Publications by authors named "Kerney Jebrell Glover"

22 Publications

  • Page 1 of 1

Preparation and characterization of neutrally-buoyant oleosin-rich synthetic lipid droplets.

Biochim Biophys Acta Biomembr 2021 Apr 30;1863(8):183624. Epub 2021 Apr 30.

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

Lipid droplets also known as oil bodies are found in a variety of organisms and function as stores of high-energy metabolites. Recently, there has been interest in using lipid droplets for protein production and drug delivery. Artificial lipid droplets have been previously prepared, but their short lifetime in solution and inhomogeneity has severely limited their applicability. Herein we report an improved methodology for the production of synthetic lipid droplets that overcomes the aforementioned limitations. These advancements include: 1) development of a methodology for the expression and purification of high-levels of oleosin, a crucial lipid droplet component, 2) preparation of neutrally-buoyant synthetic lipid droplets, and 3) production of synthetic lipid droplets of a specific size. Together, these important enhancements will facilitate the advancement of lipid droplet science and its application in biotechnology.
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http://dx.doi.org/10.1016/j.bbamem.2021.183624DOI Listing
April 2021

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

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

Evidence for Surface Recognition by a Cholesterol-Recognition Peptide.

Biophys J 2016 Jun 6;110(12):2577-2580. Epub 2016 Jun 6.

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

Two cholesterol recognition/interaction amino-acid consensus peptides, N-acetyl-LWYIKC-amide, and N-acetyl-CLWYIK-amide, have been coupled to exchangeable mimics of Chol (cholesterol) and Phos (1,2-dipalmitoyl-sn-glycerol-3-phospho-(1'rac-glycerol)) via disulfide bond formation. Equilibration between Chol and Phos via thiolate-disulfide interchange reactions has revealed that both peptides favor Chol as a nearest-neighbor in liquid-disordered (ld) bilayers to the same extent. In contrast, no Chol- or Phos-recognition could be detected by these peptides in analogous liquid-ordered (lo) bilayers. Fluorescence measurements of the tryptophan moiety have shown that both peptides favor the membrane-water interface. Taken together, these results provide strong evidence that the recognition behavior of the LWYIK motif is, fundamentally, a surface phenomenon but that partial penetration into the bilayer is also necessary.
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http://dx.doi.org/10.1016/j.bpj.2016.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919587PMC
June 2016

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

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

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

Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure.

Biochemistry 2012 May 25;51(18):3911-8. Epub 2012 Apr 25.

Department of Chemistry, Lehigh University, 6 E. Packer Ave, Bethlehem, Pennsylvania 18015, USA.

Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1(62-178) was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1(82-178), Cav1(96-178), Cav1(62-136), Cav1(82-136), Cav1(96-136)) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96-136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav1(96-136) P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111-129; mutant, residues 111-133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1(62-178) (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1(62-178), which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.
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http://dx.doi.org/10.1021/bi3001853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396432PMC
May 2012

The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.

Biochim Biophys Acta 2012 May 4;1818(5):1158-64. Epub 2012 Jan 4.

Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA.

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96-136 (the entire intact transmembrane domain). Caveolin-1(96-136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57-65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix-break-helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix-break-helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix-break-helix structure.
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http://dx.doi.org/10.1016/j.bbamem.2011.12.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319262PMC
May 2012

Discoid Bicelles as Efficient Templates for Pillared Lamellar Periodic Mesoporous Silicas at pH 7 and Ultrafast Reaction Times.

Nanoscale Res Lett 2010 Oct 6;6(1):61. Epub 2010 Oct 6.

Department of Chemistry, Lehigh University, 6 East Packer Avenue, Bethlehem, PA 18015, USA.

We report the first synthesis of periodic mesoporous silicas templated by bicelles. The obtained materials form novel pillared lamellar structures with a high degree of periodic order, narrow pore size distributions, and exceptionally high surface areas.
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http://dx.doi.org/10.1007/s11671-010-9813-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212208PMC
October 2010

Lag periods during the self-assembly of {Mo(72)Fe(30)} macroions: connection to the virus capsid formation process.

J Am Chem Soc 2009 Oct;131(42):15152-9

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015, USA.

The kinetic properties of the self-assembly of hydrophilic Keplerate-type polyoxometalate (POM) {Mo(72)Fe(30)} macroanions into single-layer, vesicle-like blackberry structures in solutions were monitored by the static and dynamic laser light scattering techniques. In the presence of additional electrolytes, an obvious lag period at the initial stage of self-assembly was observed, followed by a fast increase of the scattered intensity. The whole kinetic curve is sigmoidal with a lag phase. A two-step nucleation-growth mechanism is proposed to explain this lag phase: the {Mo(72)Fe(30)} macroanions slowly associate into oligomers (mostly dimers), which are the thermodynamically unfavorable intermediates, at the initial stage; once the oligomers reach a critical concentration, the blackberry formation process is accelerated. Analytical ultracentrifugation (AUC) was used to confirm the oligomeric state in {Mo(72)Fe(30)} solution during the lag period. The length of the lag period is dependent on temperature, ionic strength, and the valent states of the additional salts, as well as the solvent content. The kinetics (including the lag period) of the blackberry formation of the {Mo(72)Fe(30)} macroanions show similarities to the self-assembly of virus capsid proteins (which are also soluble macroions) into spherical capsid shells, suggesting possible connections between the self-assembly behaviors of inorganic species and biological macromolecules.
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http://dx.doi.org/10.1021/ja903548mDOI Listing
October 2009

Reliable expression and purification of highly insoluble transmembrane domains.

Anal Biochem 2009 Jan 1;384(2):274-8. Epub 2008 Oct 1.

Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA.

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.
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http://dx.doi.org/10.1016/j.ab.2008.09.038DOI Listing
January 2009

From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni.

Biochemistry 2007 May 17;46(18):5579-85. Epub 2007 Apr 17.

Department of Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.
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http://dx.doi.org/10.1021/bi602633nDOI Listing
May 2007

Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway.

Biochemistry 2006 Apr;45(16):5343-50

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'-diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.
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http://dx.doi.org/10.1021/bi0602056DOI Listing
April 2006

Chemoenzymatic synthesis of glycopeptides with PglB, a bacterial oligosaccharyl transferase from Campylobacter jejuni.

Chem Biol 2005 Dec;12(12):1311-5

Department of Chemistry and Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PgIB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PgIB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GaINAc-alpha1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PgIB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PgIB to transfer a wide variety of saccharides. With the demonstration of PgIB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.
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http://dx.doi.org/10.1016/j.chembiol.2005.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519243PMC
December 2005

Investigating bacterial N-linked glycosylation: synthesis and glycosyl acceptor activity of the undecaprenyl pyrophosphate-linked bacillosamine.

J Am Chem Soc 2005 Oct;127(40):13766-7

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

The chemical synthesis and biological activity of undecaprenyl pyrophosphate bacillosamine (Und-PP-Bac), an obligatory intermediate in the asparagine-linked glycosylation pathway of Campylobacter jejuni, are reported. The key transformation involves the coupling of bacillosamine phosphate and undecaprenyl phosphate. The synthetic Und-PP-Bac can be used to investigate the activity of the enzyme PglA, which catalyzes the first glycosyl transfer in substrate biosynthesis for N-linked protein glycosylation in the pathogenic gram-negative bacterium. The availability of this synthetic substrate makes it possible to access polyprenyl-linked oligosaccharides, such as the GalNAc-alpha-1,3-bacillosamine-alpha-1-PP-Und intermediate, that will enable exploration of the remaining enzymes in the prokaryotic glycosylation pathway. Study of the bacterial glycosylation system will provide insight into the corresponding eukaryotic process, which is currently poorly understood.
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http://dx.doi.org/10.1021/ja054265vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1351108PMC
October 2005

In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation.

Proc Natl Acad Sci U S A 2005 Oct 26;102(40):14255-9. Epub 2005 Sep 26.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

Campylobacter jejuni has a general N-linked glycosylation pathway (encoded by the pgl gene cluster), which culminates in the transfer of a heptasaccharide: GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac [where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] from a membrane-anchored undecaprenylpyrophosphate (Und-PP)-linked donor to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Herein we report, the cloning, overexpression, and purification of four of the glycosyltransferases (PglA, PglH, PglI, and PglJ) responsible for the biosynthesis of the Und-PP-linked heptasaccharide. Starting with chemically synthesized Und-PP-linked Bac and various combinations of enzymes, we have deduced the precise functions of these glycosyltransferases. PglA and PglJ add the first two GalNAc residues on to the isoprenoid-linked Bac carrier, respectively. Elongation of the trisaccharide with PglH results in a hexasaccharide revealing the polymerase activity of PglH. The final branching glucose is then added by PglI, which prefers native lipids for optimal activity. The sequential activities of the glycosyl transferases in the pathway can be reconstituted in vitro. This pathway represents an ideal venue for investigating the integrated functions of a series of enzymatic processes that occur at a membrane interface.
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http://dx.doi.org/10.1073/pnas.0507311102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1242339PMC
October 2005

Position of residues in transmembrane peptides with respect to the lipid bilayer: a combined lipid Noes and water chemical exchange approach in phospholipid bicelles.

J Biomol NMR 2002 Jan;22(1):57-64

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0314, USA.

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH2) was incorporated into small unaligned phospholipid bicelles, which provide a 'native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the 'bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively 15N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.
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http://dx.doi.org/10.1023/a:1013817818794DOI Listing
January 2002