Subunit dimers of alpha-hemolysin expand the engineering toolbox for protein nanopores.
ABSTRACT Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane β-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.
[Show abstract] [Hide abstract]
ABSTRACT: Post-transcriptional modifications of the 3'-ends of RNA molecules have a profound impact on their stability and processing in the cell. Uridylation, the addition of uridines to 3'-ends, has recently been found to be an important regulatory signal to stabilize the tagged molecules or to direct them towards degradation. Simple and cost-effective methods for the detection of this post-transcriptional modification are not yet available. Here, we demonstrate the selective and transient binding of 3'-uridylated ssRNAs inside the β barrel of the staphylococcal alpha-hemolysin (αHL) nanopore, and investigate the molecular basis of uridine recognition by the pore. We show the discrimination of 3'-oligouridine tails on the basis of their lengths and propose the αHL nanopore as a useful sensor for this biologically relevant RNA modification.ACS Nano 12/2013; 8(2). DOI:10.1021/nn4050479 · 12.03 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: The aim of this article is to educate neonatal caregivers about metagenomics. This scientific field uses novel and ever changing molecular methods to identify how infants become colonized with microbes after birth. Publications using metagenomics appear infrequently in the neonatal literature because clinicians are unaccustomed to the analytical techniques, data interpretation, and illustration of the results. This review covers those areas. After a brief introduction of neonatal citations forthcoming from metagenomic studies, the following topics are covered: (1) the history of metagenomics, (2) a description of current and emerging instruments used to define microbial populations in human organs, and (3) how extensive databases generated by genome analyzers are examined and presented to readers. Clinicians may feel like they are learning a new language; however, they will appreciate this task is essential to understanding and practicing neonatal medicine in the future. © 2013 S. Karger AG, Basel.Neonatology 11/2013; 105(1):14-24. DOI:10.1159/000354944 · 2.37 Impact Factor
Article: Nanoscale Electrochemistry[Show abstract] [Hide abstract]
ABSTRACT: This review reports recent advances in the field of nanoscale electrochemistry. We specifically focus on new electrochemical phenomena, properties, and technological capabilities essential to reducing the dimensions of an electrochemical probe to the nanometer scale, as well as electrochemical properties of new nanoscale electrode materials. Here we adapt the conventional definition of nanoscale to refer to lengths between 1 and 100 nm. Nanoscale electrochemistry is critically important for modern electrochemical science as well as many other key research areas, such as energy conversion and storage, catalysis, sensor development, and environmental science. Nanoscale electrochemical investigations have provided unique information unattainable using traditional methods. For example, nanoelectrodes can measure ultrafast electron-transfer kinetics that is often too fast to investigate with conventional electrodes. Nanoscale electrochemical materials, such as metal/semiconductor nanoparticles, have unique chemical and physical properties, and nanoscale electrochemical methods can be used to prepare advanced electrocatalytic materials. In addition, the use of nanoscale electrode probes has enabled electrochemical imaging with nanoscale spatial resolution, yielding unique information for better understanding heterogeneous electrode/solution interfaces.Analytical Chemistry 11/2012; 85(2). DOI:10.1021/ac3031702 · 5.83 Impact Factor