Supporting Information for: Mechanism of Reversible Peptide-Bilayer. Attachment: Combined Simulation and Experimental Single-Molecule Study

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1 Supporting Information for: Mechanism of Reversible Peptide-Bilayer Attachment: Combined Simulation and Experimental Single-Molecule Study Nadine Schwierz a,, Stefanie Krysiak c, Thorsten Hugel c,d, Martin Zacharias b a Chemistry Department, University of California, Berkeley, CA 97, USA b Physik-Department, Technische Universität München, 878 Garching, Germany c NIM and IMETUM, Technische Universität München, 878 Garching, Germany d Institute of Physical Chemistry, University of Freiburg, 79 Freiburg, Germany nadine.schwierz@ph.tum.de Force-extension curves at the bilayer from simulations (A) -, v=. m/s, v=. m/s (C), v=. m/s (D), v=. m/s - 3 -, v=. m/s, v=. m/s - 3 -, v=. m/s, v=. m/s (E), v=. m/s, v=. m/s (F), v=. m/s, v=. m/s (G), v=. m/s, v=. m/s (H), v=. m/s, v=. m/s (I), v=. m/s, v=. m/s Figure S: Simulated force-extension curves for pulling velocity v =. m/s (solid lines) for (A) glu, gln, (C) lys, (D) ala, (E) val, (F) trp, (G) tyr, (H) leu and (I) phe. Open points denote the results from the static simulations. Each dynamic simulation is repeated twice with two different starting configurations. S

2 The simulated desorption forces of the 9 different homopolypeptides, consisting of - amino acids, are determined using dynamic simulations with constant pulling velocity (v=. m/s) and static pulling simulations (v= m/s). Each dynamic simulation is repeated at least five times with different initial configurations. For the subsequent static simulations, two dynamic pulling curves were selected at random to extract the starting points for the static simulations. The results of these simulations, i.e. the force F in dependence of the separation between the surface and the AFM tip z AFM, are shown in Figure S. The separation is measured from the surface defined by the mean position of the phosphate groups and the center of mass of the last peptide residue (C-terminus). The lines result from the dynamic pulling. Open symbols result from 9 ns static simulations in which the cantilever is held at a fixed separation from the surface. Velocity dependence of the desorption forces Figure S shows the dependence of the desorption force of polyalanine on the pulling velocity and different starting configuration. For better comparison, the results from the static simulations are shown to the left. The results for the lowest pulling velocity (v=. m/s) are in good agreement with the results for the static simulations. For low pulling velocities dissipation is small and we reach a stationary non-equilibrium where the desorption force is independent of the pulling velocity and the initial configuration. Therefore, the simulation results can be compared to the experimental results even though the pulling velocity is several orders of magnitude higher in the simulations compared to the experiments. With increasing pulling velocity the desorption forces increase and depend on the initial configuration. 3 Static v=... v [m/s] Figure S: Dependence of the desorption force of polyalanine on the pulling velocity and starting configuration in comparison to the results from the static simulations (left). Different colors indicate different initial configurations. 3 Internal energy and free energy of desorption The internal energy and the different contributions are obtained as time average over the last ns of the static simulations. The errors are determined by block averaging. The static simulations are split into two sets according to surface adsorbed and desorbed configurations. The energy difference is calculated as weighted average between the two sets. Figure S3A, B show the result for tyrosine for the peptide-surface interaction and the total internal energy. The free energy is obtained by integrating the force along the pulling path A = zb z A < F > dz () where z A and z B are the surface separations of the adsorbed state and the desorbed bulk state and < F > is the weighted average desorption force. Figure S3C shows the desorption force and the free energy A (gray area). Figure S shows the internal energy difference U = U(bulk) U(surface) between the surface adsorbed configurations and the desorbed bulk configurations. The individual interactions of peptide (P), water (W) and surface (S) are combined to represent the sum of all solvent mediated interactions (WW + PW + SW), the sum of all peptide related contributions (PP + PS + PW) and the sum of all terms due to surface solvation and solvent-solvent interactions (WW + SW + SS). Charged peptides are shown to the left, uncharged peptides to the right. The uncharged peptides are ordered according to increasing side-chain hydrophobicity. The sum of solvent mediated interaction is always negative and dominant for the charged and polar amino acids. The sum of all peptide related contributions is negative for the charged peptides and positive for the uncharged peptides. These contributions are canceled by the interaction due to surface solvation and the solvent-solvent interaction. The sum of WW + SW + SS shows the opposite sign than the peptide contributions. To obtain the correct sign of the total energy the peptide related terms (PP + PS + PW) are decisive. The only exception from this trend is the polar amino acid gln. The total energy of the polar gln is negative (see Figure 8 in the main text). Therefore, the polar amino acid is grouped rather with charged amino acids since they are all weakly adsorbing and disfavor the adsorbed configuration energetically. Note that the sums of individual contributions are much larger in magnitude than the resulting energy. All contributions are therefore required for a quantitatively correct predictions. S

3 U PS [kj/mol] (A) surface (C) bulk z [nm] U [*e+ kj/mol] z [nm] A z [nm] Figure S3: Evaluation of the internal energy and the free energy for the tyrosine with residues: (A) Internal energy of the peptide-surface interaction and the total internal energy in dependence of the peptide-surface separation (open points). The resulting average energy in the adsorbed state is shown as red line, the average energy in the desorbed state is shown as blue line. (C) Mean desorption force obtained from the static simulations (open points) in dependence of the peptide-surface separation. The free energy A is obtained from integration over the desorption path (gray area). The red line is the weighted average desorption force. 8 Charged peptides Uncharged peptides WW+SW+PW PP+PW+PS WW+SW+SS U [kj/mol] - -8 glu lys gln ala val trp tyr leu phe Figure S: Internal energy difference between the surface adsorbed state and the desorbed bulk state for the sums of different contributions: The sum of all solvent mediated interactions (WW + PW + SW), the sum of all peptide related contibutions (PP + PS + PW) and the sum of all terms due to surface solvation and solvent-solvent interactions (WW + SW + SS). The peptides to the left are charged, the peptides to the right are uncharged and are ordered according to increasing side-chain hydrophobicity. Details of peptides, desorption forces and free energies The simulation results of the 9 peptides are summarized in table. Determining experimental desorption forces and the force loading rate Experimental force-distance traces were obtained for polytryptophan, polytyrosine and polyglutamic acid on a DOPC bilayer. The forces were obtained with a trigger force of 3- pn, a retraction speed of µm/s and a surface dwell time of s. As a control also tips with PEG only were prepared to be able to exclude unspecific adhesion effects of the tip and the effects of the PEG linkers from the measurements of the polypeptides. SA shows a sample retraction curves of the PEG functionalized control tip DOPC. On DOPC only a short range interaction of PEG with the surface is observed. The force retraction curves of the PEG tip on DOPC were analyzed for force peaks larger than the noise level. Out of 33 curves with the standard experimental parameters (3- pn trigger force, s dwell on the surface, µm/s retraction speed) desorption events were found and peak force and length were extracted. Those are plotted in SB. The longest peak length out of those events was.8 nm. To exclude unspecific adhesion effects of the tip and the effects of the PEG linkers from the measurements taken with polypeptides functionalized to the AFM tip, force peaks of less than nm distance from the surface are classified as unspecific adhesion and only force peaks further than nm from the surface were recorded as desorption events of the polypeptides. In SC a sample force curve of polytryptophan on DOPC is shown and the maximal peak force (blue dot) and the detach peak force (green dot) are marked, the nm cutoff is shown as a dashed line. Furthermore an average force was calculated over the distance starting with the nm cutoff and ending at the detachment as illustrated by a solid line in SC.The same sample force trace is plotted against time in SD. For the maximal peak forces the instantaneous loading rate was determined by fitting the last points before the maximal force peak with a line and extracting the slope. S3

4 Table : Details of the 9 peptides used in simulations: glutamic acid (glu), glutamine (gln), lysine (lys), alanine (ala), valine (val), tryptophan (trp), tyrosine (tyr), leucine (leu) and phenylalanine (phe). The table shows the side-chain, residue volume v r from [], scaled hydrophobicity h S from [], the average desorption force FDes from the first configuration (shown in black in Figure S) and the average desorption force FDes from the second configuration (shown in cyan in Figure S) obtained from the static simulations. F Des is the resulting averaged desorption force of FDes and F Des and A the free energy of desorption per residue. F Exp Des is the experimentally measured desorption force. Note that for gnutamic acid most desorption events in the experiments occur at a force lower than the indicated value (see section ). Residue vr h S FDes FDes F Des A F Exp Des [Å 3 ] [pn] [pn] [pn] [kj/mol] [pn] glu 9.3.3±3.3 3.±.9 3.7±.7.38±.3 3 ±/3 gln. 7.9±. 9.7±. 3.±.37.9±.3 lys ±.88.99±. 3.±..8±.8 ala 7..±3.8.98± ±.87.±. val.8.37±3..9± 8.9.3±.3 3.7±. trp ±..9± ± ±.8 ± 8 tyr ±.3 37.±.3 3.8±.3.±.8 ± leu ± ±. 9.8±.98.78±.7 phe 3. 7.±.8 8.3±.7 7.9±. 8.±.7 Table : Details of the energy decomposition for the 9 peptides used in simulations: glutamic acid (glu), glutamine (gln), lysine (lys), alanine (ala), valine (val), tryptophan (trp), tyrosine (tyr), leucine (leu) and phenylalanine (phe). The table lists the values of the decomposition of the internal energy difference U = U(bulk) U(surface) of the different homopeptides into interaction contributions involving peptide (P), surface (S), and water (W) as shown in Figure 8 of the main text. In addition, the total internal energy U and entropic contribution T S are listed. All values correspond to the energy per monomer in units kj/mol. The values for the free energy A are listed in Table. U PP U WW U PS U SW U PW U SS U T S glu -8.±3.7 8.±.9.±8.9 -.±9. -7.±. -7.±. -.9±.9.9 ±.7 gln -.7±.7.± ± ±3. -8.±. 7.±.8 -.7±.3.8±.8 lys.7±. 9.73±.9 9.9± ± ± ±.7-7.7±.3 8.±. ala -.83±.7.± ±.8 -.±.9 -.7±3. -.±.7.7±.79 -.±.3 val.±.3 9.7±.8 9.± ±. -8.±.3.±.3 -.7±..37±.3 trp -.77±.9 9.7±. 3.3±.7 -.±7. -.±.9.±3.3.±.3.3±. tyr -.± ±7.9 7.±.9-8.± ±3. 7.3±8..±..±. leu -.± ±9. 8.± ± ±.9.±.3.±.8.3±.7 phe -3.±.3.7±.8 8.3±.3-9.± ±.3 -.±.3.±..±. Averaged experimental desorption forces At least force-distance traces were recorded for each experiment ( for glutamic acid, 99 for polytryptophan and 78 for polytyrosine). The probability to observe a measurable event was one order of magnitude lower for polyglutamic acid (.7%) than for polytyrosine (3%) or polytryptophan (7%). For polyglutamic acid and polytyrosine there is only one peak in the detachment length histogram (see S7) on DOPC (7 nm ± 3 nm and nm ± 7 nm). In the case of polytryptophan there are two polyhomopeptides with different detachment length (9 nm ± 3 nm and nm ± 3 nm). Since we compare the averaged forces of polyhomopeptides with similar detachment length only traces with 9 nm ± 3 nm were further evaluated in the case of polytryptophan (9 out of 7 curves). The force of each trace with an event was averaged over the distance starting at nm distance from the surface until the force dropped to zero. Those values were plotted in the histograms depicted in S. Polytryptophan and polytyrosine histograms were fitted with a Gaussian. This was not done for polyglutamic acid due to insufficient sample size. For polytryptophan and polytyrosine desorption force values of pn ± 8 pn and pn ± pn were extracted from the peak of the Gaussian fit. The errors of the measurement consist of the statistical standard deviation and additionally a % error due to the calibration of the spring constant by the thermal noise method. For polyglutamic acid the force at the maximum of the histogram was taken (3 pn). This value probably overestimates the desorption force since the low number of events indicates that most desorption events occur at forces that we cannot resolve S

5 (A) force [pn] 3 peak force [pn] 3 (C) - distance [nm] - (D) peak length [nm] 3 3 force [pn] force [pn] - distance [nm] -. time [s]. Figure S: (A) Depicts a sample retraction force distance trace in green of a kda PEG functionalized tip on DOPC. The collected sample traces of the PEG tip on DOPC were analyzed for force peaks. Shows the maximal force peaks of PEG on DOPC plotted against the peak length. No peak was further away from the surface than nm. This was taken as cutoff value separating unspecific interaction from specific interactions. (C) Illustrates how the polypeptide desorption curves are analyzed. Peaks with a distance larger nm ( nm cutoff denoted as dotted line) are determined. The peak with the highest force is extracted as maximal force (blue dot), the value of the max force is averaged over measurement points to make it independent of the noise level. The last peak (largest distance to the surface) is extracted as detach peak (green dot). The distance of the detach peak to the surface is called detach length. An average force value is extracted averaging the force over the distance beginning at the nm cutoff until the detach length, the average force value is denoted by a solid line. (D) Shows a force time trace of the same measurement. The max peak is indicated by a blue dot and the fit to determine the instantaneous loading rate is plotted as a black line. in our experiment. Therefore, a large error towards zero force is given (3 pn), while the error towards higher force should be similar to the other amino acids ( pn). S

6 7 Experimental desorption length The distance between the surface and the detach peak is determined for every force event of the polypeptides on DOPC. This detachment length on DOPC (red histograms) and the plateau length of the force plateaus on the hydrophobic control surface (blue histograms) are depicted in S7 for polyglutamic acid, polytyrosine and polytryptophan. For all three polyhomopeptides the detachment length on DOPC differs from the plateau length on the hydrophobic control surface. In S8 A the histograms of the plateau force on the hydrophobic surface and the averaged force on DOPC are plotted for polytyrosine and in S8 B the corresponding length histograms of polytyrosine are shown again. The data on the hydrophobic surface is shown in blue and on DOPC in red. For polytyrosine on both surfaces only one length peak is present, indicating that only one polypeptide was bound to the tip. The detach length on DOPC is drastically shorter (factor of about ) than the plateau length on PTFE. The average force on DOPC is about 3. times smaller than the plateau force on PTFE. For force plateaus it has been demonstrated, that the plateau length is a function of the plateau force respectively the adsorption free energy per monomer of the polypeptide [3]. This can be explained when looking at the already desorbed part of the polypeptide. Since at typical plateau forces below pn the polypeptide is not fully stretched the tip surface distance is strictly smaller than the contour length, the ratio between tip surface distance and contour length depends on the force (as described by freely jointed chain or worm like chain models). When looking at the free energy of the chain, it is energetically favorable to desorb once the stretching free energy of the desorbed part is larger than the adsorption free energy of the adsorbed part. This again is a function of plateau force. On DOPC the curve shapes are more complex than the flat plateau forces seen on PTFE, but still the relation between force and detach length seems to be similar to hydrophobic surfaces. (A) probability of event % probability of event % (C) average force [pn] 8 average force [pn] probability of event % average force [pn] Figure S: Histograms for averaged experimental desorption forces for polyglutamic acid (A), polytyrosine and polytryptophan (C) measured on a DOPC bilayer. Polytryptophan and polytyrosine histograms were fitted with a Gaussian shown in black. S

7 (A) (C) nm ± 3nm 3 length [nm] 3 length [nm] 3 length [nm] nm ± nm 88nm ± nm nm ± 7nm nm ± 8nm 9nm ± 3nm 3nm ± 3nm nm ± 3nm 3nm ± nm 9nm ± 8nm Figure S7: The probability distribution of the detachment length on DOPC (red) and the plateau length on a hydrophobic control surface (blue) for polyglutamic acid (A), polytyrosine and polytryptophan (C) respectively. The length peaks are fitted with a Gaussian and the values are given as insets. (A) force [pn] length [nm] 3 Figure S8: The histograms of plateau forces on a hydrophobic control surface (PTFE) as shown in blue are compared to the average force histograms on DOPC shown in red for polytyrosine (A). For the same experiment the plateau length on PTFE (blue) and detach length on DOPC (red) are shown in. The peaks are fitted with a Gaussian. Both the force ( pn vs. 83 pn) as well as the length ( nm vs. nm) are smaller on the DOPC bilayer compared to the hydrophobic surface. S7

8 References [] T. E. Creighton, Proteins Structure and Molecular Properties (W. H. Freeman & Co, NY, 99). [] S. Black and D. Mould, Anal. Biochem. 99, 93, 7 [3] S. Krysiak, S. Liese, R. Netz, T. Hugel, Journal of the American Chemical Society 3, 3, S8