Dataset Information

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

ABSTRACT: Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å ? barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance and biolayer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from 7.5 to 5.0, mirroring acidification of the endosome. Once it had undergone the transition, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto electron microscopy grids, where PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early (pH 5.5) or late (pH 5.0) endosomal pH conditions. Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and the soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.

SUBMITTER: Naik S

PROVIDER: S-EPMC3790466 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

Naik Subhashchandra S Brock Susan S Akkaladevi Narahari N Tally Jon J McGinn-Straub Wesley W Zhang Na N Gao Phillip P Gogol E P EP Pentelute B L BL Collier R John RJ Fisher Mark T MT

Biochemistry 20130909 37

Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å β barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH-dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor, from the endosome to the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time pre ...[more]

PMID: 23964683

Similar Datasets

Project description:Surface plasmon microscopy has been of interest to the science and engineering community and has been utilized in broad aspects of applications and studies, including biochemical sensing and biomolecular binding kinetics. The benefits of surface plasmon microscopy include label-free detection, high sensitivity, and quantitative measurements. Here, a theoretical framework to analyze and compare several non-interferometric surface plasmon microscopes is proposed. The scope of the study is to (1) identify the strengths and weaknesses in each surface plasmon microscopes reported in the literature; (2) quantify their performance in terms of spatial imaging resolution, imaging contrast, sensitivity, and measurement accuracy for quantitative and non-quantitative imaging modes of the microscopes. Six types of non-interferometric microscopes were included in this study: annulus aperture scanning, half annulus aperture scanning, single-point scanning, double-point scanning, single-point scanning, at 45 degrees azimuthal angle, and double-point scanning at 45 degrees azimuthal angle. For non-quantitative imaging, there is a substantial tradeoff between the image contrast and the spatial resolution. For the quantitative imaging, the half annulus aperture provided the highest sensitivity of 127.058 rad/μm2 RIU-1, followed by the full annulus aperture of 126.318 rad/μm2 RIU-1. There is a clear tradeoff between spatial resolution and sensitivity. The annulus aperture and half annulus aperture had an optimal resolution, sensitivity, and crosstalk compared to the other non-interferometric surface plasmon resonance microscopes. The resolution depends strongly on the propagation length of the surface plasmons rather than the numerical aperture of the objective lens. For imaging and sensing purposes, the recommended microfluidic channel size and protein stamping size for surface plasmon resonance experiments is at least 25 μm for accurate plasmonic measurements.

Dataset Information

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

Publications

Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets