Publications by authors named "Monica D Rieth"

4 Publications

  • Page 1 of 1

Preparation of DPPC liposomes using probe-tip sonication: Investigating intrinsic factors affecting temperature phase transitions.

Biochem Biophys Rep 2020 Jul 20;22:100764. Epub 2020 Apr 20.

Department of Chemistry, Southern Illinois University Edwardsville, 44 South Circle Drive, Box 1652, Science West, Edwardsville, IL, 62026, USA.

Liposomes are an important tool and have gained much attention for their promise as an effective means of delivering small therapeutic compounds to targeted sites. In an effort to establish an effective method to produce liposomes from the lipid, dipalmitoyl-phosphatidylcholine or DPPC, we have found important aspects that must be taken into consideration. Here, we used probe-tip sonication to prepare liposomes on a batch scale. During this process we uncovered interesting steps in their preparation that altered the thermodynamic properties and phase transitions of the resulting liposome mixtures. Using differential scanning calorimetry to assess this we found that increasing the sonication time had the most dramatic effect on our sample, producing almost an entirely separate phase transition relative to the main phase transition. This result is consistent with reports from the current literature. We also highlight a smaller transition, which we attribute to traces of unincorporated lipid that seems to gradually disappear as the total lipid concentration decreases. Overall, sonication is an effective means of producing liposomes, but we cannot assert this method is optimal in producing them with precise physical properties. Here we highlight the physical effects at play during this process.
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http://dx.doi.org/10.1016/j.bbrep.2020.100764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176824PMC
July 2020

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

Adenosine A2a receptors form distinct oligomers in protein detergent complexes.

FEBS Lett 2016 09 2;590(18):3295-306. Epub 2016 Sep 2.

Department of Chemical Engineering, University of California Santa Barbara, CA, USA.

The human adenosine A2a receptor (A2aR) tunes its function by forming homo-oligomers and hetero-oligomers with other G protein-coupled receptors, but the biophysical characterization of these oligomeric species is limited. Here, we show that upon reconstitution into an optimized mixed micelle system, and purification via an antagonist affinity column, full-length A2aR exists as a distribution of oligomers. We isolated the dimer population from the other oligomers via size exclusion chromatography and showed that it is stable upon dilution, thus supporting the hypotheses that the A2aR dimer has a defined structure and function. This study presents a crucial enabling step to a detailed biophysical characterization of A2aR homodimers.
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http://dx.doi.org/10.1002/1873-3468.12367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039092PMC
September 2016

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