Publications by authors named "Kyle T Root"

7 Publications

  • Page 1 of 1

Reconstitution of full-length human caveolin-1 into phospholipid bicelles: Validation by analytical ultracentrifugation.

Biophys Chem 2020 04 26;259:106339. Epub 2020 Feb 26.

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

A significant hurdle in obtaining biophysical information on membrane proteins is developing a successful strategy for their reconstitution into a suitable membrane mimic. In particular, utilization of the more 'native-like' membrane mimics such as bicelles is generally more challenging than simple micellar solubilization. Caveolin-1, an integral membrane protein involved in membrane curvature, endocytosis, mechano-protection, and signal transduction, has been shown to be particularly recalcitrant to standard reconstitution protocols due to its highly hydrophobic characteristics. Herein we describe a robust method to incorporate recombinantly produced full-length caveolin-1 into bicelles at levels needed for biophysical experimentation. The benchmark of successful reconstitution is the obtainment of protein in a homogeneous state; therefore, we developed a validation procedure to monitor the success of the reconstitution using analytical ultracentrifugation of density-matched bicelles. Our findings indicated that our protocol produces a very homogeneous preparation of caveolin-1 associated with bicelles, and that caveolin-1 is highly α-helical (by circular dichroism spectroscopy). We believe that this methodology will serve as a general strategy to facilitate biophysical studies on membrane proteins.
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http://dx.doi.org/10.1016/j.bpc.2020.106339DOI Listing
April 2020

Secondary structure of caveolins: a mini review.

Biochem Soc Trans 2019 10;47(5):1489-1498

Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, PA 18015, U.S.A.

Caveolae are 50-100 nm invaginations found within the plasma membrane of cells. Caveolae are involved in many processes that are essential for homeostasis, most notably endocytosis, mechano-protection, and signal transduction. Within these invaginations, the most important proteins are caveolins, which in addition to participating in the aforementioned processes are structural proteins responsible for caveolae biogenesis. When caveolin is misregulated or mutated, many disease states can arise which include muscular dystrophy, cancers, and heart disease. Unlike most integral membrane proteins, caveolin does not have a transmembrane orientation; instead, it is postulated to adopt an unusual topography where both the N- and C-termini lie on the cytoplasmic side of the membrane, and the hydrophobic span adopts an intramembrane loop conformation. While knowledge concerning the biology of caveolin has progressed apace, fundamental structural information has proven more difficult to obtain. In this mini-review, we curate as well as critically assess the structural data that have been obtained on caveolins to date in order to build a robust and compelling model of the caveolin secondary structure.
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http://dx.doi.org/10.1042/BST20190375DOI Listing
October 2019

Low- q Bicelles Are Mixed Micelles.

J Phys Chem Lett 2018 Aug 25;9(15):4469-4473. Epub 2018 Jul 25.

Department of Chemistry , University of Virginia , Charlottesville , Virginia 22904 , United States.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.
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http://dx.doi.org/10.1021/acs.jpclett.8b02079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353637PMC
August 2018

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

Reconstitution and spectroscopic analysis of caveolin-1 residues 62-178 reveals that proline 110 governs its structure and solvent exposure.

Biochim Biophys Acta 2016 Apr 14;1858(4):682-8. Epub 2016 Jan 14.

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

Caveolin-1 is a membrane protein that possesses an unusual topology where both N- and C-termini are cytoplasmic as a result of a membrane-embedded turn. In particular, proline 110 has been postulated to be the linchpin of this unusual motif. Using a caveolin-1 construct (residues 62-178) reconstituted into dodecylphosphocholine micelles with and without a cholesterol mimic, the changes that occurred upon P110A mutation were probed. Using far UV circular dichroism spectroscopy it was shown that cholesterol attenuated the helicity of caveolin-1, and that mutation of P110 to alanine caused a significant increase in the α-helicity of the protein. Near UV circular dichroism spectroscopy showed significant changes in structure and/or environment upon mutation that again were modulated by the presence of cholesterol. Stern-Volmer quenching and λ(max) analysis of tryptophan residues showed that the proline mutation caused W85 to become more exposed, W98 and W115 to become less exposed, and W128 showed no change. This finding provided evidence that regions proximal and far away from the proline are buried differentially upon its mutation and therefore this residue is strongly tied to maintaining the hydrophobic coverage along the caveolin-1 sequence. In the presence of cholesterol, the accessibilities of the two tryptophan residues that proceeded position 110 were altered much more significantly upon P110A mutation than the two tryptophans aft P110. Overall, this work provides strong evidence that proline 110 is critical for maintaining both the structure and hydrophobic coverage of caveolin-1 and that cholesterol also plays a significant role in modulating these parameters.
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http://dx.doi.org/10.1016/j.bbamem.2016.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779658PMC
April 2016

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

Probing the U-shaped conformation of caveolin-1 in a bilayer.

Biophys J 2014 Mar;106(6):1371-80

Department of Molecular Biosciences and Center for Bioinformatics, The University of Kansas, Lawrence, Kansas. Electronic address:

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82-S136; Cav182-136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav182-136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182-136 insertion depth in the bilayer), corroborate that Cav182-136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav182-136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.
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http://dx.doi.org/10.1016/j.bpj.2014.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984989PMC
March 2014