Characterization of Bifunctional Spin Labels for Investigating the

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Characterization of Bifunctional Spin Labels for Investigating the Structural and Dynamic Properties of Membrane Proteins Using EPR Spectroscopy Indra Dev Sahu, Andrew F. Craig, Megan M. Dunagan, Robert M. McCarrick, and Gary A Lorigan J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07631 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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The Journal of Physical Chemistry

Characterization of Bifunctional Spin Labels for Investigating the Structural and Dynamic Properties of Membrane Proteins using EPR Spectroscopy Indra D. Sahu, Andrew F. Craig, Megan M. Dunagum, Robert M. McCarrick, and Gary A. Lorigan*

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056 * Corresponding author. Email: [email protected]

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ABSTRACT 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 lineshape 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 lineshape for all 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 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 2 ACS Paragon Plus Environment

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study demonstrates the utility of investigating the structural and dynamic properties of membrane proteins in physiologically relevant membrane mimetics using BSLs.



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INTRODUCTION Site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is a very powerful structural biology technique to study structural and dynamic properties of soluble and membrane proteins without size limitations.1-4 In SDSL experiments, all native non-disulfide bonded cysteines are eliminated by replacing them with another amino acid such as alanine. A unique cysteine residue is then introduced into a recombinant protein via site-directed mutagenesis, and subsequently reacted with a sulfhydryl-specific nitroxide reagent to generate a stable paramagnetic EPR active side-chain.5-7 The most commonly used spin label probe is methanthiosulfonate (MTSL). The flexibility of MTSL allows it to incorporate at any sites of a protein and has been very successful for determining protein topology, characterizing nanoscale backbone motion, mapping structural changes, and predicting de novo protein structures.2,

8-13

However, its application is challenging for inter-spin distances, orientation

measurements, and slow protein dynamics (micro-to-milli second).14 These problems can be resolved by utilizing more restricted spin-labels with fewer rotamers than MTSL. An approach for using bulky substituents in the nitroxide ring has been explored using EPR studies by Prof. Hubbell group.15 Another highly restricted spin-label called 2,2,6,6-tetramethylpiperidine-1oxyl-4-amino-4-carboxylic acid (TOAC) can be used for the inter-electron distances, orientation measurements and slow backbone motion, can be attached at the backbone level of amino acid in protein sequences.16 The incorporation of the TOAC spin-label into globular proteins using molecular biology techniques is very challenging.

Recently, a very successful potentially

restricted spin-label known as bifunctional spin-label (BSL) has been introduced for measuring distances and slower backbone protein motion.2,

12, 17-21

Bifunctional spin-labels can be

introduced by a facile cross-linking reaction of a bifunctional methanethiosulfonate reagent with pairs of cysteine residues at i and i + 3 or i and i+ 4 in an α-helix, at i and i+1 or i + 2 in a β4 ACS Paragon Plus Environment

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strand.17 These spin-labels are rigid and thus very useful for obtaining tighter DEER distance distributions when compared to the traditional flexible MTSL.17, 18 DEER studies incorporating the unique BSL have been recently conducted on soluble and membrane protein systems.12, 17, 18, 20, 22

However, a detailed characterization of BSLs for investigating the structural and dynamic

properties of membrane proteins is still lacking. In this study, we characterized BSLs by temperature dependent EPR measurements on an integral membrane protein KCNE1. KCNE1 is a single-pass transmembrane protein consisting of 129 amino acids that modulates the function of certain voltage gated potassium ion channels (Kv) including KCNQ1.23-25 Recent biochemical and electrophysiological studies indicated that the transmembrane domain (TMD) of KCNE1 binds to the pore domain of the KCNQ1 channel modulating the channel’s gating.26-29 Mutations in the genes encoding these proteins result in an increased susceptibility to the genetic diseases such as congenital deafness, congenital long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death.24, 30, 31 Three double cysteine mutations were generated including two mutations (53/57, 63/67) on the TMD of KCNE1 and one mutation (102/106) on the extracellular domain (Figure 1) and further attached with BSLs. CW-EPR data were collected at various temperatures (296 K to 325 K). CW-EPR spectral lineshape analysis was performed to determine structural dynamic information such as side-chain mobility and rotational correlation times. This study also reports for the first time the distance measurement between two BSLs attached on the KCNE1 TMD in POPC/POPG lipid bilayers using CW-EPR line broadening spectroscopy. MATERIALS AND METHODS Site-directed mutagenesis



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The His-tag expression vectors (pET-16b) containing the wild type and a cysteine-less mutant of KCNE1 were transformed into XL1-Blue Escherichia coli cells (Stratagene). Plasmid extracts from these cells were obtained using the QIAprep Spin Miniprep Kit (Qiagen). Site-directed cysteine mutants were introduced into the cysteine-less KCNE1 gene using the Quickchange lightning site-directed mutagenesis kit (Stratagene). The KCNE1 mutations were confirmed by DNA sequencing from XL10-Gold E. coli (Stratagene) transformants using the T7 primer (Integrated DNA Technologies). Successfully mutated vectors were transformed into BL21(DE3) CodonPlus-RP E. coli cells (Stratagene) for protein overexpression. Single BSL mutants (Phe53/Phe57, Leu63/Arg67, Ser102/Cys106) were generated by introducing a pair of Cys residues at the i and i+4 positions (53, 57, 63, 67, 102 and 106). These mutants were chosen to cover KCNE1 TMD as well as one in the extracellular region of KCNE1. Double BSL mutants (Phe53-Phe57/Leu63-Arg67) were generated by introducing two pairs of Cys residues at the i and i+4 positions (53, 57, 63 and 67). Overexpression and purification: The overexpression and purification of E. coli BL21 cells carrying mutated KCNE1 genes were carried out by using previously described protocol.25 E. coli cells carrying mutants of choice were grown in an M9 minimal medium with 50 µg/mL ampicillin. The cell culture was incubated at 37 °C and 240 rpm until the OD600 reached to 0.8, at which point protein expression was induced using 1 mM IPTG (isopropyl-1-thio--D-galactopyranoside), followed by continued rotary shaking at 37°C for 16 h. Purification of KCNE1 from inclusion bodies was carried out according to a previously described method24 and eluted using 0.05% LMPG detergent. Protein samples were concentrated by using Microcon YM-3 (molecular weight cutoff, 3,000) (Amicon). Protein concentration was determined from the OD280 using an extinction coefficient of 1.2


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mg/mL protein per OD280 on a NanoDrop 200c (Thermo Scientific). The purity of KCNE1 from overexpression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Spin labeling and reconstitution into proteoliposomes: Spin labeling and proteoliposomes reconstitution were carried out following the protocol previously

described.12,

18,

32

The

bifunctional

spin-label

(BSL)

(3,4-Bis-

(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy Radical) (HO1944) was obtained from Toronto Research Chemicals Inc., Toronto, Canada. The spin-label was dissolved in methanol to a concentration of 125 mM and added directly to the concentrated protein in elution buffer at a 10:1 spin label:protein molar ratio for single BSL mutants with double-Cys and 20:1 spin-label:protein molar ratio for dual BSL mutants with four-Cys and then reacted for 24 hours with gentle shaking at room temperature in the dark to complete the labeling. Excess/unreacted free spin labels were removed by an extensive dialysis. Dialysis was carried out at room temperature in a regenerated cellulose dialysis tubing (Fisher brand MW cutoff 3.5 kDa) against 1L dialysis buffer (100 mM NaH2Po4, pH 7.8) and 0.2% SDS without reducing agent for a week with buffer changes twice daily. The spin labeling efficiency was determined by comparing the nano-drop UV A280 protein concentration with the spin concentration obtained from CW-EPR spectroscopy. The protein concentration for all KCNE1 samples was ~ 75 µM, and the spin labeling efficiency for all samples was ~ 75 %.

The reconstitution of spin-labeled protein into POPC/POPG (3:1) proteoliposomes was carried out via dialysis methods following a similar protocol in the literature.18, 32 The concentrated spin labeled KCNE1 protein was mixed with stock lipid slurry (400 mM SDS, 75 mM POPC and 25 mM POPG, 0.1 mM EDTA, 100 mM IMD, pH 6.5). The lipid slurry was pre-equilibrated to a 7 ACS Paragon Plus Environment


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clear mixed micelles via an extensive freeze thaw cycles. The final protein:lipid molar ratio was set to 1:400. The KCNE1-lipid mixture was then subjected to an extensive dialysis to remove all SDS present, during which process KCNE1/POPC/POPG vesicles spontaneously formed. The 4L dialysis buffer (10 mM imidazole and 0.1 mM EDTA at pH 6.5) was changed twice daily. The completion of SDS removal was determined when the KCNE1-lipid solution became cloudy and the surface tension of the dialysate indicated complete removal of detergent. Reconstitution into lipodisq nanoparticles: Lipodisq nanoparticles (pre-hydrolyzed styrene-maleic anhydride copolymer 3:1 ratio) were obtained from Malvern Cosmeceutics Ltd. (Worcester, United Kingdom). The protein-lipid complex was incorporated into SMA-lipodisq nanoparticles following published protocols.12, 18, 33-35

A 500 µl aliquot of proteoliposome-reconstituted protein sample (~30 mM POPC/POPG

lipid) was added with the same amount (500 µl) of 2.5% of lipodisq solution prepared in the same dialysis buffer (10 mM Imidazole, 0.1 mM EDTA at pH 6.5) dropwise over 3-4 minutes. The protein-lipodisq solution was allowed to equilibrate overnight at 4o C. The resulting solution was centrifuged at 40,000xg for 30 minutes to remove the non-solubilized protein. The supernatant was further concentrated to desired volume and concentration for EPR measurements. EPR Spectroscopic Measurements: EPR experiments were conducted at the Ohio Advanced EPR Laboratory. CW-EPR spectra were collected at X-band on a Bruker EMX CW-EPR spectrometer using an ER041xG microwave bridge and ER4119-HS cavity coupled with a BVT 3000 nitrogen gas temperature controller. Each spin-labeled CW-EPR spectrum was acquired by signal averaging 20 42-s field scans with a central field of 3315 G and sweep width of 100 G, modulation frequency of 100 kHz, 8 ACS Paragon Plus Environment

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modulation amplitude of 1 G, and microwave power of 10 mW at different temperatures (296 K -325 K). CW-dipolar broadening EPR data were analyzed using the Short Distances LabVIEW program developed by Dr. Christian Altenbach following the instruction provided by the developer (http://www.biochemistry.ucla.edu/biochem/Faculty/Hubbell/). A Gaussian function was used to obtain the distance distribution. EPR Spectral Simulations: EPR spectra were simulated using the non-linear least squares (NLSL) program including the macroscopic order, microscopic disorder (MOMD) model developed by the Freed group.36,

37

The principle components of the hyperfine interaction tensor A = [5.5±0.5, 5.5±0.5, 34.8±0.8] G and g-tensors g=[2.0088±0.0002, 2.0070±0.0002, 2.0023±0.0001] were obtained from a leastsquares fit to the spectrum of 63/67 KCNE1 in a frozen state at 150 K. During the simulation process, the A and g tensors were held constant and the rotational diffusion tensors were varied. A two-site fit was used to account for both rigid/slower and higher/faster motional components of the EPR spectrum. The best fit rotational correlation times and the relative population of both components were determined using the Brownian diffusion model. Molecular Dynamics modeling of KCNE1 bearing BSLs in POPC/POPG Lipid Bilayers: Molecular dynamics simulations on KCNE1 (PDB ID: 2K21) in POPC/POPG lipid bilayers were performed using NAMD 38 version 2.9 with the CHARMM36 force field. 39, 40 CHARMM-GUI 41

(http://www.charmm-gui.org) was used for simulation set up and input generation, and visual

molecular dynamics software, VMD42 version 1.9.1 was used for MD trajectory analysis. The membrane, composed of a pre-equilibrated bilayer of POPC/POPG molecules with a 60 Å2 surface, was built using membrane builder protocol under CHARMM-GUI.

41, 43

The dual BSLs 9

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labeled KCNE1 (PDB ID: 2K21) 44 was inserted into the membrane and the system was solvated into a water box and ionized to add bulk water above and below the membrane and to neutralize the system with KCl using the membrane builder protocol.41,

43

The final assembled system

comprised waters, POPC/POPG lipids, ions and the protein (a total of 18,793 atoms). Six steps of equilibration and minimization of the system were performed under NAMD using the input files generated by CHARMM-GUI before running production run following the instructions provided in the membrane builder protocol. Starting from this equilibrated system, NAMD production run was collected out to 20 ns using Langevin dynamics.45 Electrostatic interactions were computed using the Particle-Mesh Ewald algorithm with a 12 Å cutoff distance 46 and Van der Waals interactions were computed with a 12 Å cutoff distance and a switching function to reduce the potential energy function smoothly to zero between 10-12 Å. Periodic-boundary conditions were used and constant temperature (303 K) and pressure (1 atm) were maintained. Equations of motion were integrated with a time step of 2 fs and trajectory data were recorded in 2 ps increments.

45

Probability distance distribution was obtained from trajectory data using the

script (distance.tcl) provided in the VMD software package. All molecular dynamics simulations were run on a home-built 24-node Linux Beowulf style cluster installed in our lab.

RESULTS The characterization of BSL was performed using EPR spectroscopy on the integral membrane protein KCNE1 in two membrane mimetics (POPC/POPG liposomes and POPC/POPG lipodisq nanoparticles). Figure 1 shows the schematic representation of the BSL spin-label probe, the locations of the spin-labels mapped onto the NMR structure of the KCNE1 membrane protein in LMPG micelles, and a predicted topology of KCNE1 in lipid bilayers.23 These sites were chosen 10 ACS Paragon Plus Environment

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to include the TMD and extracellular region of KCNE1. A POPC:POPG (3:1) lipid bilayer was used to mimic phospholipids typically found in mammalian membranes.18, 25, 32, 47 CW-EPR Spectral Measurements: CW-EPR spectra of site-directed spin-labels are used to probe dynamic properties of a protein.1, 2 CW-EPR spectral data for a series of spin-labeled protein sequences were used to model the KCNE1 protein structure with a spatial resolution at the residue-specific level.35, 48-52 CW-EPR experiments were performed on three individual positions (53/57 and 63/67 on the TMD, and 102/106 on the outside of the membrane) on KCNE1 at various temperatures from 296 K to 325 K. CW-EPR spectra of KCNE1 bearing BSLs are shown in Figures 2 (A-C) for the mutants 53/57, 63/67, and 102/106 in POPC/POPG lipid bilayers (left panel) and POPC/POPG lipodisq nanoparticles (right panel) at temperatures 296 K to 325 K. Inspection of the CW-EPR spectra indicates that the spectral lineshapes are similar for both inside TMD mutants 53/57 and 63/67 in POPC/POPG lipid bilayers obtained at 296 K having a characteristic immobilized lineshape. However, the line broadenings decrease with increase in temperature up to 325 K. Similarly, the EPR spectrum of the outside mutant 102/106 in POPC/POPG lipid bilayers also has a characteristic immobilized lineshape at 296 K and the line broadening decreases with increase in temperature up to 325 K. The line broadenings for these BSL labeling probes in lipodisq nanoparticles are higher than that of the proteoliposome samples indicating that the motion of BSLs labeled sites is slower in lipodisq nanoparticle samples. The decrease in line broadening pattern with increase in temperature is consistent with the liposome samples. The CW-EPR spectra of all three mutants contain two motional components with a slower/rigid component (left arrow) and a more motional/less rigid component (right arrow). CW-EPR spectra obtained for these BSL samples are consistent with previously reported EPR spectra for BSL samples.17, 11 ACS Paragon Plus Environment


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18, 21

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The minor component of more motional/less rigid component (right arrow) for the

bifunctional spin label samples observed in this study has been also documented in previous studies.17,

53-55

A decrease in the line broadening pattern with increasing temperature is also

consistent with the previously reported temperature dependent SDSL CW-EPR spectra.35, 56 In order to more quantitatively describe the motion of the BSLs of KCNE1, spectral linewidths (2Azz) were plotted against the temperatures for 53/57, 63/67 and 102/106 for both POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles in Figure 3. Figure 3(A) indicates that the spectral width (2Azz) for the site 53/57 decreases (66 G to 64 G) with increase in the temperature for POPC/POPG liposomes. The decrease in spectral width pattern (69 G to 67 G) for lipodisq nanoparticle samples is also similar to the liposome samples. However, the 2Azz values for lipodisq nanoparticle samples are higher than that of liposome samples. From the spectral width profile plotted for the site 63/67 and the site 102/106 (see Figure 3(B) and 3(C)), the 2Azz values for the site 63/67 decrease from 67 G to 65 G for liposome samples and 70 G to 67 G for lipodisq nanoparticle samples, and 2Azz values for the site 102/106 decrease from 70 G to 67 G for liposome samples and 71 G to 69 G for lipodisq nanoparticle samples. The 2Azz values for all three BSL labeling sites indicated that the dynamics of BSLs labeled sites increases with increase in temperature. Interestingly, the 2Azz values for the outside probe 102/106 are slightly higher than that for the inside TMD probes (53/57 and 63/67). The slight increase in 2Azz values for the site 102/106 may be due to the polarity effect of the solvent as the site 102/106 is in contact with the extracellular region of KCNE1.57 The other possible reason may be that the region around the site 102/106 might be approaching to the membrane surface so that the BSL spin label can be trapped into the head group region of the lipid bilayers causing decrease in the motion of the BSL spin labels.52 Figure 3(C) also revealed that the 2Azz pattern between 12 ACS Paragon Plus Environment

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liposomes and lipodisq nanoparticle samples for the outside probe (102/106) is relatively narrower than that for the inside probes (53/57 and 63/67). This is expected as the lipodisq nanoparticles samples have no significant or minor effect on the motion of extracellular regions of the protein.35 The inverse central linewidths were also plotted against the temperatures for all three BSL labeled KCNE1 mutants as shown in Figure 4. From Figure 4, it is clear that the inverse central linewidth of the site 53/57 in liposome remains similar from temperature 296 K to 318 K and then increases at 325 K. This inverse central linewidth pattern is similar for the BSL labeled site 63/67 in liposomes from temperature 296 K to 308 K and then increases from 318 K to 325 K. This pattern is different for the site 102/106 indicating that the inverse central linewidth increases almost linearly with increase in temperature from 296 K to 325 K. However, the inverse central linewidth remains similar for lipodisq nanoparticle samples for all the temperatures for the sites 53/57 and 63/67, but becomes higher for the site 102/106 at a temperature of 325 K. The inverse central linewidth data indicate that the side-chain dynamics of the BSL labeled residues located inside the membrane bilayer behave differently than that of BSL labeled residue located outside the membrane bilayer. This motional behavior is consistent with the previously published KCNE1 side-chain dynamics in lipid bilayers.35, 52 In order to further quantify the spin-label side-chain motion of KCNE1, non-linear least squares (NLSL) MOMD EPR spectral simulations were carried out for the representative EPR spectra of BSL labeled sites 53/57, 63/67 from the KCNE1 TMD, and 102/106 from extracellular domain of KCNE1 at 296 K and 325 K to determine the rotational correlation times and the relative population of the two motional components (see Table 1). The previously described method of simulation under NLSL program was used for this study.17, 21, 35-37, 52, 58 The Zeeman interaction 13 ACS Paragon Plus Environment


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tensors (gxx, gyy, gzz) and hyperfine interaction tensors (Axx, Ayy, Azz) were obtained from a leastsquares fit to the spectrum of BSL labeled site 63/67 in a frozen state at 150 K and held constant during the fitting process and the rotational correlation times and the relative population of the two components were determined from the best fit EPR spectra (see Figure 5, red lines). Table 1 shows rotational correlation times and the corresponding population of the slower/rigid (site 1) and the more motional/less rigid component (site 2) components of EPR spectra. The EPR spectra were simulated using a two-site fit for the data at both temperatures 296 K and 325 K to obtain the best fit simulations. The simulation results indicated that the side-chain spin-label motion of transmembrane mutants at 296 K in POPC/POPG liposome is immobilized with longer correlation times of 139 ns for slower/rigid component with a relative population of 92 % and 2.6 ns for the more motional/less rigid component with a relative population of 8 % for the site 53/57, 135 ns for slower/rigid component with a relative population of 82 % and 2.9 ns for more motional/less rigid component with a relative population of 18 % for the site 63/67, while that of the extracellular mutant having 132 ns for slower/rigid component with a relative population of 76 % and 2.1 ns for the more motional/less rigid component with a relative population of 24 % for the site 102/106. Similarly, the side-chain spin-label motion of KCNE1 mutants at 296 K in POPC/POPG lipodisq nanoparticles are highly immobilized with longer correlation times of 332 ns for slower/rigid component with a relative population of 76 % and 3.2 ns for the more motional/less rigid component with a relative population of 24 % for the site 53/57, 227 ns for slower/rigid component with a relative population of 84 % and 4.2 ns for the more motional/less rigid component with a relative population of 16 % for the site 63/67, while that of the extracellular mutant having 225 ns for slower/rigid component with a relative population of 70 % and 3.4 ns for the more motional/less rigid component with a relative


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population of 30 % for the site 102/106. The simulation results indicated that the rotational correlation times decreased to 37 ns for slower/rigid component with a relative population of 78 % and 1.7 ns for the more motional/less rigid component with a relative population of 22 % for the site 53/57, 20 ns for slower/rigid component with a relative population of 90 % and 0.8 ns for the more motional/less rigid component with a relative population of 10 % for the site 63/67, while that of the extracellular mutant decreasing to 17 ns for slower/rigid component with a relative population of 91 % and 0.3 ns for the more motional/less rigid component with a relative population of 9 % for the site 102/106 with increasing temperature to 325 K in POPC/POPG lipid bilayers. Similarly, the rotational correlation times decreased to 58 ns for the slower/rigid component with a relative population of 97 % and 0.7 ns for the more motional/less rigid component with a relative population of 3 % for the site 53/57, 60 ns for slower/rigid component with a relative population of 96 % and 0.8 ns for the more motional/less rigid component with a relative population of 4 % for the site 63/67, while that of the extracellular mutant decreased to 43 ns for slower/rigid component with a relative population of 95 % and 0.4 ns for the more motional/less rigid component with a relative population of 5 % for the site 102/106 with increasing temperature to 325 K in lipodisq nanoparticles. The decreasing pattern of rotational correlation times for all the lipodisq nanoparticle samples is similar to that of the liposome samples with increasing temperature from 296 K to 325 K. However, the values of the rotational correlation times are longer for lipodisq nanoparticle samples when compared to that of the KCNE1 liposome samples. The decrease in the motional behavior of rigid spin-labeled KCNE1 lipodisq nanoparticle samples are consistent with the previously published literature.35 Distance Measurement between Dual Bifunctional Spin-labels using CW Dipolar Broadening EPR Experiment: 15 ACS Paragon Plus Environment


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Distance measurements between two spin labels on a protein are a very powerful tool to probe structural and conformational dynamics.2,

59, 60

EPR spectroscopic techniques can estimate the

distance between two spin-labels in the form of dipolar interactions between them in sitedirected spin labeling experiments. The electron-electron dipolar interaction produces a significant broadening or dipolar splitting of the CW-EPR spectrum when compared to the individual non interacting spins.59 CW-EPR dipolar broadening technique can measure intermediate range distances between 8 to 25 Å while double electron-electron resonance (DEER)/pulsed electron-electron double resonance (PELDOR) spectroscopy can measure long range distances between 20 to 80 Å,2,

18, 61-63

and provide valuable information and distance

restraints for structural modelings.64, 65 In this study, we measured a distance between two BSLs (53/57 to 63/67) representing the transmembrane domain of KCNE1 at a temperature of 296 K in POPC/POPG proteoliposomes as shown in Figure 6. Figure 6 clearly indicates that a significant dipolar broadening is observed in the EPR spectrum of the double-labeled BSL mutants when compared to the spectrum of the singly labeled BSL mutant. The dipolar line broadening observed between the single BSL labeled and the double BSL labeled spectra is consistent with a recently published dipolar broadening study on the integral membrane sulfurtransferase.66 The analysis of the dipolar broadening data resulted a distance of (15.3 ± 2.0) Å. The uncertainty observed in this distance measurement may be due to the spin label rotamers and the backbone fluctuation of the KCNE1 protein in the presence of lipid bilayers.52 A Gaussian function was used to obtain distance distribution during the spectral analysis under Short Distances LabVIEW program (http://www.biochemistry.ucla.edu/biochem/Faculty/Hubbell/). In order to validate the experimental results, molecular dynamics modeling of KCNE1 bearing BSLs (53/57-63/67) was


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performed in POPC/POPG lipid bilayers. A 20 ns molecular dynamics simulation data revealed 17 Å distance which is closely agreed with the experimental value of 15.3 Å. DISCUSSIONS We previously utilized bifunctional spin labels (BSLs) to improve DEER distance measurements and validate the three-dimensional structure refinement of transmembrane domain of KCNE1 membrane protein in lipid bilayers and lipodisq nanoparticles.12, 18 Bifunctional spin-labels are able to restrict the motion of spin-labels reducing the corresponding number of conformational rotamers and hence improving DEER measurements with tighter distance distributions.17, 19 This improvement can be very helpful for determining the structural model of membrane proteins which is difficult or nearly impossible to obtain by using other biophysical methods.12,

22

Recently, BSLs have been utilized to measure the helical tilt of the membrane protein phospholamban (PLB) in magnetically-aligned bicelles using EPR spectroscopy.21 Spectral changes observed in the EPR spectra of BSLs are due to the changes in backbone motion of proteins and hence it is very suitable for measuring the dynamic properties of the backbone of the protein.19 There is no significant perturbation in structure and function of proteins due to the attachment of bifunctional spin-labels.

17, 19, 67

Recently, Fleissner et al. compared the crystal

structure of T4 Lysozyme bearing BSL with the structure of the wild-type T4 Lysozyme and suggested that the attachment of bifunctional spin label doesn't distort the secondary structure of the protein. 17 In this study, we used EPR spectral lineshape analysis on an integral membrane protein KCNE1 bearing BSLs for both inside and the outside mutants against different temperatures and reported spectral widths, side-chain mobilities, and rotational correlation times. The rotational correlation



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times corresponding to the spin-labeled sites are expected to reflect the backbone motion of the protein because the BSLs are rigidly attached having limited motional rotamers.19 These rotational correlation times clearly differentiate EPR spectra of the inside and the outside probes of KCNE1. There is a significant differentiable effect of temperature on both the inside and the outside BSL probes (see Figure 2). This is expected as the backbone motion is relatively slower for the transmembrane domain of KCNE1 when compared to the extracellular region of the protein.52 Our previous CW-EPR studies on KCNE1 in liposome indicated that the slower/rigid and more motional/less rigid components for the transmembrane mutant V50C bearing the MTSL spin label have rotational correlation times of 13.7 ns with a relative population of 26% and 1.2 ns with a relative population of 74% respectively.52 The rotational correlation times for KCNE1 transmembrane mutants bearing the bifunctional spin label (BSL) reported in this study are longer with a higher relative population for the slower/rigid components (see Table 1) when compared to that of the MTSL spin labeled mutants.52 The rotational correlation times reported in this study are consistent with previously reported rotational correlation times of BSL spin labeled proteins.17, 21 The multiple motional components observed in the EPR spectra may be due to the heterogeneity in the population of incorporated proteins into liposomes. Our recent CWEPR studies on KCNE1 in POPC/POPG lipid bilayers and POPC/POPG lipodisq nanoparticles indicated that the multiple motional components observed in EPR spectra is due to the fact that the protein undergoes multiple conformations when interacting with lipid bilayers 35, 52 and hence it is a characteristic of KCNE1 protein in lipid bilayers. There may be some possible minor contribution from the uncoupled BSLs or BSL bound to a single cysteine (see Figure S2) towards more motional/less rigid components. 17, 21, 55 The variation in the relative population of the two motional components at higher temperature (see Table 1) may be due to the increase in


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the heterogeneity in the population of incorporated proteins into liposomes due to the effect of increased temperatures causing conformational exchange for the stability of the membraneprotein complex. . Intermediate range distance measurements (8 Å - 25 Å) of globular or membrane proteins are usually carried out at lower temperature or applying osmolytes such as sucrose or glycerol using CW-dipolar line broadening in order to reduce the global motion of protein or peptide. This study reports the distance measurement at a room temperature which is very important for functional activities of proteins. In order to validate the usage of BSLs for line broadening distance measurements, molecular dynamics modeling of dual BSL-labeled KCNE1 was performed in POPC/POPG lipid bilayers and distance between the two BSLs was determined by analyzing 20 ns molecular dynamics trajectory data. The experimentally measured distance agrees closely with the molecular dynamics modeling. The dynamic information and distance measurement reported in this study are consistent with the previously reported structural and dynamic properties of KCNE1 in POPC/POPG lipid bilayers.12, 35, 52 Future experiments such as EPR saturation recovery will be very helpful to investigate the relaxation properties of BSL labeled KCNE1 in lipid bilayer environment.68 Distance measurement on membrane proteins in a native membrane environment is very challenging due to the motion of spin label probes and heterogeneity observed during the sample preparation. In this study, we reported for the first time the utilization of restricted BSLs for intermediate range distance measurements using CW dipolar broadening EPR on an integral membrane protein KCNE1. Our current and past studies have provided a strong database for utilizing BSLs for EPR studies of membrane proteins to answering several structural and dynamic questions.12, 18 19 ACS Paragon Plus Environment


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CONCLUSION We successfully characterized rigid BSLs attached to the KCNE1 membrane protein at various temperatures (296 K-325 K) for structural dynamics and distance measurements. BSLs can be used to determine intermediate distances at physiological temperature using dipolar broadening CW-EPR without using any osmolytes. Distance measurement at physiological temperature is very important for functional activities of proteins. This study also reports the feasibility of BSLs for measuring the backbone motion of membrane proteins. This study opens a path for measuring more accurate intermediate range distances of membrane proteins at physiological temperature suitable for protein functions. SUPPORTING INFORMATION AVAILABLE

Overlay of CW-EPR spectra of KCNE1 bearing BSLs at 102/106 in liposome and lipodisq at 296 K (Figure S1) and a cartoon representation of KCNE1 with a BSL attached at a single Cys at the site 53 (Figure S2). This information is available free of charge via the Internet at http://pubs.acs.org

ACKNOWLEDGEMENTS We would like to thank Dr. Christian Altenbach at the University of California Los Angeles for assisting with the Short Distances LabVIEW program. This work was supported by National Institutes of Health Grants R01 GM108026. Funding was also provided by National Science Foundation (NSF) Grant CHE-1305664. Prof. Gary A. Lorigan would like to acknowledge support from the John W. Steube Professorship.


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Figure 1: Chemical structure of the bifunctional spin-label probe (A), Cartoon representation of the NMR structure of KCNE1 (PDB ID: 2k21) with BSL spin labeling sites represented by green sphere at their alpha carbons (B), A predicted topology of KCNE1 in lipid bilayers based on previous solution NMR studies (C). 23


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Figure 2: CW-EPR spectra of KCNE1 bearing BSLs at 53/57 (A), 63/67 (B), and 102/106 (C) in POPC/POPG liposomes (left panel) and POPC/POPG lipodisq nanoparticles (right panel) as a function of temperature. The arrows represent the two motional components in the spectra.



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Figure 3: Spectral width (2Azz) as a function of temperature for KCNE1 bearing BSLs at sites 53/57 (A), 63/67 (B), and 102/106 (C) calculated from EPR spectra in Figure 2.



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Figure 4: Inverse central linewidth as a function of temperature for KCNE1 bearing BSLs at sites 53/57 (A), 63/67 (B), and 102/106 (C) calculated from EPR spectra in Figure 2.



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Figure 5: CW-EPR spectral simulations of KCNE1 mutants bearing BSLs at 296 K and 325 K at the sites 53/57 (A), 63/67 (B), and 102/106 (C) using NLSL MOMD program developed by Freed and Co-workers.


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(A)

(B)

53/57

63/67

Figure 6: Distance measurement on KCNE1. (A) CW dipolar broadening EPR spectra of KCNE1 bearing BSLs at sites 53/57 and 63/67 in POPC/POPG liposomes (left panel) and the corresponding distance distribution (right panel) obtained from data analysis by using the Short Distances LabVIEW program. (B) Cartoon representation of KCNE1 bearing two BSLs at sites 53/57 and 63/67 (left panel) and the corresponding distance distribution obtained from 20 ns molecular dynamics trajectory data analysis (right panel). 31 ACS Paragon Plus Environment


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Table 1: Rotational correlation times (τ) and the relative population (P) of two spectral components of the EPR spectra (Figure 5) obtained from the best fit NLSL MOMD spectral simulation. The uncertainty in correlation time (τ) value is ± 0.2-5 ns for liposome samples and ± 0.3-15 ns for lipodisq samples respectively. Similarly, the uncertainty in relative population (P) value is ± 3-6 % for liposome samples and ±2-4 % for lipodisq samples. These uncertainties were estimated based on the multiple batch for sample preparation and multiple simulation run.

Sites

Temperature (K)

Liposomes τ, ns (site 1)

τ, ns (site 2)

P, % (site 1)

P, % (site 2)

Lipodisq τ, ns (site 1)

τ, ns (site 2)

P, % (site 1)

P, % (site 2)

53/57

296

139

2.6

92

8

332

3.2

76

24

325

37

1.7

78

22

58

0.7

97

3

296

135

2.9

82

18

227

4.2

84

16

325

20

0.8

90

10

60

0.8

96

4

296

132

2.1

76

24

225

3.4

70

30

325

17

0.3

91

9

43

0.4

95

5

63/67 102/106


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TOC Graphic


Characterization of Bifunctional Spin Labels for Investigating the

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