Molecular Dynamic Simulations

Supporting Material for: Tethered spectroscopic probes estimate dynamic distances with sub-nanometer resolution in voltage-dependent potassium channels Brian W. Jarecki, Suqing Zheng, Leili Zhang, Xiaoxun Li, Xin Zhou, Qiang Cui, Weiping Tang, Baron Chanda

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INDEX:

PAGES

1. Supporting Text and Figures

2-7

2. Experimental Methods and Instrumentation (Biochemistry)

8-9

3. Experimental Methods (Molecular Dynamic Simulations)

10

4. Experimental Methods and Instrumentation (Electrophysiology)

11-12

5. Strategies and Procedures for Synthesis (Chemistry)

13-16

6. Supporting References

17

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1. Supporting Text and Figures

Figure S1. Chemical library of the novel tethered quenchers. Synthetic compounds are grouped into two categories (Type 1 and 2), then further sub-divided (Series a and b) based on the experiment. Type 1 quenchers were used for the model peptide studies and contained a cysteine-reactive tail. Type 2 quenchers that contained a tetraethylammonium group were used for the potassium channel experiments. Quenchers in the Series a subgroup were used for tethering experiments and contained either a cysteinereactive group (Type 1, Series a) or a tetraethylammonium group (Type 2, Series a) to anchor the compounds to their target site. Series b quenchers were used for the freely diffusing studies and did not contain an anchoring moiety.

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Analyzing and Interpreting the Distance Distribution between the Fluorophore and Quencher: The observation that the OO distance distribution peaks at the same distance (~7.5 Å) regardless of the length of the polyproline and PEG chains suggests that the interaction between TAMRA and the quencher group is important. To investigate how the treatment of this interaction impacts the qualitative trends observed in the simulations, we carried out additional molecular dynamics (MD) simulations (Fig. S2) in which the van der Waals attraction between TAMRA and the quencher group were scaled with a factor that ranges from 0 to 0.9 (0, 0.3, 0.7 and 0.9). We focused on the van der Waals attraction because it is the dominant interaction between these two largely non-polar moieties.

Figure S2. OO distance distribution from implicit solvent simulations using different VDW attraction scaling factors. If the scaling factor goes to 0, the interactions between TAMRA/NO• and the rest of the system are hard sphere-like. Attraction scaling factor is set to 0.3 for (A) Pro 6 and (D) Pro 10; 0.7 for (B) Pro 6 and (E) Pro 10; and 0.9 for (C) Pro 6 and (F) Pro 10. An upper value of 0.9 was chosen as suggested by Juneja et al.(1) who observed that the scaling factor of 0.9 is required to sample the proper end-to-end distance distribution and radius of gyration for PEG chains with the GBSW model. The simulations with a scaling factor of 0, referred to as “hardsphere” simulations, were carried out with the WCA module of CHARMM; for other scaling factors, the appropriate scaling of interactions is done with the scalar module of CHARMM. As a qualitative evaluation of the impact of sampling, we also compared the results of the hard-sphere simulations to those from an analytical model in which the polyproline is assumed to be a rigid rod and the PEG chain is described as a Gaussian chain with a bond length of b=4.7 Å. Following straightforward

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derivations, the cumulative distribution for the distance between one end of the polyproline and the end of PEG chain (see Fig. S3A) is given by,

𝐸𝑞𝑛1.

2

𝐹(𝑑 < 𝑧

2)

�𝑙𝑎+√𝑙 2 𝑎2 −4𝑙 2 +4𝑧 2 �⁄2

2 3𝑅2 3 � �� �− ⋅� 𝑑𝑅 � exp = � 𝑑𝑎 � 2𝑚𝑏 2 2𝜋𝑚𝑏 2 𝜋√1 − 𝑎2 �𝑙𝑎−√𝑙2 𝑎2 −4𝑙2 +4𝑧 2 �⁄2 −2 2

1

where l is the length of the rigid polyproline rod, and F is the accumulative contact probability for squared end-to-end distance (i.e., d2) smaller than z2. As shown in Fig. S3, the results of the hard-sphere simulations and the analytical Gaussian chain model are in qualitative agreement in terms of peak position in the distribution, suggesting that the degree of conformational sampling is adequate for our purpose. Both give the relevant distance distribution (OO distance for the hard sphere simulations and end-to-end distance for the Gaussian chain) peaks at rather long PEG lengths, especially for Pro 10. Clearly, without any attractive interactions between TAMRA and the quencher, the chain entropy of the PEG chain dominates and the probability that TAMRA and the quencher form a close contact is very low.

Figure S3. OO distance distribution comparison between Gaussian chain model and hard sphere simulations. (A) Schematic representation of various tethered quenchers covalently attached to a polyproline substrate and the respective variables used for simulations and distance calculations. The cyan cylinder represents the rigid-rod substrate (l; length) with a fluorophore (F; blue sphere) attached at one end. Tethered quenchers are attached at the opposing end of the substrate with varying number of PEG units (pink spheres) with a bond length (b), assuming a Gaussian chain distribution. A quenching moiety B.W. Jarecki et al.

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(Q; red sphere) is attached at the end of the PEG chain. The contact distance (d) between Q and F can be used to calculate the collisional frequency as a function of the PEG chain length. End-to-end distances in (B) Pro 6 and (C) Pro 10 are estimated by Equation 1 for a Gaussian chain theory model. OO distances in (D) Pro 6 and (E) Pro 10 are calculated from simulations using WCA potential between TAMRA/NO• and other groups, assuming a hard sphere model. With a low degree of attraction between TAMRA and the quencher (a scaling factor of 0.3), the OO distance distribution is almost the same as that from the hard sphere model. As the attraction becomes larger, a peak at ~7.5 Å starts to emerge, which eventually dominates the distribution at the scaling factor of 0.9. Among the four sets of scaling factors tested here, only results with the scaling factor of 0.9 led to a qualitative agreement with the experimental observation for the fluorescence intensity. This is satisfying because the same scaling factor was found to give reliable structural properties for PEG with the GBSW implicit solvent model. These simulations highlight that the quenching behavior is not simply governed by the chain properties (e.g., chain entropy) of PEG, and the interaction between the fluorophore and quencher plays a major role. Together with the importance of PEG/fluorophore interaction discussed in the primary text, the simulation studies emphasize that when designing fluorescence probes, it is not always appropriate to regard fluorophores, quenchers and the intervening motifs as merely spectators. Considering additional interactions between these groups and the substrate might be critical to accurately interpret the data.

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Figure S4. TAMRA labeled Pro 6 in the presence of freely diffusing quenching compounds. (A) Emission spectra of Pro 6 in the presence of increasing concentrations of TEMPO. This version of TEMPO includes a nitroxide radical and lacks a PEG spacer and maleimide group. (B) A synthetic quenching compound, with a nitroxide radical (PEG5-NO•; S-A3), used in tethering experiments with a spacer of 5 PEG units without the maleimide moiety.

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2. Experimental Methods and Instrumentation (Biochemistry) Peptide Preparation and Labeling: Polypeptides of defined lengths containing 6 or 10 proline residues (Pro 6 or 10) flanked by an Nterminal glycine and C-terminal cysteine were synthesized on an automated synthesizer (Protein Technologies Inc.; Prelude model, Tucson, AZ) by the University of Wisconsin-Madison Biotechnology Facility. Bulk solvents utilized for peptide synthesis were obtained from Fisher Scientific. The general method of synthesis follows principles initially described by Merrifield (1963)(2) with modification subsequently introduced by Meienhofer et al.(3) and Fields et al.(4). The syntheses were carried out on H-Cys(Trt)-2ClTrt resin (EMD Biosciences; Billerica, MA). Each synthetic cycle results in the addition of one amino acid residue to that already linked to the resin, so that synthesis proceeds from the Cterminal to N-terminal direction. Free amino acids were protected at the α-amino group with Fmoc (9fluorenylmethoxycarbonyl, EMD Biosciences). Each synthetic cycle involved four steps: (1) Deprotecting the α-amino Fmoc with 2% DBU, 20% piperidine in DMF (dimethylformamide). Routine deprotection reactions used 2 x 2.5 min reaction periods. A third 2.5 min reaction period was employed for each peptide beginning with the seventh coupling in anticipation that deprotection of the proline residues would become more difficult at this point. (2) After draining away the piperidine the resin was rinsed six times with DMF. A 4-fold excess of the appropriate Fmoc-amino acid (100 mM in DMF) was delivered to the reaction vessel and converted to an active ester by the addition of 1 eq. HCTU (1HBenzotriazolium)-1-[bis(dimethylamino)methylene]-5-chloro-hexafluorophosphate(1-),3-oxide) hexafluorophosphate) and 2 eq. NMM (N-methylmorpholine). (3) The activated Fmoc amino acid was coupled to the free amino group bound to the resin using short double couplings (2 x 10 min). (4) After completion of the coupling reaction, the resin is rinsed with DMF and the cycle of deprotection, activation and coupling is repeated for the addition of each subsequent amino acid. Following coupling of the Nterminal residue, a final deprotection cycle was used to remove the N-terminal Fmoc from the resinpeptide. Polypeptides were labeled manually with TAMRA (carboxytetramethylrhodamine; Anaspec, Fremont, CA) by coupling to the free N-terminal glycine amino group using a 3-fold excess of TAMRA activated with HCTU (Protein Technologies; Tucson, AZ)/NMM (1:2 with respect to TAMRA). After four hours the reagent was removed by filtration and the resin was rinsed with DMF and methylene chloride. The dried resin-peptide was simultaneously cleaved from the resin and deprotected (removal of side-chain protective groups) by reacting with TFA (trifluoroacetic acid) containing 2.5% ethanedithiol and 2.5% water. Following cleavage, the TFA solution was filtered to remove the resin and precipitate the deprotected peptide in a large excess of cold t-butylmethylether. The peptide was sedimented by B.W. Jarecki et al.

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centrifuge and washed twice with additional volumes of ether. The ether precipitate was vacuum dried and the resulting product was dissolved in 40% ACN (acetonitrile). The peptide solution was frozen and lyophilized, which acts as an additional purification step to remove trace amounts of volatile chemicals. Freeze drying converted the peptide to a crystalline solid. The peptides were dissolved a second time in a defined volume of solvent, then 0.5% of the peptide solution was removed for quality control evaluations (analytical HPLC, mass determination). The peptides were then freeze-dried a second time and used for downstream reactions. Peptides were stored as lyophilized powders at -80oC. Crosslinking, Purification, and Analysis of Tethered Quencher Polypeptide Complexes: Synthetic compounds with either a nitroxide radical or 3,4-dibromo quenching groups and the commercially available 4-maleimido-TEMPO (4-Maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy, free radical; Sigma-Aldrich; St. Louis, MO) both with a free maleimide group attached, were reacted (10 mM, DMSO) with the available cysteine on the polypeptide substrates (2.5 mM, buffered water, pH 7.6) for 12 h at room temperature and in a total volume of 100-150 µL and then stored at -80oC. Compounds were purified on a reversed-phase HPLC (Varian, ProStar; Agilent Technologies, Inc.; Santa Clara, CA) equipped with a ProStar 210 delivery module and ProStar 335 photo diode array detector using a C18 column (Zorbax SB-C18; Agilent Technologies, Inc.; Santa Clara, CA) at a flow rate of 1 mL/min over 30-60 min using a linear gradient from 0.1% TFA in 10% ACN / 90% H2O to 90% ACN / 10% H2O. Collected fractions were lyophilized and stored as a powder at -80oC. Fractions were reconstituted in buffered water (pH 7.6) and analyzed using MALDI-MS (Voyager DE Pro, Applied Biosystems-Life Technologies; Grand Island, NY) to determine molecular weight. Concentrations were determined using data from a Nanodrop 2000c spectrophotometer (ThermoScientific; Waltham, MA) for each sample. Samples were diluted to final concentration of 10 nM in fresh TFE (2,2,2-trifluoroethanol) from frozen aliquots and stored at -80oC. Fluorescence Spectroscopy: Fluorescent counts were measured using a QuantaMaster Model C-60/2000 Spectrofluorimeter (Photon Technologies International; Birmingham, NJ) with an excitation range 450-562 nm emitting at 572 nm to determine the relative excitation spectrum. The emission spectrum was assayed over a wavelength range from 525-700 nm, and excited at 500 nm. Peak emission wavelength of TAMRA was reliably observed at 565-572 nm. Excitation and emission spectra were obtained at 25oC. Collected data represents averaged (3-5 times) runs for each condition.

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3. Experimental Methods (Molecular Dynamic Simulations) The molecules of interest were constructed with the general sequence: NO-(PEG) m -linker-Cys(Pro) n -Gly-TAMRA, where the linker consists of triazole and succinimide. To compare with the experimental quenching data, two different polyproline lengths (n = 6, 10) and four different PEG lengths (m = 3, 5, 7, 9) were studied in the simulations. The CHARMM c37a1 package(5) was used for all simulations. To efficiently sample the conformational space of the system, an implicit solvent model, Generalized Born with a simple Switching function (GBSW)(6), was used. The CGenFF online interface(7) was used to generate force field parameters for groups in the linker, the fluorophore (TAMRA) and the quencher (NO•). To obtain correct end-to-end distance and radii of gyration distributions for the PEG chain, we adopted the PEG parameters revised by Juneja et al.(1) specifically for GBSW simulations. CHARMM22 parameters(8) were used for all amino acid residues. All simulations started from extended initial structures where TAMRA and NO• were at least 35 Å apart (measured with n = 6 and m = 3). 8000 steps of steepest descent energy minimization and 8000 steps of adapted basis Newton-Raphson minimization were followed by 100 ps of molecular dynamics equilibration steps before the production runs. Three independent production runs were done over 50 ns at constant temperature (300 K). As recommended by early works of Juneja et al.(1), the surface tension term in GBSW was set to 0, and the attraction term in the van der Waals interactions was scaled by a factor of 0.9. For further discussion see the Supporting Text and Figures. The van der Waals interactions within the peptide were not scaled to ensure that proper conformations of the peptide were sampled. To estimate the quenching probability, we measured the cumulative contact probability between the oxygen in the aromatic ring of TAMRA and oxygen in the quencher group (NO•). For simplicity, a linear relationship(9) was assumed between the cumulative contact probability for the OO distance up to a cutoff value of 7.5 Å and the quenching probability. For example, the intensity plotted in Fig. 3G and H is one minus the estimated quenching probability.

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4. Experimental Methods and Instrumentation (Molecular Biology and Electrophysiology) Molecular Biology and Oocyte Expres sion: Single residue cysteines [S1-S2 (T276); S3-S4 (M356); S3-S4 (A359C); S5-S6 (E422)] were engineered into select sites within the extracellular loops of a high-affinity TEA / cysteine-removed (T449F, C245V, C301S, C308S, C462A) inactivation removed (Δ6-46) Shaker potassium channel background using Quikchange mutagenesis kits (Stratagene; La Jolla, CA). Engineered mutations were confirmed by gene sequencing. For cRNA preparation, plasmids were linearized by NotI digestion. cRNA was generated by in vitro transcription using T7 RNA polymerase kit (mMessage mMachine, Ambion, Inc; Grand Island, NY) and reconstituted in nuclease-free water then stored at -80oC until use. Xenopus laevis oocytes (stage IV or V) were surgically removed in a manner consistent with the guidelines of the Animal Care and Use Committee at the University of Wisconsin-Madison and stored in 5 mM HEPES, pH 7.6 adjusted with NaOH, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 100 mg/mL of bovine serum albumin, and 100 µg/mL of gentamicin. Selected oocytes were placed into an injection chamber filled with the above buffer lacking calcium. Oocytes were microinjected near the vegetal pole equator with 50 nL of cRNA at concentrations between 0.1-0.5 µg/µL. Injected oocytes were incubated at for 2-4 d at 18oC in 5 mM HEPES, pH 7.6 adjusted with NaOH, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 100 µg/mL gentamicin, 50 U/mL penicillin and 50 µg/mL streptomycin supplemented with fresh EDTA (100 µM) DTT (200 µM) each day. On the day of experiments, the injected oocyte solution was spiked with fresh DTT and EDTA and incubated at 18oC for 1 h. Electrophysiology and Fluorescence Measurements: Depending on the labeling site, oocytes were washed in hyperpolarizing (10 mM HEPES, pH 7.5 adjusted with NaOH, containing 120 mM NMG-MES, and 1.8 mM CaCl 2 ) or depolarizing (10 mM HEPES, pH 7.5 adjusted with NaOH, containing 110 mM KCl, 1.5 mM MgCl 2 , 0.8 mM CaCl 2 ) solutions. Oocytes were labeled at 18oC or on ice with 5-50 µM TMRM (tetramethylrhodamine maleimide, Invitrogen; Grand Island, NY) from a 10 mM DMSO stock. Labeled oocytes were washed and stored in 5 mM HEPES, pH 7.6 adjusted with NaOH, containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 until use for fluorescence experiments. For ionic current and fluorescence measurements the extracellular recording solution was comprised of 10 mM HEPES, pH 7.4 adjusted with MES, containing 100 mM NaOH, 5 mM KOH, 2 mM Ca(OH) 2 . The intracellular solution was comprised of 10 mM HEPES, pH 7.4 adjusted with MES, containing 100 mM KOH, 5 mM NaOH, 2 mM EGTA. Tetraethylammonium chloride (TEA) and the quenching

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compounds (tethered TEA-PEGn-NO•; n=0-4 (C0-C4) and freely diffusing PEGn-NO• (S-C1)) were prepared fresh for each experiment and bath applied to yield a final concentration of 2-3 mM for fluorescence quenching and pharmacology experiments. Ionic conductance and fluorescent signals were obtained using a customized cut-open voltage-clamp fluorometry setup (CA-1B; Dagan Instruments; Minneapolis, MN). The cut-open setup was placed on a stage of an upright microscope (BX50WI, Olympus; Center Valley, PA). Light from a halogen lamp source (Hammamatsu Photonics; Bridgewater, NJ) was filtered with a HQ535/50 bandpass filter and split using a Q565LP dichroic mirror (Chroma Technology Corp.; Bellows Falls, VT). Emitted light was filtered with a HQ610/75 bandpass filter (Chroma Technology Corp.; Bellows Falls, VT) and focused onto a PIN-020A Photodiode (OSI Optoelectronics; Hawthorne, CA) by a condenser lens. The photodiode was connected to the headstage of an integrating Axopatch 1B patch clamp amplifier (MDS Analytical Technologies; Sunnyvale, CA). To ensure that the photocurrent was within the dynamic range of the amplifier, additional current was fed into the summing junction of the headstage. Data Acquisition and Analysis: Electrical and fluorescence signals were sampled at 250 kHz with a Digidata 1440 interface (MDS Analytical Technologies; Sunnyvale, CA). Oocytes were voltage-clamped and held at -80 mV for all experiments. For ionic current measurements, linear leak and membrane capacitive current were subtracted online using a P/4 protocol with a subtraction holding potential of -130 mV. Each fluorescence recording represents an average of 5-10 traces generated by a 20-ms test potential to 40 mV from -120 mV. This voltage pulse was chosen because the channels are at maximal Po at this potential. Ionic current and fluorescence signals were low-pass filtered at 20 and 10 kHz respectively. For analysis of fluorescence quenching, signals were subsequently low-pass filtered offline at 5 kHz. Fluorescence intensity decay due to photobleaching was corrected by fitting the data before the pulse to a straight line and subtracting it from the full record in Clampfit (Molecular Devices; Sunnyvale, CA) or Origin (OriginLab; Northampton, MA). Fluorescence signals were obtained for the same oocyte before and after application of the controls or quenching compounds to determine the quenching efficiency. Percent change was determined by examining the change in fluorescence (ΔF/F) before and after quencher. Fluorescence and electrophysiological data were acquired using Clampex (Molecular Devices; Sunnyvale, CA) and analyzed with Clampfit, Excel (Microsoft; Redmond, WA), or Origin. Statistical analyses were performed using Origin and Excel. Statistical significance using an independent two tailed Student’s t-test was accepted at p