Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

Metal ion - Dehydrin interactions investigated by affinity capillary electrophoresis and computer models.

Dehydrins are specialized proteins which are related to environmental stress tolerance in plants. The proteins can bind different metal ions and have versatile other functions such as reduction of reactive oxygen species and acting as transcription factor. The structure determination of proteins from this family is challenging, since they have a high number of disordered structure elements. Consequently, to determine the functionality of these proteins on a molecular basis a computed model is helpful. This work focuses on a model for the Arabidopsis thaliana dehydrin AtHIRD11. To develop a model which reflects experimental data from literature and own binding data from affinity capillary electrophoresis experiments, a more rigid state of this protein was chosen. The Cu2+-complex of this protein was formed and evaluated. The model explains some of the properties of the complexes. Possible Cu2+-bindings site were found and the change of conformations were investigated via molecular dynamics simulation. The AtHIRD11-Cu2+-complex is a first approach towards a complex model for a structural versatile protein, which is already sufficient to explain binding data and possible structure elements.

The Arabidopsis KS-type dehydrin recovers lactate dehydrogenase activity inhibited by copper with the contribution of His residues.

Dehydrin, which is one of the late embryogenesis abundant (LEA) proteins, is involved in the ability of plants to tolerate the lack of water. Although many reports have indicated that dehydrins bind heavy metals, the physiological role of this metal binding has not been well understood. Here, we report that the Arabidopsis KS-type dehydrin (AtHIRD11) recovered the lactate dehydrogenase (LDH) activity denatured by Cu(2+). The LDH activity was partially inhibited by 0.93 µM Cu(2+) but totally inactivated by 9.3 µM Cu(2+). AtHIRD11 recovered the activity of LDH treated with 9.3 µM Cu(2+) in a dose-dependent manner. The recovery activity of AtHIRD11 was significantly higher than those of serum albumin and lysozyme. The conversion of His residues to Ala in AtHIRD11 resulted in the loss of the Cu(2+) binding of the protein as well as the disappearance of the conformational change induced by Cu(2+) that is observed by circular dichroism spectroscopy. The mutant protein showed lower recovery activity than the original AtHIRD11. These results indicate that AtHIRD11 can reactivate LDH inhibited by Cu(2+) via the His residues. This function may prevent physiological damage to plants due to heavy-metal stress.

Ca(2+) -complex stability of GAPAGPLIVPY peptide in gas and aqueous phase, investigated by affinity capillary electrophoresis and molecular dynamics simulations and compared to mass spectrometric results.

Strong, sequence-specific gas-phase bindings between proline-rich peptides and alkaline earth metal ions in nanoESI-MS experiments were reported by Lehmann et al. (Rapid Commun. Mass Spectrom. 2006, 20, 2404-2410), however its relevance for physiological-like aqueous phase is uncertain. Therefore, the complexes should also be studied in aqueous solution and the relevance of the MS method for binding studies be evaluated. A mobility shift ACE method was used for determining the binding between the small peptide GAPAGPLIVPY and various metal ions in aqueous solution. The findings were compared to the MS results and further explained using computational methods. While the MS data showed a strong alkaline earth ion binding, the ACE results showed nonsignificant binding. The proposed vacuum state complex also decomposed during a molecular dynamic simulation in aqueous solution. This study shows that the formed stable peptide-metal ion adducts in the gas phase by ESI-MS does not imply the existence of analogous adducts in the aqueous phase. Comparing peptide-metal ion interaction under the gaseous MS and aqueous ACE conditions showed huge difference in binding behavior.

Precise small volume sample handling for capillary electrophoresis.

CE is one of the most important analytical techniques. Although the injected sample volume in CE is only in the nanoliter range, most commercial CE instruments need approximately 50 µL of the sample in the injection vial to perform the analysis. Hence, in order to fully profit from the low injection volumes, smaller vial volumes are required. Thus experiments were performed using silicone oil, which has higher density than water (1.09 g/mL) to replace sample dead volume in the vial. The results were compared to those performed without using the silicone oil in the sample vial. As an example five standard proteins namely beta-lactoglobulin, BSA, HSA, myoglobin, and ovalbumin, and one of the coagulation cascade involved proteins called vitonectin were investigated using CE. Mobility ratios and peak areas were compared. However, no significant changes were observed (RSDs% for mobility ratios and peak areas were better than 0.9 and 5.8%, respectively). Afterwards, an affinity CE method was used to investigate the interactions of two proteins, namely HSA and vitronectin, with three ligands namely enoxaparin sodium, unfractionated heparin, and pentosan polysulfate sodium. Mobility shift precision results showed that the employment of the filling has no noticeable effect on any of the protein-ligand interactions. Using a commercial PrinCE instrument and an autosampler the required sample volume is reduced down to 10 µL, and almost this complete volume can be subsequently injected during repeated experiments.

Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.

To facilitate the implementation of affinity capillary electrophoresis into routine binding screening studies of proteins with metal ions, method acceleration, transfer and precision improvement were investigated. Affinity capillary electrophoresis was accelerated by using shorter capillaries, employing lower sample concentrations and smaller injection volumes. Intra- and inter-instrument method transfers were investigated considering the temperature setting of the capillary cooling system. For intra-instrument method transfer, similar results were obtained when transferring a method from a long (62 cm) to a short (31 cm) capillary. The analysis time was reduced from 9 to 4 min. In case of inter-instrument method transfer, interaction results showed small variation on the capillary electrophoresis instrument with inefficient capillary cooling system. Binding measurement precision was enhanced by slightly pushing the sample above the beginning of the capillary. Changing the buffer vials after each 30 runs and employing extra flushing after each 60 subsequent runs further enhanced the precision. The use of 0.1 molar ethylenediaminetetraacetic acid in the rinsing solution successfully desorbs the remaining metal ions from the capillary wall. Excellent precision for apparent mobility ratio measurements was achieved for different protein-metal ion interactions (relative standard deviation of 0.16-0.89%, 15 series, 12 runs for each).

Data quality in drug discovery: the role of analytical performance in ligand binding assays.

Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, ß-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

Recent advances in affinity capillary electrophoresis for binding studies.

The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry.

Assessment of a computerised decision support system for allergic rhino-conjunctivitis counselling in German pharmacy.

OBJECTIVE: In a prospective two-phase intervention study we evaluated a newly developed computerised pharmacy decision support system (PDSS) for the counselling of patients with allergic rhinitis and conjunctivitis. METHOD: Community pharmacists were invited to counsel a virtual patient in a first interview. Immediately thereafter, we trained pharmacists to operate the PDSS, which they applied in a second patient interview immediately following the training. We assessed the completeness of mandatory questions asked as defined by national guidelines. RESULTS: Participating pharmacists (n = 50, 78% female, mean age 34 [IQR: 27-40] years, 8 [2-14.5] years of practical experience, 32% specialised in community pharmacy) asked considerably more mandatory questions to confirm appropriateness of self-medication with 7 (5.25-9; 78%) from 9 questions compared to 2 (1-3; 22%) without PDSS (median; P < 0.001). In particular, using the PDSS more than doubled mandatory questions (9/12, 6.25-10; 75% vs. 4/12, 3-5; 33%; P < 0.001) relevant for appropriate drug selection. CONCLUSION: Pharmacists omitted many questions mandatory to assess whether self-medication is appropriate. Using the newly developed PDSS more than doubled the number of mandatory questions asked. The results suggest that the PDSS is ready for evaluation of its impact in real patients.