indra sahu | Campbellsville University (original) (raw)
Papers by indra sahu
Biochimica et Biophysica Acta (BBA) - Biomembranes
2,4-Dinitrophenol’s (2,4-DNP) effect on synthetic membrane proteins was investigated to test if i... more 2,4-Dinitrophenol’s (2,4-DNP) effect on synthetic membrane proteins was investigated to test if it is selective to cancer-mimic synthetic cell membranes, which are more bountiful in phosphatidylserine (PS) lipids compared to normal cells that have much more phosphatidylcholine (PC) lipids. Three undergraduate students prepared the untreated synthetic membranes and incorporated 2,4-DNP into a second set of treated samples. Students analysed the samples via solid-state 31P NMR. The results indicate that 2,4-DNP breaks down 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) synthetic membranes, but not 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphocholine (POPC) membranes that mimic the normal cell membrane composition. The 31P NMR spectrum of the head groups of the membrane suggests that the 2,4-DNP is disturbing the multilamellar vesicles (MLVs) and forming small micelles only in the POPS synthetic membranes.
Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infectio... more Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S 21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S 21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S 21 68) and inactive antipinholin (S 21 68 IRS) in DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron−electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S 21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S 21 68 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S 21 68 IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S 21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.
Biochimica et Biophysica Acta (BBA) - Biomembranes
Biochimica et Biophysica Acta (BBA) - Biomembranes
Biochimica et Biophysica Acta (BBA) - Biomembranes
Membranes, Apr 27, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Biophysical Journal, 2022
Biophysical Journal, 2022
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021
There have recently been advances in methods for detecting local secondary structures of membrane... more There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S21 pinholin. This ESEEM approach uses a combination of site-directed spin labeling and 2H-labeled side chains. Pinholin S21 is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S21 pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S21 pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 310-helix which have been observed in other channel forming proteins.
Biophysical Journal, 2021
Biophysical Journal, 2021
Biophysical Journal, 2021
Biomacromolecules, 2020
Peak assignments of the Fourier-transform infrared spectra and solution NMR spectra of the synthe... more Peak assignments of the Fourier-transform infrared spectra and solution NMR spectra of the synthesized SMADs and representative TEM images of SMADLPs made from POPC liposomes (PDF)
Biophysical Journal, 2020
Biophysical Journal, 2020
have not been studied yet. Since voltage-gated potassium (Kv) channels regulate a great variety o... more have not been studied yet. Since voltage-gated potassium (Kv) channels regulate a great variety of biological processes, direct effects of CDs on these channels can contribute to the known side effects (such as immunosuppression via Kv1.3) of numerous drugs. Our aim was to characterize direct, ligand-like effects of CDs with different sizes and side chain substitutions on various Kv channels and to demonstrate that these are independent from their effects on membrane cholesterol depletion. We carried out patch-clamp measurements to characterize the direct effects of CDs on Kv channels. Most of the tested CDs at concentrations of 1 and 5 mM partially reversibly inhibited Kv1.3 currents within 15 seconds, while some of them had no such effect. To examine the potential membrane biophysical and cholesterol depleting effects of CDs, after 1 hour incubation we measured membrane fluidity, hydration, lipid order and the extent of cholesterol depletion. We also performed a cell viability assay on Jurkat cells using flow cytometry, where in parallel with direct inhibitory effects on Kv1.3 and independent from cholesterol depletion we detected cell death after 24-hour-long CD treatments.Based on our results we can conclude that CDs exert previously unknown direct inhibitory effects on Kv ion channels independent from cholesterol depletion, which can play an important role in side effects of drugs containing CDs.
Chemistry and Physics of Lipids, 2019
Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing... more Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing homogenous and functional lipid membrane mimetic systems into which membrane proteins can properly fold and function. It has recently been shown that styrene-maleic acid (SMA) copolymers act as a macromolecular surfactant and therefore facilitate the formation of disk-shaped lipid bilayer nanoparticles (styrene-maleic acid copolymer-lipid nanoparticles (SMALPs)) that retain structural characteristics of native lipid membranes. We have previously reported controlled synthesis of SMA block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization, and that alteration of the weight ratio of styrene to maleic acid affects nanoparticle size. RAFT-synthesis offers superior control over SMA polymer architecture compared to conventional radical polymerization techniques used for commercially available SMA. However, the interactions between the lipid bilayer and the solubilized RAFT-synthesized SMA polymer are currently not fully understood. In this study, EPR spectroscopy was used to detect the perturbation on the acyl chain upon introduction of the RAFT-synthesized SMA polymer by attaching PC-based nitroxide spin labels to the 5 th , 12 th , and 16 th positions along the acyl chain of the lipid bilayer. EPR spectra showed high rigidity at the 12 th position compared to the other two regions, displaying similar qualities to commercially available polymers synthesized via conventional methods. In addition, central EPR linewidths and correlation time data were obtained that are consistent with previous findings.
Biochimica et Biophysica Acta (BBA) - Biomembranes
2,4-Dinitrophenol’s (2,4-DNP) effect on synthetic membrane proteins was investigated to test if i... more 2,4-Dinitrophenol’s (2,4-DNP) effect on synthetic membrane proteins was investigated to test if it is selective to cancer-mimic synthetic cell membranes, which are more bountiful in phosphatidylserine (PS) lipids compared to normal cells that have much more phosphatidylcholine (PC) lipids. Three undergraduate students prepared the untreated synthetic membranes and incorporated 2,4-DNP into a second set of treated samples. Students analysed the samples via solid-state 31P NMR. The results indicate that 2,4-DNP breaks down 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) synthetic membranes, but not 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphocholine (POPC) membranes that mimic the normal cell membrane composition. The 31P NMR spectrum of the head groups of the membrane suggests that the 2,4-DNP is disturbing the multilamellar vesicles (MLVs) and forming small micelles only in the POPS synthetic membranes.
Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infectio... more Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S 21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S 21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S 21 68) and inactive antipinholin (S 21 68 IRS) in DMPC (1,2-dimyristoyl-snglycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron−electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S 21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S 21 68 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S 21 68 IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S 21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.
Biochimica et Biophysica Acta (BBA) - Biomembranes
Biochimica et Biophysica Acta (BBA) - Biomembranes
Biochimica et Biophysica Acta (BBA) - Biomembranes
Membranes, Apr 27, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Biophysical Journal, 2022
Biophysical Journal, 2022
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021
There have recently been advances in methods for detecting local secondary structures of membrane... more There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S21 pinholin. This ESEEM approach uses a combination of site-directed spin labeling and 2H-labeled side chains. Pinholin S21 is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S21 pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S21 pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 310-helix which have been observed in other channel forming proteins.
Biophysical Journal, 2021
Biophysical Journal, 2021
Biophysical Journal, 2021
Biomacromolecules, 2020
Peak assignments of the Fourier-transform infrared spectra and solution NMR spectra of the synthe... more Peak assignments of the Fourier-transform infrared spectra and solution NMR spectra of the synthesized SMADs and representative TEM images of SMADLPs made from POPC liposomes (PDF)
Biophysical Journal, 2020
Biophysical Journal, 2020
have not been studied yet. Since voltage-gated potassium (Kv) channels regulate a great variety o... more have not been studied yet. Since voltage-gated potassium (Kv) channels regulate a great variety of biological processes, direct effects of CDs on these channels can contribute to the known side effects (such as immunosuppression via Kv1.3) of numerous drugs. Our aim was to characterize direct, ligand-like effects of CDs with different sizes and side chain substitutions on various Kv channels and to demonstrate that these are independent from their effects on membrane cholesterol depletion. We carried out patch-clamp measurements to characterize the direct effects of CDs on Kv channels. Most of the tested CDs at concentrations of 1 and 5 mM partially reversibly inhibited Kv1.3 currents within 15 seconds, while some of them had no such effect. To examine the potential membrane biophysical and cholesterol depleting effects of CDs, after 1 hour incubation we measured membrane fluidity, hydration, lipid order and the extent of cholesterol depletion. We also performed a cell viability assay on Jurkat cells using flow cytometry, where in parallel with direct inhibitory effects on Kv1.3 and independent from cholesterol depletion we detected cell death after 24-hour-long CD treatments.Based on our results we can conclude that CDs exert previously unknown direct inhibitory effects on Kv ion channels independent from cholesterol depletion, which can play an important role in side effects of drugs containing CDs.
Chemistry and Physics of Lipids, 2019
Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing... more Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing homogenous and functional lipid membrane mimetic systems into which membrane proteins can properly fold and function. It has recently been shown that styrene-maleic acid (SMA) copolymers act as a macromolecular surfactant and therefore facilitate the formation of disk-shaped lipid bilayer nanoparticles (styrene-maleic acid copolymer-lipid nanoparticles (SMALPs)) that retain structural characteristics of native lipid membranes. We have previously reported controlled synthesis of SMA block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization, and that alteration of the weight ratio of styrene to maleic acid affects nanoparticle size. RAFT-synthesis offers superior control over SMA polymer architecture compared to conventional radical polymerization techniques used for commercially available SMA. However, the interactions between the lipid bilayer and the solubilized RAFT-synthesized SMA polymer are currently not fully understood. In this study, EPR spectroscopy was used to detect the perturbation on the acyl chain upon introduction of the RAFT-synthesized SMA polymer by attaching PC-based nitroxide spin labels to the 5 th , 12 th , and 16 th positions along the acyl chain of the lipid bilayer. EPR spectra showed high rigidity at the 12 th position compared to the other two regions, displaying similar qualities to commercially available polymers synthesized via conventional methods. In addition, central EPR linewidths and correlation time data were obtained that are consistent with previous findings.