Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/81230
Title: Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability
Authors: Surya, Wahyu
Torres, Jaume
Keywords: Chemistry; Issue 98; Analytical ultracentrifugation; sedimentation equilibrium; molecular weight; membrane proteins; small hydrophobic; respiratory syncytial virus; detergent; density matching; oligomeric size; histidine protonation; oligomer stability
Issue 98
Sedimentation equilibrium
Analytical ultracentrifugation
Molecular weight
Membrane proteins
Small hydrophobic
Issue Date: 2015
Source: Surya, W., & Torres, J. (2015). Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability. Journal of Visualized Experiments, 98, e52404-.
Surya, W., & Torres, J. (2015). Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability. Journal of Visualized Experiments, 98, e52404-. Remove
Series/Report no.: Journal of Visualized Experiments
Abstract: Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.
URI: https://hdl.handle.net/10356/81230
http://hdl.handle.net/10220/39183
ISSN: 1940-087X
DOI: 10.3791/52404
Rights: © 2015 Journal of Visualized Experiments. This paper was published in Journal of Visualized Experiments and is made available as an electronic reprint (preprint) with permission of Journal of Visualized Experiments. The published version is available at: [http://dx.doi.org/10.3791/52404]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.
Fulltext Permission: open
Fulltext Availability: With Fulltext
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