For SDS PAGE, sample buffer was added to the lyophilized ...



Supplementary Material.

Site specific infrared dichroism. For each of the labeled sites, one in K2-ETM-K2 (F23) and five in ETM, we obtained a pair of dichroic ratios (Rhelix, Rsite). Rhelix corresponds to the amide A dichroism, and Rsite to the labeled amino acid (1). These dichroisms are shown in Table 1 (three independent measurements per labeled amino acid). As described before (2), these values were combined for consecutive labels (i, i+1), assuming a canonical α-helix periodicity, i.e, Δω =100˚. These combinations were used to obtain helix tilt, ( and rotational orientation ω at each i labeled residue, as well as the contribution of disorder (f) in the samples, which ranged from 0.7 to 0.9.

|Residue |Rhelix |Rsite |

|F23* |3.6 |3.3 |

| |3.8 |3.6 |

| |3.6 |2.7 |

|L21 |3.7 |5.5 |

| |3.5 |6.5 |

| |3.7 |5.2 |

|A22 |3.4 |2.5 |

| |3.6 |2.6 |

| |3.3 |2.6 |

|F23 |3.6 |2.4 |

| |3.4 |2.3 |

| |3.5 |2.5 |

|V24 |3.6 |5.4 |

| |4.0 |5.0 |

| |3.7 |5.4 |

|V25 |3.6 |2.7 |

| |3.6 |2.6 |

| |3.3 |2.7 |

Table 1. Dichroic ratios obtained for ETM, with or without flanking lysines. F23*: amide I and site dichroic ratios for F23 in the peptide K2-ETM-K2. The values for L21, A22, F23, V24 and V25 correspond to ETM, i.e., without flanking lysines.

NMR. Synthetic ETM was analysed by solution NMR, after solubilization in DPC (dodecylphosphocholine, Avanti Polar Lipids) micelles at a peptide-to-lipid molar ratio of 1:60, with a peptide concentration of 0.1 mM and 20 mM sodium phosphate buffer (pH 6.5). Residue-specific assignment of 1H resonances was performed using a combination of 2D homonuclear NOESY (mixing time 60 ms, total acquisition time 5 days) and TOCSY (mixing time 45 ms, total acquisition time 3 days) at 37 °C using a Bruker Avance spectrometer operating at 1H frequency of 900 MHz. The same NOESY was used to collect NOE constraints.

[pic]

Figure 1. Solution NMR results for ETM without (left) or with (right) terminal lysines, solubilized in DPC micelles. Top: Bundle of 20 lowest energy structures of ETM, generated from CYANA(3) showing the peptide backbones in cyan, and residue sidechains in gold. The α-helices are oriented so that the phenyl ring of F23 (center of helix), faces down. The parameter θ indicates the extent of bending of the ETM α-helix (the value for no bending θ would be 180°). Middle: secondary structure plots depicting sequential and medium range NOE connectivities shown as bands of varying thicknesses; dNN amide backbones and dαN(i,i+3), dαN(i,i+4) connectivities are mostly continuous throughout the length of the peptide, indicating that ETM adopts a predominantly (-helical conformation. Bottom: Amide connectivities are shown in 2D homonuclear NOESY spectra.

Analytical Ultracentrifugation. Sedimentation equilibrium experiments were performed using a Beckman XL-I analytical ultracentrifuge at 25ºC (4). Two different samples were used: (1) ETM non labeled, where absorbance was measured at 254 nm (ε254 = 600 M-1 cm-1), (2) ETM labeled with 7-nitrobenz-2-oxa-1,3-diazole (NBD, Sigma), for use in more diluted conditions (see below), where absorbance was measured at 450 nm (ε450 = 21,000 M-1 cm-1). The buffer composition was 20mM MOPS (3-N-morpholino-propanesulfonic acid), 100 mM KCl, 1mM MgCl2 at pH 7.4, and 10 mM dodecylphosphocholine (DPC). To match the density of DPC, D2O was added to the buffer to a final volume ratio of 50.5% (5). The density-matched buffer was added to the dry peptide.

The samples were centrifuged in three-compartment carbon-epoxy centerpieces with quartz windows for lengths of time sufficient to achieve equilibrium, typically 20 h, and run at 28K, 34.5K and 42K rpm. The equilibrium data sets were analysed using the program ULTRASCAN (6). At the end of each run, absorbance data at 254 nm (ETM) or 450 nm (for NBD labeled ETM) was obtained. The monomeric molecular mass of ETM and its partial specific volume of the peptides were calculated with the program SEDNTERP (7).

For unlabeled ETM (Fig. 2), the monomeric mass was determined to be 3,403 Da, whereas the partial specific volume, calculated after correction for partial hydrogen/deuterium exchange (4), was 0.810 cm3/g. Three different concentrations, 135, 200 and 270 μM of ETM were used, i.e., peptide-to-detergent ratios were 1:75, 1:50 and 1:37, respectively. The log plot (ln A vs. r2-r02) was not linear, indicating the presence of more than one species at these peptide-to-detergent ratios. The data was best fitted to a monomer-pentamer equilibrium (variance 5 x 10-5), although the fit could be marginally improved by the addition of a higher order oligomer (n >10), which is probably due to pentamer aggregation and not representing more than 10 % of the material. The decimal logarithm of the monomer-pentamer association constant was 17.1, i.e., at this DPC concentration, when the concentration of peptide is ca. 75 μM, 50% of the ETM population is pentameric.

For the NBD-labeled ETM (Fig. 3), the calculated monomeric molecular mass was 3,535 Da, and the partial specific volume was 0.797 cm3/g. The use of the label NBD allowed the use of lower peptide-to-detergent ratios. The concentrations of NBD-labeled peptide were 7 μM, 14 μM and 28 μM, i.e., peptide-to-detergent ratios were 1:1400, 1:700 and 1:350, respectively. The log plot was linear (not shown), and the data could be fitted to a single (monomeric) component (3,439 Da), which indicates that the density matching with D2O is adequate.

[pic]

Figure 2. Sedimentation equilibrium results for non-labeled ETM in DPC micelles. The data was best fitted to a monomer-pentamer equilibrium. (A) residuals; (B) 1:37 at 42K rpm (green), 1:50 at 34.5K rpm (blue), 1:50 at 28K rpm (red); (C) 1:75 ratio at 3 different velocities 42K (green), 34.5K (blue) and 28K (red). The three curves that are absent were deemed unsuitable for fitting by the software Ultrascan and were not included in the fit.

[pic]

Figure 3. Sedimentation equilibrium results for NBD-labeled ETM in DPC micelles. The data was fitted to a single (monomeric) component. (A) residuals of the fit, with variance 3 x 10-5 ;(B) absorbance traces at the 3 concentrations and 3 velocities indicated in the text, and the corresponding fit (line).

Electrophoresis. The electrophoretic mobility of the peptide was assessed using SDS PAGE and PFO (perfluoro-octanoic acid) PAGE (Fluorochem Ltd, UK). Sample buffer was added to the lyophilized peptide to a final concentration of 2μg/μl. After vortexing for 1 min the sample was heated at 70° C for 5 to 15 minutes and loaded on a 15% SDS PAGE (Tris-Glycine) gel. The loading volumes were 5, 10 and 20 μl. The sample was electrophoresed at room temperature at a constant voltage of 100V until the dye front reached the bottom of the gel. After completion, the SDS/PAGE gel was stained with Coomassie blue or Silver stain-Plus kit (Bio-rad). For PFO-PAGE, the running buffer and the sample solubilizing buffer were prepared with PFO. Briefly, tricine-based running buffers were made with a concentration of 0.1% (w/v) PFO and 4% (w/v) PFO for sample solubilizing buffer. Gradient gels (10-20%) were used. The sample was electrophoresed at room temperature at a constant voltage of 40V, until the dye front reached the bottom of the gel. After completion, the PFO gels were thoroughly washed with deionised water to remove the excess PFO, followed by staining with Coomassie blue.

Figure 4. Electrophoresis of SARS ETM in SDS (left) and PFO (right). Left panel: lanes 1, myoglobin; lane 2, K2-ETM-K2; lane 3, ETM. Arrows indicate dimers, trimers and pentamers. Right panel: lane 1, molecular weight markers, lanes 2−3, same as in left panel.

Conductance studies. A lipid mixture POPE:POPS:POPC, molar ratio 5:3:2, with a lipid concentration of 50 mg/ml was used to form the artificial lipid bilayer. The working volume was 1 ml in each cis and trans chambers. The buffer composition in assymmetric conditions was: cis, 500 mM NaCl, 50 mM HEPES, pH 7.2, and trans, 50 mM NaCl, 50 mM HEPES, pH 7.2. Small aliquots of peptide-containing liposomes (about 5-10 µg of peptide) were added to the cis chamber with continuous stirring.

Electrical currents were recorded using a Bilayer Clamp BC-525D amplifier (Warner Instruments) as described. The trans chamber was set as reference and the cis chamber was held at different potentials ranging from -100 mV to +20 mV in 20 mV increments. Data were filtered at 50 Hz with an 8-pole Bessel filter, and analogue output signal was digitized at a sampling rate of 1 kHz by using an A/D converter (Digidata 1322A, Axon Instruments). Data processing was performed using pClamp 9.2 software (Axon Instruments). Single channel conductance was calculated from the corresponding Gaussian fits using SigmaPlot 9.0 software (Systat Software, Inc.) to current histograms by using data from segments of continuous recordings lasting longer than 10 s. Openings shorter than 0.5 ms were ignored. The recording temperature was at the room temperature. Given voltages have been corrected for calculated liquid junction potential using pClamp 9.2 software (Axon Instruments). Throughout, mean data is presented graphically with error bars reflecting standard deviation of the mean.

[pic]

Figure 5. Conductance of peptides ETM (left) and K2-ETM-K2 (right). Left column: A, single channel currents recorded at the membrane potentials indicated on the right. The short dash line indicates the closed state of the channel; B, all-points amplitude histograms representing single channel current amplitude and open probability at different holding potentials; C, mean unitary current-voltage relationships for the predominant, fully open state of the channel ranging from 0 to 80 mV. The slope of the fit from 20 mV to 80 mV, is 25.5 pS; D, open probability of the channel as a function of potential from 20 mV to 80 mV. Each data point in C and D represents the average and standard deviation of 5 recordings. Right column: Same as above, for K2-ETM-K2. The conductance calculated was 53.4 pS.

[pic]

Figure 6. Effect of amantadine addition (100 μM, see arrow) on the conductance of the ETM peptides with (K2-ETM-K2), or without (ETM) terminal lysines.

1. Torres, J., J. A. Briggs, and I. T. Arkin. 2002. Multiple site-specific infrared dichroism of CD3-z, a transmembrane helix bundle. J. Mol. Biol. 316:365-374.

2. Arkin, I. T., K. R. MacKenzie, and A. T. Brunger. 1997. Site-directed dichroism as a method for obtaining rotational and orientational constraints for oriented polymers. J. Am. Chem. Soc. 119:8973-8980.

3. Guntert, P. 2004. Automated NMR structure calculation with CYANA. Methods Mol Biol 278:353-378.

4. Kochendoerfer, G. G., D. Salom, J. D. Lear, R. Wilk-Orescan, S. B. Kent, and W. F. DeGrado. 1999. Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly 1. Biochemistry 38:11905-11913.

5. Li, R., C. R. Babu, J. D. Lear, A. J. Wand, J. S. Bennett, and W. F. DeGrado. 2001. Oligomerization of the integrin aIIbb3: roles of the transmembrane and cytoplasmic domains. Proc. Nat. Sci. USA 98:12462-12467.

6. Demeler, B. 2005. ULTRASCAN, a comprehensive data analysis software package for analytical ultracentrifuge experiments. In Modern analytical Ultracentrifugation: Techniques and Methods, Cambridge. 210-229.

7. Laue, T. M., and W. F. Stafford, 3rd. 1999. Modern applications of analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct. 28:75-100.

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download