Conformational changes of the <i>Hs</i>DHODH N-terminal Microdomain via DEER Spectroscopy

Vicente, Eduardo Festozo; Sahu, Indra D.; Costa-Filho, Antonio J.; Cilli, Eduardo Maffud; Lorigan, Gary A.

doi:10.1021/acs.jpcb.5b01706

Cited by 18 publications

(20 citation statements)

References 42 publications

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“…The success of this measurement opens the door for more in depth EPR structural and dynamic studies to be conducted in the future, such as pulsed EPR measurements. [36][37][38][39][40] 31 P SS-NMR spectroscopy allowed for the study of the pinholin system from the lipid perspective and showed interactions of both forms of the pinholin with the lipid membrane, to varying degrees, through decreases in the CSA width when compared to the empty DMPC MLVs. These differences in the way the active S 21 68 and inactive S 21 IRS forms of the pinholin interact with the membrane suggest differences in the externalization of TMD1, and ultimately the role each form plays in the bacteriophage lytic pathway.…”

Section: Discussionmentioning

confidence: 99%

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein

Drew

Ahammad

Serafin

et al. 2019

Analytical Biochemistry

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The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S 21 protein of phage φ21 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S 21 68, and negativedominant mutant form, S 21 IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [θ] 222 / [θ] 208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31 P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31 P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.

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Section: Discussionmentioning

confidence: 99%

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein

Drew

Ahammad

Serafin

et al. 2019

Analytical Biochemistry

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“…Unique structural and dynamic information on membrane proteins can be gleaned using these powerful techniques. Table 1 shows recent examples of some biologically important membrane protein systems studied by using EPR spectroscopy [43,44,49,64,74,75,78,79]. EPR spectroscopy has become complementary to existing biophysical methods to study membrane proteins.…”

Section: Resultsmentioning

confidence: 99%

“…A recent example of using DEER spectroscopy is the study of N-terminal microdomain of human dihydroorotate dehydrogenase enzyme (HsDHODH) [75]. The HsDHODH enzyme is an attractive drug target for the potential treatment of several proliferative diseases such as cancer and rheumatoid arthritis.…”

Section: Introductionmentioning

confidence: 99%

“…The HsDHODH enzyme is an attractive drug target for the potential treatment of several proliferative diseases such as cancer and rheumatoid arthritis. Eduardo et al used DEER distance data in combination with CD spectroscopy to determine the different conformational states of HsDHODH peptide in micelles and liposomes [75]. The results revealed that the peptide undergoes different conformational states in micelles and liposomes suggesting this mircodomain acts in specific regions or areas of the mitochondria, which can be related with the control of the quinone access to the HsDHODH active site.…”

Section: Introductionmentioning

confidence: 99%

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Biophysical EPR Studies Applied to Membrane Proteins

Sahu¹,

Lorigan²

2015

J Phys Chem Biophys

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Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented.

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“…Nitroxide based SDSL DEER spectroscopy has been applied to study several membrane protein systems including KvAP voltage-sensing domain, pentameric ligand-gated channel, E. coli integral membrane sulfurtransferase (YgaP), bacteriorhodopsin, KCNE1, KCNE3, C99 amyloid precursor protein, human dihydroorotate dehydrogenase enzyme (HsDHODH), influenza A M2 protein, outer membrane cobalamin transporter BtuB in intact E. coli, cardiac Na + /Ca 2+ exchange (NCX1.1) protein, Na + /Proline transporter PutP Escherichia coli, tetrameric potassium ion channel KcsA, α-synuclein, membranefusion K/E peptides, ABC transporter MsbA, HCN channels, YetJ membrane protein, ectodomain of gp41, multimeric membrane transport proteins, and multidrug transporter LmrP [32,78,150,[161][162][163][167][168][169][170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186].…”

Section: Distance Measurement On Membrane Proteins Using Dual Sdsl Epmentioning

confidence: 99%

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Sahu

Lorigan

2021

Biophysica

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Membrane proteins are essential for the survival of living organisms. They are involved in important biological functions including transportation of ions and molecules across the cell membrane and triggering the signaling pathways. They are targets of more than half of the modern medical drugs. Despite their biological significance, information about the structural dynamics of membrane proteins is lagging when compared to that of globular proteins. The major challenges with these systems are low expression yields and lack of appropriate solubilizing medium required for biophysical techniques. Electron paramagnetic resonance (EPR) spectroscopy coupled with site directed spin labeling (SDSL) is a rapidly growing powerful biophysical technique that can be used to obtain pertinent structural and dynamic information on membrane proteins. In this brief review, we will focus on the overview of the widely used EPR approaches and their emerging applications to answer structural and conformational dynamics related questions on important membrane protein systems.

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Conformational changes of the HsDHODH N-terminal Microdomain via DEER Spectroscopy

Cited by 18 publications

References 42 publications

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein

Biophysical EPR Studies Applied to Membrane Proteins

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

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