Quantifying the Distribution of the Stoichiometric Composition of

Subscriber access provided by Chicago State University

Article

Quantifying the Distribution of the Stoichiometric Composition of Anti-cancer Peptide Lycosin-I on Lipid Membrane with Single Molecule Spectroscopy Huaxin Tan, Wenjuan Luo, Lin Wei, Bo Chen, Wenxuan Li, Lehui Xiao, Sergei Manzhos, Zhonghua Liu, and Songping Liang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b12618 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 6, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Quantifying the Distribution of the Stoichiometric Composition of Anti-cancer Peptide Lycosin-I on Lipid Membrane with Single Molecule Spectroscopy Huaxin Tan,†,‡ Wenjuan Luo,† Lin Wei,† Bo Chen,† Wenxuan Li,§ Lehui Xiao,†,* Sergei Manzhos,§,* Zhonghua Liu,‡,* and Songping Liang‡

†. Dynamic Optical Microscopic Imaging Laboratory, Key Laboratory of Chemical

Biology & Traditional Chinese Medicine Research, Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, 410081, P. R. China. ‡. College of Life Sciences, Hunan Normal University, Changsha, 410081, P. R. China. §. National University of Singapore, 117576, Singapore. E-mail: [email protected], [email protected], [email protected]

Abstract Lycosin-I, a peptide toxin derived from spider venom, has been demonstrated to be a promising candidate for the inhibition of tumor cell growth in-vitro and in-vivo by interacting with and penetrating the cell membrane. Owing to the shortage of efficient characterization strategy, however, there is still lacking of detailed knowledge about the distribution of the stoichiometric composition information of this peptide in solution and on lipid membrane prior to the cellular uptake process, which is fundamentally important for the understanding of the anti-cancer mechanism. In this work, with an objective-type total internal reflection fluorescence microscopy (TIRF), the distribution of the stoichiometric composition of lycosin-I in different solutions as well as on the lipid membrane was explored extensively based on the statistical single

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

molecule fluorescence intensity analysis for the first time. It was found that lycosin-I mainly displayed in a monomer state in diverse physiological solutions regardless of the concentration of the peptide and the incubation time. However, on the lipid membrane, the fraction of small size oligomers increased as a function of time. Fusion of movable peptide molecules to those peptide oligomers with restricted motion on the lipid membrane was also observed.

Introduction A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells.1,2 Our recent work also showed that the spider peptide toxin, named lycosin-I (a kind of AMP that derived from the venom of the spider Lycosa singorensis and whose amino acid sequence was determined as RKGWFKAMKSIAKFIAKEKLKEHL), is able to potently inhibit tumor cell growth in-vitro, and suppresses various tumor growth in-vivo when tested in human cancer xenograft models.3 The preliminary experimental results demonstrated that the lycosin-I peptide can indeed interact with the cell membrane and then be internalized into the cytoplasm of cancer cells to initiate the programmable cell death. However, it remains elusive why only some host defense peptides are able to kill cancer cells where others do not.4 In addition, it is still not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs is the same or different.5


Page 2 of 31

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


According to the distinct fatal targets, the function of AMPs or anticancer peptides (ACPs) can be generally classified into two major groups, membrane disruptive and non-membrane disruptive peptides.6,7 The first class functions through interaction with the cell membrane, permeabilizing the phospholipids bilayer and eventually causing cell death. The non-membrane disruptive peptides may go through the membrane and interact with variable intracellular macromolecular targets and then initialize cell death. From this account, regardless of the ultimate anti-cancer mechanism of the peptide, to fully address the two fundamental issues as mentioned above, the urgent yet essential step is to comprehend in detail the stoichiometry of the ACPs in physiological environments and how they interact with cell membrane, because the cytotoxic effect inevitably involves an initial interaction of the monomers or oligomers with the plasma membrane. In this work, we explored the distribution of the stoichiometric composition of the lycosin-I peptide molecules before and after adsorption to the lipid membrane with total internal reflection fluorescence microscopy (TIRF).8 Each lycosin-I molecule was firstly conjugated with single fluorescent molecule Rhodamine B in the N-terminal during the peptide synthesis process. Since the evanescent field generated by TIRF illumination only excites the fluorescent molecules close to the solid/liquid interface (generally less than 200 nm), the diffusion and adsorption dynamics of the peptide molecules on the lipid membrane could thus be tracked in real time. Based on the quantitative statistical single molecule fluorescence intensity analysis, the stoichiometric clustering state of lycosin-I in different buffer solutions and on lipid



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 31

membrane could be elucidated unambiguously. It was noted that the majority of lycosin-I displayed in a monomer state in diverse solutions regardless of the concentration of the peptide and the incubation time. However, on the lipid membrane, lycosin-I was found to gradually aggregate once been added onto the lipid membrane. The fraction of monomer decreased dramatically from 61.3% (in PBS) to 43.3% after the incubation time of 0.5 h. The growth rate of oligomers was dependent on the incubation time as well as the concentration of peptide in the bulk solution. These results would considerably improve the knowledge on the mechanism of how ACPs interacts with cancer cell membrane, which is of great importance for an increased understanding of the peptide/membrane interaction mechanism and for their potential as clinical anti-cancer drugs.

Experimental Section Chemicals and materials. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Life Bioscience. Lycosin-I was synthesized using an Fmoc (N-(9-fluorenyl) methoxycarbonyl)/tert-butyl strategy and HOBt/TBTU/NMM coupling method on an automatic peptide synthesizer (PerSeptive Biosystems) as we previously described. conjugated

to

the

3

Rhodamine B, from Sigma-Aldrich, was covalently

N-terminal

of

lycosin-I

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

at

a

(POPC)

mole and

ratio

of

1:1.

sulforhodamine

1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine (Texas-Red DHPE, 0.001%


Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


wt) were purchased from Avanti Polar Lipids. All other chemicals not mentioned here were purchased from Sigma-Aldrich.

Preparation of lipid bilayer. The procedure for lipid bilayer fabrication was based on the self-assembly of small unilamellar vesicles (SUVs) on the surface of a clean glass slide. The detailed method was described in our previous work.9 In brief, for SUVs preparation, POPC was dissolved in chloroform/methanol (1:1) for 15 min. Then the solvent was removed by evaporation under vacuum condition. The lipid film was swelled with PBS buffer for 30 min and suspended by vortex. The SUVs solution was diluted to 0.5 mg/mL with PBS buffer and then extruded 21 times through a porous polycarbonate membrane (with pore size of 100 nm) with a mini-extruder apparatus. The resulted 100 nm SUVs solution was stored at 4 oC prior to use. The lipid bilayer was prepared inside a customer-built flow channel on a clean cover glass slide. The flow channel was firstly washed with PBS and then 50 µL of freshly prepared SUVs solution was injected to the channeled and incubated for 30 min at 37 oC. Additional SUVs suspended in the solution were washed away with PBS. To confirm the integrity of the lipid bilayer made by this method, Texas-Red DHPE was mixed with POPC during the SUVs fabrication process. The homogeneously intercalated dye molecules on the lipid bilayer should then result in evenly distributed fluorescence signal within the flow channel provided the lipid bilayer is successfully generated. Furthermore, fluorescence recovery after



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

photobleaching (FRAP) was performed to characterize the integrity and fluidity of the lipid membrane as shown in Figure S1.

Imaging of individual lycosin-I with TIRF. Single molecule fluorescence imaging experiments were performed on an inverted optical microscope (Ti-U, Nikon, Japan). The mercury lamp was replaced with a solid diode laser (532 nm, CNI laser Changchun, China). The expanded laser line was reflected with a dichroic mirror and focused onto the back port of an oil immersion objective (NA 1.49, 100 ×, Nikon, Japan). The emitted fluorescence from the sample was then filtered by a band pass filter (605/55, Semrock, U.S.A.) In order to reduce background noise from the sample, the incidence angle of the laser line was slightly adjusted to achieve total internal reflection illumination. The fluorescence image of Rhodamine B-labeled lycosin-I was captured by an EMCCD (Ultra 897, Andor, UK). The exposure time was set to 20 ms. Under this condition, the data acquisition rate can reach around 47 Hz for dynamic tracking. All of the fluorescence images were then processed with Image J (http://rsb.info.nih.gov/ij/). Additional experimental details about peptide characterizations and molecular dynamics simulations please refer to the supplementary information.

Results and discussion Quantification of the stoichiometry of lycosin-I with statistical single molecule fluorescence intensity analysis.


Page 6 of 31

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To distinguish the chemical compositions of a mixture, chromatography (such as HPLC, capillary electrophoresis, size exclusion chromatography and so on) is the commonly adopted method, which can effectively resolve the compounds with different chemical structure, conformation as well as molecular weight.10,11 Since these methods typically work in specific buffer solutions, for biological samples, it is difficult to elucidate in-situ and real-time information based on the ensemble measurement strategies. Single molecule fluorescence imaging allows for direct probing of the dynamic information of target molecules and is particularly suitable for non-invasive biological sample observation in-situ.12-15 Herein we explored the distribution of the stoichiometric composition of lycosin-I in physiological buffer solutions as well as on a lipid membrane based on quantitative single molecule fluorescence intensity analysis, Figure 1. Firstly, single Rhodamine B molecule was labeled with lycosin-I at the N-terminal during the peptide synthesis process with a peptide/dye ratio of 1:1 (Figure S2). Based on the control results, the labeling process didn’t affect on the native biological activity of the peptide, Figure S3. Under TIRF illumination, Rhodamine B-labeled lycosin-I could be observed individually after adsorbed on the glass slide surface. Since the dye-labeling ratio is 1:1, one could elucidate the stoichiometric composition of the observed object directly based on the observed number of photobleaching steps from each fluorescent spot.15-18 Ideally, counting the photobleaching steps statistically according to the time-dependent fluorescence bleaching tracks can generate an accurate estimation of the distribution of the stoichiometric composition information



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for static events analysis.18,19 However, for dynamic process analysis, this strategy is typically inefficient because only parts of the observed objects will be bleached step by step within the observation window, making the quantification of the distribution of the stoichiometric composition challenging especially for large size oligomers. In addition, other associated processes might also degenerate the accuracy of the results such as blinking of the dye, dissociation of the molecule at the interface.20-22 To address these issues, we quantified the molecular stoichiometry based on statistical single molecule fluorescence intensity analysis. Rather than counting the time-consuming photobleaching steps, we used the fluorescence intensity distribution function to decode the stoichiometric composition information of oligomers in a statistical way. In this route, only few fluorescence images are required to obtain the stoichiometric information of the peptide through fitting the counted single spot fluorescence intensity information with the intensity distribution function. The fluorescence intensity distribution function from single lycosin-I molecules was determined experimentally by statistically (around 100 points) analyzing the intensity from single lycosin-I on the glass slide surface. Each data point was verified based on the time dependent fluorescence track. Only those molecules with single bleaching step were utilized for the subsequent statistical analysis. The fluorescence intensity from single lycosin-I shows well defined Gaussian distribution with mean gray value of 2651.3 and standard deviation of 353.9, Figure 1C, which is typically dependent on the orientation of the molecular dipole, the property of local physical and chemical environment and so on.


Page 8 of 31

Page 9 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For large size oligomers, the intensity distribution function is complicated because the photo-luminescence process from dyes is independent from each other. Therefore, it is not convenient to directly obtain the intensity distribution function experimentally, especially for large aggregates. In this case, we used Monte Carlo simulation to elucidate the intensity distribution function of oligomers. The simulation is based on the following assumptions: 1) the fluorescence intensity of each dye from the oligomer obeys the intensity distribution function of the monomer as determined above, 2) the photo-luminescence process of the dye is independent with each other.23 Figure 2A shows the simulated fluorescence intensity distribution functions of dimers, trimmers and tetramers. In order to verify the accuracy of the simulated results, we treated the coverslip with Piranha solution and dipped it in NH3·H2O for 1 hour. After this treatment, the surface of the coverslip is negatively charged. An obvious increased adsorption of Rhodamine B-labeled lycosin-I on the coverslip was observed, Figure S4. Through statistically measuring the fluorescence intensity from those spots with noticeable two bleaching steps, a dimer intensity distribution function could be determined experimentally. As shown in Figure 2C, in comparison with the computer simulation result, these two data sets match well with each other, confirming the reliability of the simulation strategy for intensity distribution function elucidation.

The effect of buffer solution on the stoichiometry of lycosin-I.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

Until now, there are several models have been proposed to describe the interaction mechanism of AMPs with lipid membrane, such as the widely accepted carpet mechanism, Barrel Stave mechanism and Toroidal mechanism.7,24 These mechanisms are essentially classified based on the final interaction state of the peptide with lipid membrane while little is considered about the stoichiometric composition information of the peptide prior to and during the association process.

In living cell

system, owing to the complexity of the cell membrane as well as the buffer solution, it is difficult to adopt a specific lipid interaction mechanism to explain the cytotoxicity of peptide purely based on the lipid/peptide interaction. Moreover, growing evidence suggests that the toxicity of many peptides is closely correlated with their self-assembled stoichiometric composition.25-27 It has been demonstrated that, in physiological buffer solutions or on lipid membranes, the peptides can self-assemble into oligomeric aggregates, which are significantly more toxic to cells than the monomer.25 In this regard, systematically elucidating the detailed stoichiometric composition of the peptide is the key step to rationally explain the biological function of the peptide. According to the previous experimental results, the minimum concentration of lycosin-I that will cause noticeable toxic effect to HeLa cells is in the range of 10-6 M.3 Therefore, we firstly explored the stoichiometric composition of lycosin-I in water at the threshold of toxic concentration. As shown in Figure 3A, the majority of lycosin-I peptides were staying in a monomer state with fraction around 75.4%. The ratio of dimer, trimer and tetramer is 13.2%, 7.2% and 4.2% respectively. Larger


Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecular aggregate was not found in this condition. It is interesting to note that extending the incubation time of the peptide in water did not result in obvious variation of stoichiometric distribution. Furthermore, when the concentration of the peptide was gradually decreased, such as from 10-7 to 10-9 M, the ratio between these compositions was not changed noticeably, Figure 3A. These results indicate that the peptide/peptide association force in deionized water solution is very weak. This might be owing in part to the vigorous Brownian diffusion of the molecules as well as the electrostatic repulsion between the positively charged peptides whose isoelectric point is calculated to be 10.78. To ascertain this argument, the electrostatic interaction was weakened by dispersing the lycosin-I peptides in PBS buffer. The increased ionic strength of the solvent can effectively reduce the thickness the electric double layer, resulting in moderate electrostatic interaction while other interactions will not change significantly. As shown in Figure 3B, at the threshold toxic concentration, the ratio of monomer in PBS buffer indeed decreased to 22.1% and followed by a substantial increase in the dimer fraction (from 13.2% to 28.3%). The ratio of trimer and tetramer also increased slightly (4.5% and 2.5% respectively). Larger aggregates in this condition were still negligible. According to the previous experimental results, under favorable salt solution, many types of peptides could self-assemble into super-molecular aggregates after appropriate 3D structure folding process.28 Since this self-assembly process is typically a thermodynamically favorable yet kinetically limited process, the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

self-association step between neighboring molecules in the solution is therefore a time and concentration dependent process. In this regard, we further explored the clustering state of lycosin-I as a function of time and their concentrations in PBS buffer. As illustrated in Figure 4, the stoichiometric composition of peptide in PBS buffer is still insensitive to the variation of incubation time and peptide concentration, indicative of weak peptide/peptide attractive force. Similar results were also observed in cell culture mediums with or without serum proteins, Figure S5 and S6. It is worthy of noting that the distribution of the stoichiometric composition of lycosin-I in these buffer solutions is comparable with each other. No noticeable variation was observed among these buffers. These results together demonstrate that the association interactions between peptide/peptide (such as hydrogen bonding, salt bridge and hydrophobic interaction, which are typically correlated with the 3D conformation of the peptide) are very weak, which is not efficient to compromise the thermal induced Brownian diffusion as well as electrostatic repulsion between peptides. The observed weak clustering behavior of the lycosin-I in solution is also corroborated by MD simulations. The initial monomer separation of a few Å (Figure 5A) was “non-committal” in the sense that it was small enough for the molecules to come close if the binding was strong; alternatively, they would be able to drift apart if it was weak. A representative snapshot along the MD trajectory of a dimer simulation is showed in Figure 5. What we observed is that monomers are able to repeatedly


Page 12 of 31

Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


approach each other (Figure 5B) and to drift apart (Figure 5C), which implies that any dimers formed can also be dissociated at room temperature.

Increased self-association of lycosin-I on lipid membrane. In order to understand the role of lipid membrane on the effect of peptide/peptide interaction, we further explored stoichiometric composition information of lycosin-I on the lipid membrane. In contrast to the prokaryotic membranes, which are predominantly composed of negatively charged

phosphatidylglycerol (PG),

cardiolipin (CL), or phosphatidylserine (PS), the mammalian membranes are enriched in the zwitterionic phospholipids (neutral net charge) phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin (SM).7,9 On this account, it is more interesting to explore the effect of neutral charge lipid membrane on the stoichiometry of lycosin-I rather than using negatively charged lipid bilayer. Interestingly, evident interaction between peptide and lipid membrane was observed after the peptide solution was passed through a flow channel covered with POPC lipid bilayer. Many peptides were sticking on the membrane and parts of them underwent noticeable adsorption and dissociation process. The distribution of the stoichiometric composition of the peptide on the lipid membrane was also dramatically changed in comparison with that in the buffer solutions, Figure 6. At the concentration of 10-9 M, the ratio of monomers decreased to 43%, while the ratio of oligomers greatly increased. The fraction of dimers is almost comparable with that of monomers (37%). Extremely large assemblies were not noted within 30 min. Further



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

extending the incubation time, the fraction of oligomers increased gradually, which is noticeably distinct from the case in DI water or other buffer solutions. After 3 h, the dimer became the dominant component with the ratio around 50% and the rate for large aggregates was still very slow. From these results, it is evident that the lipid membrane plays an important role in the modulation of peptide/peptide interaction. Previous works have demonstrated that the peptide helicity plays an important role in their biological activity.29-31 The alternation of the peptide secondary structure might result in a dramatic change of the interaction force between neighboring molecules, such as hydrogen bonding as well as hydrophobic interactions. In order to understand the structure information of the peptide in the presence of zwitterionic molecules, we conducted circular dichroism (CD) measurements. Figure 7A shows the CD spectra of lycosin-I in the absence and presence of POPC. Without POPC, the peptide shows a random coil structure. For the peptide to form a stable aggregation state, the association force between peptides should be strong enough to compromise the thermal induced vigorous Brownian diffusion. The disordered structure of lycosin-I is therefore not conductive to additional attractive interactions. This measurement well explains the above observations that the peptide mainly stays in a monomer state in PBS buffer and cell culture medium. However, in the presence of POPC, a noticeable peak at around 195 nm was observed, indicative of the formation of a well-defined helix structure. Based on the CD spectrum, the helical wheel projection of lycosin-I was also calculated which highlighted the most likely configuration of amphipathic and cationic alpha helix structure, Figure 7B. The value


Page 14 of 31

Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of lycosin-I hydrophobicity (H) and hydrophobic moment (mH) was determined to be 0.246 and 0.244, respectively. Hydrophobicity corresponds to the inner translocation power of the peptide from water phase to hydrophobic phase, and the hydrophobic moment is the vectorial sum of hydrophobicity of every amino acid residue in peptide. The mH value of cationic peptides has been proven to be the decisive factor of their membrane-disruptive activities. As to the neutral lipid membrane, the influence of mH is even greater than in the case of a negative lipid membrane, owing to the major hydrophobic interaction in the former situation. Based on these results, it is rational to conclude that lipid molecules play a significant role during the peptide conformation folding process. The interaction of peptide/lipid molecules might greatly reduce the energy barrier for the peptide folding process.

Diffusion dynamics of lycosin-I on the lipid membrane. According to the results in Figure 6 and Figure S7 two sets of explanations could be drawn to account for the lipid membrane effect. The first one might be owing to that the strength of the attractive force between larger peptide clusters and lipid membrane is stronger than that of monomers. As time goes on, the density of larger clusters would increase on the surface. The second scenario could be owing to the increased association probability of peptide on the lipid membrane. Because of the reduced Brownian diffusion as well as the lipid membrane induced conformation variation process, the association strength and probability of the peptides on the lipid membrane could be increased in contrast to those in the bulk solution. Therefore, a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

diffusion-limited aggregation process from those monomer or oligomers already attached on the lipid membrane and a reaction-limited association process by directly attaching of the peptide in the solution to those already anchored on the lipid membrane could both contribute to the time dependent gradual growth of peptide clusters on the lipid membrane. Provided the first scenario is the dominant process, under a fixed peptide concentration, the growth rate of larger oligomers on the lipid membrane should be faster than that of smaller ones. As shown in Figure 6 and Figure S7, the growth rate of clusters is obviously not dependent on the size of oligomers. In order to further understand the underlying mechanism of peptide clustering process on the lipid membrane, we tracked the diffusion dynamics of lycosin-I on the lipid membrane. Three types of motions on the lipid membrane were noted. 1) Transient binding with retention time within the range of less than 20 ms and then followed with desorption. This observation is reasonable because the successful adsorption of peptide onto the lipid membrane still needs to overcome the vigorous thermal diffusion, which is typically a conformation and orientation dependent process. 2) Strong interaction with nearly frozen diffusion on the lipid membrane. The 2D diffusion coefficient is around 0.021±0.016 µm2/s, which is around two orders slower than that of lipid molecules (2.5±0.05 µm2/s),32 Figure 8A. 3) Apparent diffusions with decreased movability on the lipid membrane (with 2D diffusion coefficient of 0.2±0.1 µm2/s), Figure 8B. For the second kind of motion, the observed objectives were mainly oligomers. This might be partly owing to the increased interaction points from the oligomers on the


Page 16 of 31

Page 17 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


lipid membrane, which subsequently results in a large peptide/lipid cluster. A similar observation was made in the case of other cell penetration peptides. Once the local density of peptide increased, the Brownian diffusion of peptide modified nanocargo could be greatly inhibited, which is even able to confine the diffusion of individual 60 nm gold nanoparticles (with a 2D diffusion coefficient of (8.5 ± 2.4) × 10−3 µm2/s).9 Upon Further inspection of the diffusion dynamics of individual objects on the lipid membrane, parts of mobile peptides even could fuse with those peptides with restricted motion on the lipid membrane, Supporting Movie 1. This observation agrees well with the second explanation as discussed above and is also consistent with concentration dependent experiments on the lipid membrane as shown in Figure 6.

Conclusion In conclusion, with TIRF microscopy, the stoichiometry distribution of anticancer peptide lycosin-I in different solutions and on lipid membrane was analyzed with statistical single molecule fluorescence intensity analysis. It was found that the fraction of monomer state dominates the major distribution in diverse physiological solutions, regardless of the concentration of peptide and the incubation time in buffers. However, on the lipid membrane, lycosin-I was found to gradually aggregate once been anchored onto lipid membrane. The reduced diffusion dynamics after being adsorbed on the lipid membrane and changed conformation might greatly facilitate the peptide clustering process. These new insight on the dynamic molecular stoichiometric information of lycosin-I could considerably improve the knowledge on



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the mechanism of how lycosin-I interacts with cancer cell membrane, which is of great importance for an increased understanding of the anti-cancer mechanism and for their potential as clinical anti-cancer drugs. Furthermore, the method demonstrated here also affords a convenient and robust strategy for the in-situ and real-time analysis of dynamic molecule aggregation process in complex biological surroundings.


Page 18 of 31

Page 19 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures and Figure captions

Figure 1. A) Schematic diagram of the TIRF setup for single molecule imaging. B) Representative normalized fluorescence intensity track of single lycosin-I molecule as indicated with red arrow in A). C) The measured fluorescence intensity distribution of single lycosin-I on the cover slip surface (green bars) and the corresponding Monte Carlo simulation result (red line).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. A) The simulated fluorescence intensity distribution functions of lycosin-I monomer (red), dimer (green), trimer (blue) and tetramer (brown). B) Representative normalized fluorescence intensity track of dimer on cover slip surface; C) The measured (green bars) and simulated fluorescence intensity distribution (red line) of lycosin-I dimers.


Page 20 of 31

Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. The distribution of the stoichiometric composition information of lycosin-I in different solutions. A) Lycosin-I in DI water; B) Lycosin-I in PBS; C) Lycosin-I in phenol red-free DMEM cell medium without serum; D) Lycosin-I in phenol red-free DMEM cell medium with 10% fetal bovine serum. The x-coordinate represents the concentration of peptide. The distribution information was calculated from more than 500 measurements in each experiment.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Time dependent clustering process of lycosin-I in PBS. The distribution of the stoichiometric composition information of lycosin-I at concentration of A) 10-6 M, B) 10-7 M, C) 10-8 M and D) 10-9 M.


Page 22 of 31

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. A snapshot of a point along the MD trajectory of a peptide dimer simulation: initial position A), bonded B) and dissociated C) state. Water molecules are omitted for clarity.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Self-association process of lycosin-I on lipid membrane. The time-dependent stoichiometric composition information of lycosin-I on lipid membrane at concentration of A) 10-9 M and B) 10-10 M.


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Secondary structure of lycosin-I. A) Circular dichroism spectrometry of lycosin-I in PBS buffers with or without or POPC; B) Helical wheel structure of lycosin-I as derived from the circular dichroism. The hydrophobic residues are yellow, positively charged residues are blue and negatively charged residues are red. The downward arrow points the hydrophobic face of lycosin-I. The physico-chemical properties and residue composition statistics are presented in the table below.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Representative 2D diffusion trajectories of greatly inhibited A) and slightly reduced B) lycosin-I peptides on lipid membrane.


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ASSOCIATED CONTENT

Supporting Information

Supplementary figures and additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected], [email protected].

Author Contributions H. T. and W. L. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was supported by NSFC (21205037, 21405045, 21522502), Program for New Century Excellent Talents in University (China, NCET-13-0789), Hunan Natural Science Funds for Distinguished Young Scholar (14JJ1017), and the aid program for science and technology innovation research team in higher education institutions of Hunan Province.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

References (1)

(2) (3)

(4) (5) (6) (7)

(8)

(9)

(10)

(11)

(12) (13) (14) (15) (16)

Perez, S. A.; Hofe, von, E.; Kallinteris, N. L.; Gritzapis, A. D.; Peoples, G. E.; Papamichail, M.; Baxevanis, C. N. A New Era in Anticancer Peptide Vaccines. Cancer 2010, 116, 2071–2080. Schweizer, F. Cationic Amphiphilic Peptides with Cancer-Selective Toxicity. Eur. J. Pharm. 2009, 625, 190–194. Liu, Z.; Deng, M.; Xiang, J.; Ma, H.; Hu, W.; Zhao, Y.; Li, D. W. C.; Liang, S. A Novel Spider Peptide Toxin Suppresses Tumor Growth Through Dual Signaling Pathways. Curr. Mol. Med. 2012, 12, 1350–1360. Papo, N.; Shai, Y. Host Defense Peptides as New Weapons in Cancer Treatment. Cell Mol. Life Sci. 2005, 62, 784–790. Hoskin, D. W.; Ramamoorthy, A. Studies on Anticancer Activities of Antimicrobial Peptides. BBA-Biomembranes 1778, 357–375. Powers, J.-P. S.; Hancock, R. E. W. The Relationship Between Peptide Structure and Antibacterial Activity. Peptides 2003, 24, 1681–1691. Huang, Y.; Huang, J.; Chen, Y. Alpha-Helical Cationic Antimicrobial Peptides: Relationships of Structure and Function. Protein Cell 2010, 1, 143– 152. Wei, L.; Liu, C.; Chen, B.; Zhou, P.; Li, H.; Xiao, L.; Yeung, E. S. Probing Single-Molecule Fluorescence Spectral Modulation Within Individual Hotspots with Subdiffraction-Limit Image Resolution. Anal. Chem. 2013, 85, 3789–3793. Wei, L.; Zhao, X.; Chen, B.; Li, H.; Xiao, L.; Yeung, E. S. Frozen Translational and Rotational Motion of Human Immunodeficiency Virus Transacting Activator of Transcription Peptide-Modified Nanocargo on Neutral Lipid Bilayer. Anal. Chem. 2013, 85, 5169–5175. Fekete, S.; Beck, A.; Veuthey, J.-L.; Guillarme, D. Theory and Practice of Size Exclusion Chromatography for the Analysis of Protein Aggregates. J. Pharmaceut. Biomed. 2014, 101, 161–173. Hong, P.; Koza, S.; Bouvier, E. A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates. J. Liq. Chromatogr. Relat. Technol. 2012, 35, 2923–2950. Kisley, L.; Landes, C. F. Molecular Approaches to Chromatography Using Single Molecule Spectroscopy. Anal. Chem. 2015, 87, 83–98. Weiss, S. Fluorescence Spectroscopy of Single Biomolecules. Science 1999, 283, 1676–1683. Lu, H. P.; Xun, L.; Xie, X. S. Single-Molecule Enzymatic Dynamics. Science 1998, 282, 1877–1882. Moerner, W. E.; Orrit, M. Illuminating Single Molecules in Condensed Matter. Science 1999, 283, 1670–1676. Moerner, W. E. A Dozen Years of Single-Molecule Spectroscopy in Physics, Chemistry, and Biophysics. J. Phys. Chem. B 2002, 106, 910–927.


Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17)

(18)

(19)

(20)

(21)

(22) (23) (24) (25) (26)

(27)

(28)

(29)

(30)

(31)

Zondervan, R.; Kulzer, F.; Kol'chenk, M. A.; Orrit, M. Photobleaching of Rhodamine 6G in Poly(Vinyl Alcohol) at the Ensemble and Single-Molecule Levels. J. Phys. Chem. A 2004, 108, 1657–1665. Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Single Molecule Detection and Photochemistry on a Surface Using Near-Field Optical Excitation. Phys. Rev. Lett. 1994, 72, 160–163. Zijlstra, N.; Blum, C.; Segers-Nolten, I. M. J.; Claessens, M. M. A. E.; Subramaniam, V. Molecular Composition of Sub-Stoichiometrically Labeled Α-Synuclein Oligomers Determined by Single-Molecule Photobleaching. Angew. Chem. Int. Ed. Engl. 2012, 51, 8821–8824. Gensch, T.; Böhmer, M.; Aramendía, P. F. Single Molecule Blinking and Photobleaching Separated by Wide-Field Fluorescence Microscopy. J. Phys. Chem. A 2005, 109, 6652–6658. Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E. On/Off Blinking and Switching Behaviour of Single Molecules of Green Fluorescent Protein. Nature 1997, 388, 355–358. Panzer, O.; Göhde, W.; Fischer, U. C.; Fuchs, H.; Müllen, K. Influence of Oxygen on Single Molecule Blinking. Adv. Mater. 1998, 10, 1469–1472. Lounis, B.; Moerner, W. E. Single Photons on Demand From a Single Molecule at Room Temperature. Nature 2000, 407, 491–493. Brogden, K. A. Antimicrobial Peptides: Pore Formers or Metabolic Inhibitors in Bacteria? Nat. Rev. Microbiol. 2005, 3, 238–250. Sakono, M.; Zako, T. Amyloid Oligomers: Formation and Toxicity of Aβ Oligomers. FEBS J. 2010, 277, 1348–1358. Haass, C.; Selkoe, D. J. Soluble Protein Oligomers in Neurodegeneration: Lessons From the Alzheimer's Amyloid Β-Peptide. Nat. Rev. Mol. Cell Biol. 2007, 8, 101–112. Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis. Science 2003, 300, 486–489. Ozbas, B.; Kretsinger, J.; Rajagopal, K.; Schneider, J. P.; Pochan, D. J. Salt-Triggered Peptide Folding and Consequent Self-Assembly Into Hydrogels with Tunable Modulus. Macromolecules 2004, 37, 7331–7337. Dathe, M.; Schümann, M.; Wieprecht, T.; Winkler, A.; Beyermann, M.; Krause, E.; Matsuzaki, K.; Murase, O.; Bienert, M. Peptide Helicity and Membrane Surface Charge Modulate the Balance of Electrostatic and Hydrophobic Interactions with Lipid Bilayers and Biological Membranes. Biochemistry 1996, 35, 12612–12622. Chen, Y.; Mant, C. T.; Farmer, S. W.; Hancock, R. E. W.; Vasil, M. L.; Hodges, R. S. Rational Design of Α-Helical Antimicrobial Peptides with Enhanced Activities and Specificity/Therapeutic Index. J. Biol. Chem. 2005, 280, 12316–12329. Huang, Y.; He, L.; Li, G.; Zhai, N.; Jiang, H.; Chen, Y. Role of Helicity of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32)

Α-Helical Antimicrobial Peptides to Improve Specificity. Protein Cell 2014, 5, 631–642. Ciobanasu, C.; Harms, E.; Tünnemann, G.; Cardoso, M. C.; Kubitscheck, U. Cell-Penetrating HIV1 TAT Peptides Float on Model Lipid Bilayers. Biochemistry 2009, 48, 4728–4737.


Page 30 of 31

Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC

Quantifying the Distribution of the Stoichiometric Composition of Anti-cancer Peptide Lycosin-I on Lipid Membrane with Single Molecule Spectroscopy Huaxin Tan, Wenjuan Luo, Lin Wei, Bo Chen, Wenxuan Li, Lehui Xiao, Sergei manzhos, Zhonghua Liu, and Songping Liang


Quantifying the Distribution of the Stoichiometric Composition of

Recommend Documents