Flexible, water-soluble hosts are capable of selective molecular recognition in cellular environments and can detect neurotransmitters such as choline in cells. Both cationic and anionic water-soluble self-folded deep cavitands can recognize suitable styrylpyridinium dyes in cellular interiors. The dyes selectively accumulate in nucleotide-rich regions of the cell nucleus and cytoplasm. The hosts bind the dyes and promote their relocation to the outer cell membrane: the lipophilic cavitands predominantly reside in membrane environments but are still capable of binding suitable targets in other cellular organelles. Incubating the cells with structurally similar biomarkers such as choline, cholamine, betaine, or butyrylcholine illustrates the selective recognition. Choline and butyrylcholine can be bound by the hosts, but minimal binding is seen with betaine or cholamine. Varying the dye allows control of the optical detection method, and both "turn-on" sensing and "turn-off" sensing are possible.
However, extracting those rate constants from complex HXMS behavior requires fitting the experimental HXMS data to modeled HXMS spectra using a numerical simulations approach. Here we build on our success in using numerical simulations to fit intact protein HXMS data and adapt our approach to also fit peptide HXMS data for the first time. CcdB is a bacterial toxin that interferes with DNA gyrase's activity, leading to DNA breakage in plasmids and chromosomes, and has been proposed to fold through parallel pathways. Our results for both the intact protein and peptide data suggest that HX in CcdB occurs through sub-global and global unfolding events. This complements the previous folding mechanism derived from traditional folding kinetics measurements. In addition, these results open the door to more quantitative interpretation of complex HXMS peptide data through the application of our numerical simulations algorithm. The starting point of any protein refolding transition, whether via well defined pathway or by multiple pathways, is initiated in an ensemble of fully disordered polypeptides under folding conditions. The first intramolecular interactions in the disordered polypeptides have major effect of reduction of the search for the native configuration. In order to detect the earliest formed intramolecular contacts in the refolding ensembles by detection of the intramolecular distances, we must first determine the dimensions of the transient collapsed ensemble. Using E. coli Adenylate kinase (AK) as a model protein and microfluidic mixing device combined with time resolved FRET measurements (''the double kinetics'' method) we tested the hypothesis that at the initiation of refolding, the dependence of the mean of the segmental end-to-end distance on the segment length, Dn, is weak (Dn>30). We measured the transient distributions of segmental end to end distances of seven segments of the AK molecule, with Dn from 45 to 196 residues at 50 ms after initiation of mixing. The means of these distributions range from 4552 Å , for the short segment, to 7052 Å for the long segment. The Flory exponent for the Dn dependence of the mean segmental end to end distance is 0.3250.01. Thus, this transient ensemble is indeed a collection of disordered molecules. We now have a benchmark for the segmental end to end distance distribution of unfolded AK molecules under folding conditions (poor solvent). Any intramolecular mean distance that would be significantly shorter can be considered as an indication of an early formed intramolecular contact. This experiment enables systematic detection of the time sequence of formation of non local contacts in the refolding protein molecules. A step which is essential for deciphering the mechanism of folding of globular proteins.
region forms a rod-like structure that projects the N-terminal functional domain of the protein from the bacterial cell surface. We have used SHRImP-TIRFM, a super-resolution fluorescence microscopy technique, to measure the end-to-end distance of a 7 repeat protein construct, and SEC-MALS-QELS to measure its hydrodynamic radius. Combined with crystal structures of both single and double repeat structures, we will assess the elongation of the repetitive region. Neuropilin-1 (NRP1) is the cellular growth factor that interacts with semaphorin 3A, placenta growth factor-2 and vascular endothelial growth factor (VEGF165). The interactions of NRP1 with these proteins initiate signaling pathways that have implications on fundamental cellular processes. NRP1 have been designated as a potential target for the treatment of various types of cancers. It is composed of two CUB domains (a1 and a2) that are connected with F5/8 type C1 (b1) and C2 (b2) domains, followed by a flexible linker, a MAM domain, membrane-anchored region and a cytoplasmic tail. The high-resolution structures of various domains of human NRP1 (a2, b1 and b2 and MAM domains) are available. However, the information of how the a1 domain is connected to the a2, b1 and b2 domains and how the overall assembly behaves is unavailable. Recently, a crystal structure of mouse a1, a2, b1 and b2 (PDB:4Z9) domains suggested that the a1 domain is linked with the rest of the domains via a flexible linker. We characterized the wild-type NRP1 composed of a1, a2, b1 and b2 domains and a mutant version that lacks glycosylation using the dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering techniques. The results from all three techniques suggest that the glycosylation is crucial for the stability and homogeneity of NRP1. Furthermore, all though no major difference was found between these two versions of NRP1 in terms of their low-resolution structures obtained using small angle X-ray scattering, their solution conformation differs significantly compared to the crystal structure of the deglycosylated version of mouse a1, a2, b1 and b2 structure. Based on our preliminary data, we hypothesize that the flexible linker between the a1 and a2 domains allow efficient interaction with ligands to initiate signaling pathways.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.