Biological mechanisms are often mediated by transient interactions between multiple proteins. The isolation of intact protein complexes is essential to understanding biochemical processes and an important prerequisite for identifying new drug targets and biomarkers. However, low-affinity interactions are often difficult to detect. Here, we use a newly described method called immiscible filtration assisted by surface tension (IFAST) to isolate proteins under defined binding conditions. This method, that gives a near-instantaneous isolation, enables significantly higher recovery of transient complexes as compared to current wash-based protocols, which require re-equilibration at each of several wash steps, resulting in protein loss. The method moves proteins, or protein complexes, captured on a solid phase through one or more immiscible phase barriers that efficiently exclude the passage of non-specific material in a single operation. We use a previously described polyol-responsive monoclonal antibody (PR-mAb) to investigate the potential of this new method to study protein-binding. In addition, difficult-to-isolate complexes involving the biologically and clinically important Wnt signaling pathway were isolated. We anticipate that this simple, rapid method to isolate intact, transient complexes will enable the discoveries of new signaling pathways, biomarkers, and drug targets.
The complex mixture of conformational states exhibited by oligo(ethylene glycol)-terminated alkanethiols on Ag and Au surfaces is explored by polarization-dependent X-ray absorption spectroscopy. Three self-assembled monolayers (SAMs) with known helical or all-trans conformations are used as references to characterize a SAM with unknown conformations. This case study is used as a prototype for developing a systematic framework to extract the conformations of SAMs from the polarization dependence of several orbitals. In the case at hand, these are associated with the C-H/Rydberg bonds of the alkane, the C-H/Rydberg bonds of ethylene glycol, and the C-C bonds of the backbone. The C-H/Rydberg orbitals of the alkane and ethylene glycol are distinguished via the chemical shift of the corresponding C 1s core levels.
Abstract-In this paper we use the cortical current density based inverse solution to classify Event Related Potentials, in particular for error-related potentials elicited during a BrainComputer Interface experiment. We selected discriminant cortical sources for comparing classification performance with respect to surface EEG. We found that the data from estimated cortical sources achieves higher classification accuracy for most of the subjects. In addition, the inverse method exhibits consistently discriminant activity for the sources located over the anterior cingulate cortex region for different time points. This level of neurophysiological interpretation in terms of localisation of selected cortical sources is enabled with the use of inverse solution.
Brain error processing plays a key role in goal-directed behavior and learning in human brain. Directed transfer function (DTF) on EEG signal brings unique features for discrimination between correct and error cases in brain-computer interface (BCI) system. We describe the first application of brain connectivity features for recognizing error-related signals in non-invasive BCI. EEG signal were recorded from 16 human subjects when they monitored stimuli moving in either correct or erroneous direction. Classification performance using waveform features, brain connectivity features and their combination were compared. The result of combined features yielded highest classification accuracy, 0:85. In addition, we also show that brain connectivity at theta band around 200 ms after stimuli carry highly discriminant information between error and correct trials. This paper provides evidence that the use of connectivity features improve the performance of an EEG based BCI.
Nonnative disulfide bonds have been observed among protein aggregates in several diseases like amyotrophic lateral sclerosis, cataract and so on. The molecular mechanism by which formation of such bonds promotes protein aggregation is poorly understood. Here in this work we employ previously well characterized aggregation of hen eggwhite lysozyme (HEWL) at alkaline pH to dissect the molecular role of nonnative disulfide bonds on growth of HEWL aggregates. We employed time-resolved fluorescence anisotropy, atomic force microscopy and single-molecule force spectroscopy to quantify the size, morphology and non-covalent interaction forces among the aggregates, respectively. These measurements were performed under conditions when disulfide bond formation was allowed (control) and alternatively when it was prevented by alkylation of free thiols using iodoacetamide. Blocking disulfide bond formation affected growth but not growth kinetics of aggregates which were ∼50% reduced in volume, flatter in vertical dimension and non-fibrillar in comparison to control. Interestingly, single-molecule force spectroscopy data revealed that preventing disulfide bond formation weakened the non-covalent interaction forces among monomers in the aggregate by at least ten fold, thereby stalling their growth and yielding smaller aggregates in comparison to control. We conclude that while constrained protein chain dynamics in correctly disulfide bonded amyloidogenic proteins may protect them from venturing into partial folded conformations that can trigger entry into aggregation pathways, aberrant disulfide bonds in non-amyloidogenic proteins (like HEWL) on the other hand, may strengthen non-covalent intermolecular forces among monomers and promote their aggregation.
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