate here a numerical approach by which square-wave data can be used to determine electron transfer rates. Specifically, we have developed a numerical approach in which we model the height and the shape of voltammograms collected over a range of square-wave frequencies and amplitudes to simulated voltammograms as functions of the heterogeneous rate constant and the electron transfer coefficient. As validation of the approach, we have used it to determine electron transfer kinetics in both freely diffusing and diffusionless surface-tethered species, obtaining electron transfer kinetics in all cases in good agreement with values derived using non-square-wave methods

Subsecond-Resolved Molecular Measurements in the Living Body Using Chronoamperometrically Interrogated Aptamer-Based Sensors

Electrochemical, aptamer-based (E-AB) sensors support the continuous, real-time measurement of specific small molecules directly in situ in the living body over the course of many hours. They achieve this by employing binding-induced conformational changes to alter electron transfer from a redox-reporter-modified, electrode-attached aptamer. Previously we have used voltammetry (cyclic, alternating current, and square wave) to monitor this binding-induced change in transfer kinetics indirectly. Here, however, we demonstrate the potential advantages of employing chronoamperometry to measure the change in kinetics directly. In this approach target concentration is reported via changes in the lifetime of the exponential current decay seen when the sensor is subjected to a potential step. Because the lifetime of this decay is independent of its amplitude (e.g., insensitive to variations in the number of aptamer probes on the electrode), chronoamperometrically interrogated E-AB sensors are calibration-free and resistant to drift. Chronoamperometric measurements can also be performed in a few hundred milliseconds, improving the previous few-second time resolution of E-AB sensing by an order of magnitude. To illustrate the potential value of the approach we demonstrate here the calibration-free measurement of the drug tobramycin in situ in the living body with 300 ms time resolution and unprecedented, few-percent precision in the determination of its pharmacokinetic phases

Unraveling the Conformational Landscape of Ligand Binding to Glucose/Galactose-Binding Protein by Paramagnetic NMR and MD Simulations

Protein dynamics related to function can nowadays be structurally well characterized (i.e., instances obtained by high resolution structures), but they are still ill-defined energetically, and the energy landscapes are only accessible computationally. This is the case for glucose–galactose binding protein (GGBP), where the crystal structures of the apo and holo states provide structural information for the domain rearrangement upon ligand binding, while the time scale and the energetic determinants for such concerted dynamics have been so far elusive. Here, we use GGBP as a paradigm to define a functional conformational landscape, both structurally and energetically, by using an innovative combination of paramagnetic NMR experiments and MD simulations. Anisotropic NMR parameters induced by self-alignment of paramagnetic metal ions was used to characterize the ensemble of conformations adopted by the protein in solution while the rate of interconversion between conformations was elucidated by long molecular dynamics simulation on two states of GGBP, the closed-liganded (<i>holo_cl</i>) and open-unloaded (<i>apo_op</i>) states. Our results demonstrate that, in its apo state, the protein coexists between open-like (68%) and closed-like (32%) conformations, with an exchange rate around 25 ns. Despite such conformational heterogeneity, the presence of the ligand is the ultimate driving force to unbalance the equilibrium toward the <i>holo_cl</i> form, in a mechanism largely governed by a conformational selection mechanism