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

Social behavior assessment in cognitively impaired older adults using a passive and remote smartphone application

BACKGROUND: In Alzheimer's Disease (AD), loss of social interactions has a major impact on well-being. Therefore, AD patients would benefit from early detection of symptoms of social withdrawal. Current measurement techniques such as questionnaires are subjective and rely on recall, in contradiction to smartphone applications, which measure social behavior passively and objectively. Here, we examine social interactions through passive remote monitoring with the smartphone application BEHAPP in cognitively impaired participants. This study aims to investigate (1) the association between demographic characteristics and BEHAPP outcome variables in cognitively normal (CN) older adults, (2) if social behavior as measured using the passive smartphone app BEHAPP is impaired in cognitively impaired (CI) participants compared to subjects with subjective cognitive decline (SCD), and CN older adults. In addition, we explored in a subset of individuals the association between BEHAPP outcomes and neuropsychiatric symptoms. METHOD: CN (n=209), SCD (n=55) and CI (n=22) participants, older than 45 years, installed the BEHAPP app on their own Android smartphone for 7-42 days. CI participants had a clinical diagnosis of mild cognitive impairment or AD-type dementia. The app continuously measured communication events, application usage and location. Neuropsychiatric Inventory (NPI) total scores were available from 20 SCD and 22 CI participants. RESULT: We found that older cognitively healthy participants called less frequently and made less use of apps. No sex effects were found. Linear models corrected for age, sex and education showed that compared to the CN and SCD groups, CI participants called less unique contacts and contacted the same contacts relatively more often (Figure 1). They also made less use of apps, visited less unique places and traveled less far from home. Higher total NPI scores were associated with more unique stay points and further travelling. Similar behavior patterns were found when correcting for multiple comparisons. CONCLUSION: Cognitively impaired individuals show reduced social activity, as measured by the smartphone application BEHAPP. Neuropsychiatric symptoms seemed only marginally associated with social behavior as measured with BEHAPP. This research shows that a passive and remote smartphone application is able to objectively and passively measure altered social behavior in a cognitively impaired population

The Effect of Pregnancy and Inflammatory Bowel Disease on the Pharmacokinetics of Drugs Related to Inflammatory Bowel Disease:A Systematic Literature Review

Due to ethical and practical reasons, a knowledge gap exists on the pharmacokinetics (PK) of inflammatory bowel disease (IBD)-related drugs in pregnant women with IBD. Before evidence-based dosing can be proposed, insight into the PK has to be gained to optimize drug therapy for both mother and fetus. This systematic review aimed to describe the effect of pregnancy and IBD on the PK of drugs used for IBD. One aminosalicylate study, two thiopurine studies and twelve studies with biologicals were included. Most drugs within these groups presented data over multiple moments before, during and after pregnancy, except for mesalazine, ustekinumab and golimumab. The studies for mesalazine, ustekinumab and golimumab did not provide enough data to demonstrate an effect of pregnancy on concentration and PK parameters. Therefore, no evidence-based dosing advice was given. The 6-thioguanine nucleotide levels decreased during pregnancy to 61% compared to pre-pregnancy levels. The potentially toxic metabolite 6-methylmercaptopurine (6-MMP) increased to maximal 209% of the pre-pregnancy levels. Although the PK of the thiopurines changed throughout pregnancy, no evidence-based dosing advice was provided. One study suggested that caution should be exercised when the thiopurine dose is adjusted, due to shunting 6-MMP levels. For the biologicals, infliximab levels increased, adalimumab stayed relatively stable and vedolizumab levels tended to decrease during pregnancy. Although the PK of the biologicals changed throughout pregnancy, no evidence-based dosing advice for biologicals was provided. Other drugs retrieved from the literature search were mesalazine, ustekinumab and golimumab. We conclude that limited studies have been performed on PK parameters during pregnancy for drugs used in IBD. Therefore, more extensive research to determine the values of PK parameters is warranted. After gathering the PK data, evidence-based dosing regimens can be developed

Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA

(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate-binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo-electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes

Nanoscale Porosity of High Surface Area Gadolinium Oxide Nanofoam Obtained With Combustion Synthesis

Nanoscale gadolinium oxide (Gd2O3) is a promising nanomaterial with unique physicochemical properties that finds various applications ranging from biomedicine to catalysis. The preparation of highly porous Gd2O3 nanofoam greatly increases its surface area thereby boosting its potential for functional use in applications such as water purification processes and in catalytic applications. By using the combustion synthesis method, a strong exothermic redox reaction between gadolinium nitrate hexahydrate and glycine causes the formation of crystalline nanoporous Gd2O3. In this study, the synthesis of Gd2O3 nanofoam is achieved with combustion synthesis at large scale (grams). Its nanoscale porosity is investigated by nitrogen physisorption and its nanoscale 3D structure by electron tomography, and the formation process is investigated as well by means of in situ heating inside the transmission electron microscope. The bulk nanofoam product is highly crystalline and porous with a surface area of 67 m2 g−1 as measured by physisorption, in good agreement with the electron tomographic 3D reconstructions showing an intricate interconnected pore network with pore sizes varying from 2 to 3 nm to tens of nanometers. In situ heating experiments point to many possibilities for tuning the porosity of the Gd2O3 nanofoam by varying the experimental synthesis conditions