Single-molecule studies of the conformational dynamics of ABC proteins

ATP-binding cassette (ABC) proteins are an important group of proteins that are involved in various key processes in all organisms. The working mechanism of these proteins is based on the precisely coordinated changes in their three-dimensional structure. Valuable insight into the structure and changes thereof have been obtained over the past decades. However, little is known about the dynamics as well as the existence of any heterogeneity or occurrences of rare events. To address this, methods were used and developed to study ABC proteins at the single-molecule level. These measurements revealed an unrecognized complexity in the structural changes in ABC proteins. It was shown that ABC proteins can adopt many more structural states as was initially believed. By simultaneously examining the structural changes and the binding of small molecules, direct insight into how ABC proteins switch between different structural states could be obtained. From this, we observed that structural changes in ABC proteins are not only driven by the interaction with other proteins or molecules but can also occur spontaneously. By combining the single-molecule measurements with theoretical research, we could provide new insights into how ABC proteins work. Understanding the working mechanism of ABC proteins can assist in drug development against diseases that are caused by misfunctioning of human ABC proteins, and in the development of antibiotics against pathogenic bacteria

Kinetic Modelling of Transport Inhibition by Substrates in ABC Importers

Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the ‘non-cognate’ substrates not being transported differs. Some non-cognate substrates fail to trigger the appropriate conformational change in the SBP, resulting in loss of affinity for the TMD or the inability to allosterically activate transport. In another mechanism, the SBP cannot release the bound non-cognate substrate. Here, we used rate equations to derive the steady-state transport rate of cognate substrates of an ABC importer and investigated how non-cognate substrates influence this rate. We found that under limiting non-cognate substrate concentrations, the transport rate remains unaltered for each of the mechanisms. In contrast, at saturating substrate and SBP concentrations, the effect of the non-cognate substrate depends heavily on the respective mechanism. For instance, the transport rate becomes zero when the non-cognate substrate cannot be released by the SBP. Yet it remains unaffected when substrate release is possible but the SBP cannot dock onto the TMDs. Our work shows how the different mechanisms of substrate inhibition impact the transport kinetics, which is relevant for understanding and manipulating solute fluxes and hence the propagation of cells in nutritionally complex milieus

Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues

Single-Molecule Observation of Ligand Binding and Conformational Changes in FeuA

The specific binding of ligands by proteins and the coupling of this process to conformational changes is fundamental to protein function. We designed a fluorescence-based single-molecule assay and data analysis procedure that allows the simultaneous real-time observation of ligand binding and conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-bacillibactin and delivers it to the ATP-binding cassette importer FeuBC, which is involved in bacterial iron uptake. The conformational dynamics of FeuA was assessed via Förster resonance energy transfer, whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation without the involvement of the ligand. This shows that ligand interactions are not required for conformational changes in FeuA. However, ligand interactions accelerate the conformational change 10,000-fold and temporally stabilize the formed conformation 250-fold

The right time to measure anti-Xa activity in critical illness:pharmacokinetics of therapeutic dose nadroparin

BACKGROUND: Peak anti-Xa activity of low-molecular-weight heparin nadroparin is measured 3 to 5 hours after subcutaneous injection. In critically ill patients, physiological changes and medical therapies may result in peak activities before or after this interval, possibly impacting dosing.OBJECTIVES: The primary objective was to determine the percentage of critically ill patients with adequately estimated peak activities drawn 3 to 5 hours after subcutaneous administration of a therapeutic dose of nadroparin. Adequate was defined as a peak activity of ≥80% of the actual peak anti-Xa activity. If ≥80% of patients had adequately estimated peak activities in the 3- to 5-hour interval, measurement in this interval was regarded as acceptable. The secondary objective was to determine the pharmacokinetic profile of nadroparin.METHODS: In this single-center, prospective study, we evaluated anti-Xa activities in patients admitted to a general intensive care unit. After ≥4 equal doses of nadroparin, anti-Xa activity was measured according to a 12- to 24-hour sampling scheme.RESULTS: In 25 patients, anti-Xa activities drawn between 3 and 5 hours after administration ranged 80% to 100% of the actual peak activity. Compared to the threshold level of an adequate estimation in at least 20 patients (≥80%), measuring anti-Xa activities in the 3- to 5-hour interval is an acceptable method (1-tailed binomial test; P &lt; .02). We found a large interindividual variability for nadroparin exposure (mean ± SD area-under-the-curve 0-12h, 10.3 ± 4.8 IU·h/mL) and delayed elimination (t 1/2 range, 4.0-120.9 hours) despite adequate renal function. CONCLUSION: In critically ill patients, measuring anti-Xa activity in a 3- to 5-hour interval after subcutaneous injection of therapeutic nadroparin is an acceptable method to estimate the actual peak anti-Xa activity.</p

Sedative drugs in the geriatric intensive care patient

The number of elderly patients admitted to Intensive Care Units (ICUs) has increased significantly in recent years. It has been demonstrated that the elderly are more prone to inadequate drug treatment and adverse drug effects. OBJECTIVE To perform a literature review on the effects of aging on pharmacokinetic and pharmacodynamic properties of commonly used sedative drugs in Dutch ICUs. DESIGN AND METHODS Literature review using PubMed. RESULTS Literature on the effects of aging on the pharmacology of commonly used sedative drugs in the ICU is scarce. For the general population, we found that for midazolam, propofol, fentanyl and remifentanil aging is associated with an increased susceptibility of the patients for the pharmaco-dynamic properties of these agents. This effect is confirmed for some of these drugs in ICU patients. In addition, a reduction in drug clearance was observed for propofol and remifentanil. CONCLUSION Based on these findings, we suggest to start with lower dosages for midazolam, propofol, fentanyl and remifentanil in elderly ICU patients and to re-evaluate frequently and adjust the therapy by clinical effect if necessary.</p