Indra D. Sahu

DEER EPR Measurements for Membrane Protein Structures via Bifunctional Spin Labels and Lipodisq Nanoparticles

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.

Use of electron paramagnetic resonance to solve biochemical problems.

Electron paramagnetic resonance (EPR) spectroscopy is a very powerful biophysical tool that can provide valuable structural and dynamic information about a wide variety of biological systems. The intent of this review is to provide a general overview for biochemists and biological researchers of the most commonly used EPR methods and how these techniques can be used to answer important biological questions. The topics discussed could easily fill one or more textbooks; thus, we present a brief background on several important biological EPR techniques and an overview of several interesting studies that have successfully used EPR to solve pertinent biological problems. The review consists of the following sections: an introduction to EPR techniques, spin-labeling methods, and studies of naturally occurring organic radicals and EPR active transition metal systems that are presented as a series of case studies in which EPR spectroscopy has been used to greatly further our understanding of several important biological systems.

Evidence for direct binding between HetR from Anabaena sp. PCC 7120 and PatS-5.

HetR, master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120, stimulates heterocyst differentiation via transcriptional autoregulation and is negatively regulated by PatS and HetN, both of which contain the active pentapeptide RGSGR. However, the direct targets of PatS and HetN remain uncertain. Here, we report experimental evidence for direct binding between HetR and the C-terminal RGSGR pentapeptide, PatS-5. Strains with a hetR allele coding for conservative substitutions at residues 250-256 had altered patterns of heterocysts and, in some cases, reduced sensitivity to PatS-5. Cysteine scanning mutagenesis coupled with electron paramagnetic resonance (EPR) spectroscopy showed quenching of spin label motion at HetR amino acid 252 upon titration with PatS-5, indicating direct binding of PatS-5 to HetR. Gel shift assays indicated that PatS-5 disrupted binding of HetR to a 29 base pair inverted-repeat-containing DNA sequence upstream from hetP. Double electron-electron resonance EPR experiments confirmed that HetR existed as a dimer in solution and indicated that PatS-5 bound to HetR without disrupting the dimer form of HetR. Isothermal titration calorimetry experiments corroborated direct binding of PatS-5 to HetR with a K(d) of 227 nM and a 1:1 stoichiometry. Taken together, these results indicated that PatS-5 disrupted HetR binding to DNA through a direct HetR/PatS interaction. PatS-5 appeared to either bind in the vicinity of HetR amino acid L252 or, alternately, to bind in a remote site that leads to constrained motion of this amino acid via an allosteric effect or change in tertiary structure.

Characterizing the structure of lipodisq nanoparticles for membrane protein spectroscopic studies

Membrane protein spectroscopic studies are challenging due to the difficulty introduced in preparing homogenous and functional hydrophobic proteins incorporated into a lipid bilayer system. Traditional membrane mimics such as micelles or liposomes have proved to be powerful in solubilizing membrane proteins for biophysical studies, however, several drawbacks have limited their applications. Recently, a nanosized complex termed lipodisq nanoparticles was utilized as an alternative membrane mimic to overcome these caveats by providing a homogeneous lipid bilayer environment. Despite all the benefits that lipodisq nanoparticles could provide to enhance the biophysical studies of membrane proteins, structural characterization in different lipid compositions that closely mimic the native membrane environment is still lacking. In this study, the formation of lipodisq nanoparticles using different weight ratios of POPC/POPG lipids to SMA polymers was characterized via solid-state nuclear magnetic resonance (SSNMR) spectroscopy and dynamic light scattering (DLS). A critical weight ratio of (1/1.25) for the complete solubilization of POPC/POPG vesicles has been observed and POPC/POPG vesicles turned clear instantaneously upon the addition of the SMA polymer. The size of lipodisq nanoparticles formed from POPC/POPG lipids at this weight ratio of (1/1.25) was found to be about 30 nm in radius. We also showed that upon the complete solubilization of POPC/POPG vesicles by SMA polymers, the average size of the lipodisq nanoparticles is weight ratio dependent, when more SMA polymers were introduced, smaller lipodisq nanoparticles were obtained. The results of this study will be helpful for a variety of biophysical experiments when specific size of lipid disc is required. Further, this study will provide a proper path for researchers working on membrane proteins to obtain pertinent structure and dynamic information in a physiologically relevant membrane mimetic environment.

Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes

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.

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy

Wild-type phospholamban (WT-PLB), a Ca(2+)-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca(2+)-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τ(c)); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.

Differential binding between PatS C-terminal peptide fragments and HetR from Anabaena sp. PCC 7120.

Heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120 occurs at regular intervals under nitrogen starvation and is regulated by a host of signaling molecules responsive to availability of fixed nitrogen. The heterocyst differentiation inhibitor PatS contains the active pentapeptide RGSGR (PatS-5) at its C-terminus considered the minimum PatS fragment required for normal heterocyst pattern formation. PatS-5 is known to bind HetR, the master regulator of heterocyst differentiation, with a moderate affinity and a submicromolar dissociation constant. Here we characterized the affinity of HetR for several PatS C-terminal fragments by measuring the relative ability of each fragment to knockdown HetR binding to DNA in electrophoretic mobility shift assays and using isothermal titration calorimetry (ITC). HetR bound to PatS-6 (ERGSGR) >30 times tighter (K(d) = 7 nM) than to PatS-5 (K(d) = 227 nM) and >1200 times tighter than to PatS-7 (DERGSGR) (K(d) = 9280 nM). No binding was detected between HetR and PatS-8 (CDERGSGR). Quantitative binding constants obtained from ITC measurements were consistent with qualitative results from the gel shift knockdown assays. CW EPR spectroscopy confirmed that PatS-6 bound to a MTSL spin-labeled HetR L252C mutant at a 10-fold lower concentration compared to PatS-5. Substituting the PatS-6 N-terminal glutamate to aspartate, lysine, or glycine did not alter binding affinity, indicating that neither the charge nor size of the N-terminal residues side chain played a role in enhanced HetR binding to PatS-6, but rather increased binding affinity resulted from new interactions with the PatS-6 N-terminal residue peptide backbone.

Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy.

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the α‐helical M2δ subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine‐mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the α‐helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the 2H nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an α‐helix, the i + 3 and i + 4 samples reveal a strong signal from the 2H nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest 2H‐labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (μg).

Cholesterol-Dependent Conformational Exchange Of The C-Terminal Domain Of The Influenza A M2 Protein

The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol-dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry-related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol-dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all of our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding.

Distance Measurements on a Dual-Labeled TOAC AChR M2δ Peptide in Mechanically Aligned DMPC Bilayers via Dipolar Broadening CW-EPR Spectroscopy

A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 14.6 Å from a dual TOAC-labeled AChR M2δ peptide at positions 7 and 13 that closely matches with the 14.5 Å distance obtained from a model of the labeled AChR M2δ peptide. In addition, the angles determining the relative orientation of the two nitroxides have been determined, and the results compare favorably with molecular modeling. The global analysis of the data from the aligned samples gives much more precise estimates of the parameters defining the geometry of the two labels than can be obtained from a randomly dispersed sample.