Publications by authors named "Sarah M Plucinsky"

4 Publications

  • Page 1 of 1

Preparation of Caveolin-1 for NMR Spectroscopy Experiments.

Methods Mol Biol 2020 ;2169:137-147

Department of Chemistry, Lehigh University, Bethlehem, PA, USA.

Caveolin-1 is a 20.5 kDa integral membrane protein that is involved in a myriad of cellular processes including signal transduction, relieving mechano-stresses on the cell, endocytosis, and most importantly caveolae formation. As a consequence, there is intense interest in characterizing caveolin-1 structurally. Out of the many available structural techniques, nuclear magnetic resonance (NMR) spectroscopy is particularly well suited to investigations on integral membrane proteins like caveolin-1 that have significant unstructured regions and unusual topologies. However, the technique requires relatively large amounts of protein (i.e. concentrations in the 0.5-5 mM range), and obtaining these amounts can be difficult especially for highly hydrophobic membrane proteins such as caveolin-1. Herein, we describe a robust protocol for the preparation of caveolin-1 for structural studies using NMR.
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http://dx.doi.org/10.1007/978-1-0716-0732-9_13DOI Listing
March 2021

Efficient solubilization and purification of highly insoluble membrane proteins expressed as inclusion bodies using perfluorooctanoic acid.

Protein Expr Purif 2018 03 21;143:34-37. Epub 2017 Oct 21.

Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, PA 18015, USA. Electronic address:

The purification of membrane proteins can be challenging due to their low solubility in conventional detergents and/or chaotropic solutions. The introduction of fusion systems that promote the formation of inclusion bodies has facilitated the over-expression of membrane proteins. In this protocol, we describe the use of perfluorooctanoic acid (PFOA) as an aid in the purification of highly hydrophobic membrane proteins expressed as inclusion bodies. The advantage of utilizing PFOA is threefold: first, PFOA is able to reliably solubilize inclusion bodies, second, PFOA is compatible with nickel affinity chromatography, and third, PFOA can be efficiently dialyzed away to produce a detergent free sample. To demonstrate the utility of employing PFOA, we expressed and purified a segment of the extremely hydrophobic membrane protein caveolin-1.
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http://dx.doi.org/10.1016/j.pep.2017.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750070PMC
March 2018

Secondary Structure Analysis of a Functional Construct of Caveolin-1 Reveals a Long C-Terminal Helix.

Biophys J 2015 Oct;109(8):1686-8

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania. Electronic address:

Caveolin-1 is an integral membrane protein that is the primary component of cell membrane invaginations called caveolae. While caveolin-1 is known to participate in a myriad of vital cellular processes, structural data on caveolin-1 of any kind is severely limited. In order to rectify this dearth, secondary structure analysis of a functional construct of caveolin-1, containing the intact C-terminal domain, was performed using NMR spectroscopy in lyso-myristoylphosphatidylglycerol micelles. Complete backbone assignments of caveolin-1 (residues 62-178) were made, and it was determined that residues 62-79 were dynamic; residues 89-107, 111-128, and 132-175 were helical; and residues 80-88, 108-110, and 129-131 represent unstructured breaks between the helices.
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http://dx.doi.org/10.1016/j.bpj.2015.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624155PMC
October 2015

Recent progress in the topology, structure, and oligomerization of caveolin: a building block of caveolae.

Curr Top Membr 2015 11;75:305-36. Epub 2015 Apr 11.

Department of Chemistry, Lehigh University, Bethlehem, PA, USA.

Caveolae are cholesterol-rich plasma membrane invaginations that are found in a plethora of cell types. They play many roles including signal transduction, endocytosis, and mechanoprotection. The most critical protein in caveolae is the integral membrane protein, caveolin, which has been shown to be necessary for caveolae formation, and governs the major functions attributed to caveolae. Caveolin is postulated to act as a scaffold in the high molecular weight striated coat that surrounds the caveolar bulb, stabilizing it. Caveolin interacts, both directly and indirectly, with a large number of signaling molecules, and presides over the endocytosis of molecular cargo by caveolae. However, many of the key biophysical aspects of the caveolin protein, its structure, topology, and oligomeric behavior, are just beginning to come to light. Herein is an up-to-date summary and critique of the progress that has been made in understanding caveolin on a molecular and atomic level.
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http://dx.doi.org/10.1016/bs.ctm.2015.03.007DOI Listing
March 2016