1 Supporting Information For: Preferred Side Chain Constellations at Antiparallel Coiled-Coil Interfaces Erik B. Hadley*, Oliver D. Testa, Derek N. Woolfson,, and Samuel H. Gellman*, *Department of Chemistry, University of Wisconsin, Madison, WI 53706, USA School of Chemistry, University of Bristol, Bristol, BS8 1TS, UK Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK Corresponding Author Supporting Text Synthesis of peptides The sequences of peptides, peptide thiols, and peptide thioesters prepared for this article are shown below, followed by general synthetic procedures and characterization data for each molecule. Abbreviations X = -SCH 2 C(O)- H = hydrogen on N terminus Trt = trityl-protecting group CONH 2 = C-terminal amide NHMe = C-terminal N-methyl amide Succ = N-terminal succinyl group Ac = N-terminal acetyl group Sequences. Solid-Phase Synthesis Peptides were synthesized on solid phase by using a Symphony automated synthesizer (Protein Technologies Inc.). Synthetic cycles were completed with a standard coupling time of 30 min by using O-benzotriazol-1-yl-N,N,N,N -tetramethyluronium hexafluorophosphate (HBTU, 5 equivalents). DMF was the solvent. Five equivalents of Fmoc amino acid were used for each coupling cycle. Deprotection steps used 20% piperidine in DMF for 1 5 min + 1 15 min. C-terminal amide peptides were prepared on rink amide resin. N-terminal thiols were obtained by the coupling of tritylsulfanylacetic acid as the last residue under standard conditions. Thioester peptides were prepared as described below. Thioester Peptide Synthesis Thioesters were prepared on H-Phe-2-Cl-Trt resin (30 µmol) using standard coupling conditions as described above. The protected peptide was cleaved off the resin by reacting with 8:1:1 (dichloromethane:trifluoroethanol:acetic acid) (2 ml) for 1.5 h.
2 before precipitating into hexanes (25 ml). The solvent was removed under rotary evaporation, and the remaining solid was dissolved by the addition of DMF (1 ml). DIEA (8 µl, 46 µmol), benzene thiol (5 µl, 49 µmol) and PyBop (24 mg, 46 µmol) were subsequently added. After 1 h of stirring, the DMF was removed by vacuum evaporation prior to global deprotection. After global deprotection and HPLC purification, the phenylthioester peptide intermediates were combined with peptide thiol T6 (8 mg, ca. 10-fold excess) in aqueous (ph 7, 50 mm phosphate buffer, 2 mm triscarboxyethyl phosphine) solution (4 ml), which allowed thioester exchange. After 2 h of exchange at room temperature, the desired peptide thioesters were collected by HPLC. Full-length thioester 2 was prepared by thioester exchange. The purified thioester was combined with the appropriate C-terminal thiol (prepared as described above) in aqueous (ph 7) solution, which allowed thioester exchange. The desired peptide thioester (2) was collected by HPLC. Although the yield depends on K TE (ca. 10 for this reaction), the starting materials can be collected and recycled. Disulfide Formation Approximately 1.5 mg of each cysteine containing peptide was dissolved in 2 ml of 200 mm carbonate buffer (ph 10). The solution was left exposed to open air for approximately 20 h before purifying the disulfide-linked dimer by HPLC. Cleavage and Deprotection Peptides were cleaved and/or deprotected by stirring with (CF 3 CO2H:H 2 O:triisopropylsilane, 90:5:5, v/v/v) (2 ml/ 25 µmol) for 4 h followed by precipitating into cold diethyl ether. The precipitate was collected by centrifugation/decantation prior to purification. Purification and Characterization Peptide Thiol T6 was prepared as described previously (1). CD Methods CD spectra were collected on an Aviv 202SF spectropolarimeter using 1-nm bandwidth in 1-mm quartz cells. Samples were prepared by dissolving dried peptide in H 2 O (millipore quality) to create a stock solution of ca. 0.5 mm peptide. Concentrations were determined by UV spectrophotometry in a 1-cm quartz cell by diluting the stock solution 20-fold into H 2 O and measuring the UV absorbance at 275 nm. The absorbance and a calculated extinction coefficient for the peptide ( = 1420 M -1 cm -1 no. of Tyr) (2) were used to determine the concentration of the stock solution. The displayed wavelength scan curves run through measurement points that were taken at 1-nm steps and are not smoothed. Data were collected for 5 s and averaged for each individual measurement point. The concentration of each of the species was 50 µm as measured by UV in a ph 7.0 sodium phosphate (50 mm) buffered solution. Molar ellipticities (θ)
3 were calculated by using the equation: θ = θ obsd /(10*l*c), where θobsd is the measured ellipticity (mdeg), l is the length of the cell (cm), and c is the concentration of peptide. Chemical denaturations were performed by incremental dilution of an 150 µm peptide solution with 8 M urea while monitoring the resulting CD signal at 222 nm for each dilution. The data were fit to a two-state dimer denaturation model as described in ref. 3. The fitting parameters used were as follows: where θ = the observed CD signal in deg cm 2 dmol -1 x 10-3, B Mon and B Dim = the spectroscopic signal for the monomer and dimer, respectively, F = the fraction of unfolded monomers; k = [D] 2 /[N 2 ], the equilibrium constant for concerted unfolding; p = total peptide concentration; m = a constant of proportionality relating to the solvent exposure difference between the folded and unfolded states; and x = [denaturant]. Equilibrium Test The equilibrium constant was determined starting from both the right side or the left side of a thioester exchange reaction (SI Fig. 11). Q TE is the value measured for [N T - C][T6]/[TE2][T3] at a given time. At equilibrium, Q TE = K TE. The mixture reaches equilibrium after ~ 90 min. Thioester Exchange Assays Stock solutions of 250 mm sodium phosphate buffer (ph 7.0 at 250 mm) and 20 mm tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were produced by using millipore H2O. Typically, assays were initiated by mixing approximately equal portions of a C-terminal thiol peptide and an N-terminal thioester (i.e., from the left side of the equilibrium shown in Fig. 1b). Approximately 0.06 µmol (i.e., 0.15 mg for a peptide of molecular mass 2,400) of each dry peptide was placed into a 500-µl vial insert. Most assays were conducted on a 300-µl scale by addition of liquids into the vial insert, to give initial assay concentrations of 0.1 mm peptides, 2.0 mm TCEP, and 50 mm buffer (TCEP was included in the assay solutions to prevent disulfide formation during the thioester exchange equilibration). The buffer has a slightly higher ph at 50 mm, the concentration of the assay, but this effect is offset by the extra acid groups on TCEP. H2O was used for the excess volume. Typically, 50 µl of assay solution was injected per HPLC run. Each assay was allowed to equilibrate ca. 1.5 h before HPLC injection. The HPLC column used in every case was either a C 18 Everest or a C 18 Vydac analytical column (4.6 mm i.d. 250 mm length) at a flow rate of 1.0 ml/min. A gradient of B solvent (CH 3 CN:CF 3 CO 2 H, 100:0.085, v/v) in A solvent (H 2 O:CF 3 CO 2 H, 100:0.1, v/v)
4 was used for elution. Equilibrating species were identified by HPLC retention time and MALDI mass spectrometry. Disulfide Exchange Assays Approximately 0.15 mg of each peptide was dissolved in 250 µl of Millipore H 2 O to make a stock solution of ~100 µm. A 10-µl sample of each stock solution was then injected onto an analytical HPLC column, and the resultant peak was integrated. Comparison of the HPLC integration for each stock solution was used to match the initial concentration of reactants. Assays were carried out at ~10 µm peptide concentration in 1 PBS buffer containing 125 µm oxidized glutathione and 500 µm reduced glutathione (4). The mixture was monitored over time until HPLC chromatograms were superimposable, indicating that equilibrium was reached (this generally took about an hour). Peaks were identified by comparing the retention time of the pure peptides to those in the mixture. Assays were run starting from both the left and the right side of the equilibrium to confirm that equilibrium had been reached; in each case, the measured equilibrium ratio was the same. To aid in the separation of peptides by HPLC, a GGK tag was added to the C terminus of the Ile acid peptide. It is conceivable that the presence of the tag could result in an artificial perturbation of the equilibrium. To test for this, we carried out the disulfide exchange experiments shown in SI Fig. 13. Each experiment is identical except for the location of the tag. The ratio between the two full-length disulfide-bonded peptides is similar between the two experiments, indicating that the presence of the tag does not affect the measured equilibrium ratio. Analytical Ultracentrifugation Sedimentation studies were conducted on a Beckman XLA ultracentrifuge at 25ºC. Peptide solutions of appropriate concentration were loaded into 1.2-cm cells and analyzed at 25ºC. At each speed, data were collected with a 0.001-cm step size every 2 h until two consecutive spectra were identical (typical equilibration times were 10 h). Peptide 1 was analyzed in 0.5 mm PO 4 ph 7 buffer while monitoring at 236 nm. All other peptides were analyzed in 1 PBS solution while monitoring at 280 nm. Non-linear regression was performed in accordance with the expression c r = c o exp [M(1-νρ)ω 2 (r 2 - r o 2 )/2RT] + base, where c r is the concentration (in absorbance units) at radial position r, c o is the concentration at an arbitrary reference position r o near the meniscus, ν is the partial specific volume, ρ is the solvent density, ω is the rotor speed, R is the gas constant, T is the temperature, and base is a baseline absorbance correction to account for nonsedimenting species. Molecular weight estimates were obtained from the parameter M. Global fits to the data at all speeds and concentrations were judged to be adequate by randomness of residuals. Peptides 1, 3, and 4: The data for peptides 1, 3, and 4 were well described by an ideal single-species model with a molecular weight consistent with the monomer (thioester peptides were not analyzed because they begin to hydrolyze during the several days required for the
5 measurements). A partial specific volume of 0.717 ml/g was calculated based on amino acid composition (5), and a solvent density of 0.9983 g/ml was used. Sedimentation results obtained at 50, 100 and 150 µm at different rotor speeds gave similar results, indicating that peptide aggregation is insignificant over this concentration range (SI Fig. 14). Thioester Concentration Study TE assays were run at a variety of initial concentrations to investigate the effect of concentration on the determined stability by thioester exchange. The results shown below (SI Fig. 17) indicate that the determined stabilities are independent of concentration over the range of concentrations analyzed. Discrimination Energy Calculations For each residue at d` there are 10 independent combinations involving the 5 residues at a (note that DE LI (L/I) = -DE LI (I/L), etc.). Because of the 5 residue possibilities at d`, there are five values of DE LI (L/I) for d` = Leu, Ile, Val, Asn, or Ala, five values of DE LI (L/V), etc., which provides the total of 50 discrimination energies. Shown below are the significant discrimination energies for d` = Leu, Ile, Val, Asn, and Ala (SI Fig. 18). Tables containing the DE data are shown below the figure (DE values are in kilocalories per mole). Calculation of Lateral Discrimination Energy Lateral discrimination energies may be calculated by using the data from Table 1 as described in the text. Shown in SI Fig. 19 are two representative hypothetical equilibria for such calculations. The difference in lateral DE values for this example indicates that the lateral pairing preferences depend on the vertical contacts, which is consistent with the conclusions drawn in the text. References 1. Woll MG, Gellman SH (2004) J. Am. Chem. Soc. 126: 11172-11174. 2. Edelhoch H (1967) Biochemistry 6:1948. 3. Mallam AL, Jackson SE (2005) J. Mol. Biol. 346: 1409. 4. Bilgicier B, Xing X, Kumar K (2001) J. Am. Chem. Soc. 123:11815-11816. 5. Harding SE, Rowe AJ, Harton JS eds. (1994) Modern Analytical Ultracentrifugation (Birkhauser, Boston, MA). 6. McClain DL, Woods HL, Oakley MG (2001) J. Am. Chem. Soc.123: 3151.