Megan M. Dunagan
Miami University
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Featured researches published by Megan M. Dunagan.
Biochemistry | 2013
Indra D. Sahu; Robert M. McCarrick; Kaylee R. Troxel; Rongfu Zhang; Hubbell J. Smith; Megan M. Dunagan; Max S. Swartz; Prashant V. Rajan; Brett M. Kroncke; Charles R. Sanders; Gary A. Lorigan
Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to those of globular proteins. In this study, we employed a combination of three recently developed methodologies, (1) bifunctional spin labels (BSL), (2) SMA-Lipodisq nanoparticles, and (3) Q band pulsed EPR measurements, to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated an ∼2-fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to those of proteoliposomes and narrower distance distributions for the BSL when compared to those of the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.
Biochemistry | 2014
Indra D. Sahu; Brett M. Kroncke; Rongfu Zhang; Megan M. Dunagan; Hubbell J. Smith; Andrew Craig; Robert M. McCarrick; Charles R. Sanders; Gary A. Lorigan
KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel.
Journal of Physical Chemistry B | 2017
Indra D. Sahu; Rongfu Zhang; Megan M. Dunagan; Andrew Craig; Gary A. Lorigan
EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (Tm) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the Tm titration experiment indicated an increase in Tm values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.
Journal of Physical Chemistry B | 2017
Indra D. Sahu; Andrew Craig; Megan M. Dunagan; Robert M. McCarrick; Gary A. Lorigan
Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study structural and dynamic properties of membrane proteins. The most widely used spin label is methanthiosulfonate (MTSL). However, the flexibility of this spin label introduces greater uncertainties in EPR measurements obtained for determining structures, side-chain dynamics, and backbone motion of membrane protein systems. Recently, a newer bifunctional spin label (BSL), 3,4-bis(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome the dynamic limitations associated with the MTSL spin label and has been invaluable in determining protein backbone dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing more accurate information about the structure and dynamics of several proteins, a detailed characterization of the spin label is still lacking. In this study, we characterized BSLs by performing CW-EPR spectral line shape analysis as a function of temperature on spin-labeled sites inside and outside of the membrane for the integral membrane protein KCNE1 in POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles. The experimental data revealed a powder pattern spectral line shape for all of the KCNE1-BSL samples at 296 K, suggesting the motion of BSLs approaches the rigid limit regime for these series of samples. BSLs were further utilized to report for the first time the distance measurement between two BSLs attached on an integral membrane protein KCNE1 in POPC/POPG lipid bilayers at room temperature using dipolar line broadening CW-EPR spectroscopy. The CW dipolar line broadening EPR data revealed a 15 ± 2 Å distance between doubly attached BSLs on KCNE1 (53/57-63/67) which is consistent with molecular dynamics modeling and the solution NMR structure of KCNE1 which yielded a distance of 17 Å. This study demonstrates the utility of investigating the structural and dynamic properties of membrane proteins in physiologically relevant membrane mimetics using BSLs.
Biochemistry | 2015
Indra D. Sahu; Andrew Craig; Megan M. Dunagan; Kaylee R. Troxel; Rongfu Zhang; Andrew G. Meiberg; Corrinne N. Harmon; Robert M. McCarrick; Brett M. Kroncke; Charles R. Sanders; Gary A. Lorigan
Biophysical Journal | 2015
Andrew Craig; Indra D. Sahu; Rongfy Zhang; Megan M. Dunagan; Kunkun Wang; Robert M. McCarrick; Gary A. Lorigan
Biophysical Journal | 2015
Rongfu Zhang; Indra D. Sahu; Kaylee Roy Gibson; Nefertiti Muhammad; Avnika Bali; Raven G. Comer; Andrew Craig; Megan M. Dunagan; Kunkun Wang; Carole Dabney-Smith; Gary A. Lorigan
Biophysical Journal | 2015
Indra D. Sahu; Brett M. Kroncke; Megan M. Dunagan; Rongfu Zhang; Andrew Craig; Kunkun Wang; Avnika Bali; Robert M. McCarrick; Charles R. Sanders; Gary A. Lorigan
Biophysical Journal | 2015
Megan M. Dunagan; Indra D. Sahu; Rongfu Zhang; Andrew Craig; Robert McMarrick; Gary A. Lorigan
Biophysical Journal | 2014
Megan M. Dunagan; Indra D. Sahu; Rongfu Zhang; Andrew Craig; Robert M. McCarrick; Gary A. Lorigan