Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation

Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation Nhung Thi Hong Nguyen , Shoko Fujita , Nanami Takeuchi , Ryuji Kawano , View ORCID Profile Takanari Inoue doi: https://doi.org/10.1101/2025.08.26.672490 Nhung Thi Hong Nguyen 1 Department of Cell Biology, Johns Hopkins University School of Medicine , Baltimore, MD 21205, USA 2 Center for Cell Dynamics, Johns Hopkins University School of Medicine , Baltimore, MD 21205, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shoko Fujita 3 Department of Biotechnology and Life Science , Tokyo University of Agriculture and Technology (TUAT) 2-24-16 Naka-cho Koganei-shi Tokyo 184-8588 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nanami Takeuchi 3 Department of Biotechnology and Life Science , Tokyo University of Agriculture and Technology (TUAT) 2-24-16 Naka-cho Koganei-shi Tokyo 184-8588 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ryuji Kawano 3 Department of Biotechnology and Life Science , Tokyo University of Agriculture and Technology (TUAT) 2-24-16 Naka-cho Koganei-shi Tokyo 184-8588 Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: rjkawano{at}cc.tuat.ac.jp jctinoue{at}jhmi.edu Takanari Inoue 1 Department of Cell Biology, Johns Hopkins University School of Medicine , Baltimore, MD 21205, USA 2 Center for Cell Dynamics, Johns Hopkins University School of Medicine , Baltimore, MD 21205, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Takanari Inoue For correspondence: rjkawano{at}cc.tuat.ac.jp jctinoue{at}jhmi.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Pore-forming peptides (PFPs) are powerful tools for engineering biological membranes in vitro and in living cells. They can be derived from natural sources or designed de novo , with synthetic peptides offering distinct advantages in rational design unconstrained by natural sequences. While native PFPs have been targeted to specific organelles to modulate their functions, the potential of synthetic PFPs for organelle engineering remains largely unexplored. Here, we show that the 28-amino acid synthetic peptides, SVG28 and its variant SVG28D2, can be selectively targeted to mitochondria for functional alteration. More specifically, molecular dynamics simulations first confirmed the structural stability of the SVG28 nanopore even when fused to the TOM20 transmembrane domain, a mitochondrial targeting sequence. In isolated lipid bilayers, TOM20-SVG28 formed pores with a 0.3-nS, conductance, or 1.0-nm pore. The expression efficiency was further enhanced by two amino acid substitutions (SVG28D2). In cells, both TOM20-SVG28 and TOM20-SVG28D2 localized to the mitochondria, induced morphological alterations, and reduced both membrane potential and ATP production. These observations in silico, in vitro and in cellulo collectively indicate that the synthetic SVG28 peptides self-assemble into pores in the outer mitochondrial membrane, likely permitting proton permeation. Importantly, the matrix protein Su9 remained confined within mitochondria, suggesting preserved inner membrane integrity, and thus specific effect on the target. This work establishes a framework for the design and application of genetically-encoded synthetic PFPs in living cells, opening new avenues for cellular engineering. Introduction Mitochondria are essential organelles responsible for ATP production, metabolism regulation, and apoptosis induction 1 - 5 . The functionality of mitochondria relies on the integrity and dynamic remodeling of their membranes. These membranes play a critical role in preserving the electrochemical differences necessary for energy production and in organizing distinct biochemical activities within the mitochondrion 6 - 8 . Disruptions in mitochondrial membrane structure or morphology can affect how mitochondria work, which may cause or exacerbate health problems such as neurodegenerative diseases, cancer, and metabolic disorders 9 - 11 . Pore-forming peptides (PFPs) represent a class of molecules that can integrate into lipid bilayers to trigger formation of pores, which can regulate the selective permeability of ions, signaling molecules, and macromolecules 12 - 14 . Natural and synthetic PFPs have been extensively investigated for their capacity to alter membrane properties under in vitro conditions 15 - 18 . For example, synthetic transmembrane peptides based on the pore-forming domain of TRPV4 can oligomerize to form potassium-selective channels that modulate membrane potential in vascular smooth muscle cells 19 - 22 . Poly-arginine (Arg 9 ) peptides are known for their cell-penetrating abilities and can induce formation of transient pores that in turn facilitate molecular transport 23; 24 . Voltage-dependent anion channels (VDACs), referred to as mitochondrial porins, are endogenous mitochondrial pore-forming proteins. VDACs modulate metabolite flux across the outer mitochondrial membrane and influence apoptotic pathways 25 - 28 . Despite their structural and functional diversity, the potential of synthetic PFPs engineered to selectively target mitochondria and modulate organelle integrity remains largely unexplored. This critical gap in knowledge is whether these molecules can be directed to mitochondria in living cells to precisely control membrane permeability without triggering catastrophic rupture. Overcoming this challenge through advances in design, targeting strategies, and functional regulation will be transformative for organelle engineering, disease modeling, and therapeutic innovation. In this study, we investigated if and how two artificially designed peptides, SVG28 and SVG28D2, previously demonstrated to form nanopores in planar lipid membranes without membrane rupture 29; 30 , can be targeted to an intracellular organelle for altering its properties. Results Designing pore-forming peptides targeted to mitochondria To demonstrate synthetic PFPs can function in a living-cell context, we aimed to target SVG28 synthetic peptides to mitochondria. Toward this end, the transmembrane domain of TOM20 that is known to target proteins to the mitochondrial outer membrane was fused to the N-terminus of SVG28 ( Fig. 1a ). A Flag tag was inserted with a flexible linker between TOM20 and SVG28 to enable antibody-based detection in cellular experiments without compromising functionality of TOM20 or SVG28. Download figure Open in new tab Figure 1. TOM20-Flag-SVG28 conjugate (a) Schematic of the conjugation of TOM20 and SVG28 for functional modulation of mitochondria (b) TOM20-Flag-SVG28 structures predicted by in silico methods. TOM20 in the transmembrane region is shown in purple; TOM20 outside the membrane in magenta; flag tag in orange; the linkers in green; SVG28 in cyan (c) TOM20-Flag-SVG28 pore structures after 200 ns simulation in DOPC:Chol membrane. DOPC and cholesterol molecules are shown in yellow We first predicted the structures of TOM20-Flag-SVG28 using AlphaFold3 31 and evaluated its stability through all-atom molecular dynamics (MD) simulations. TOM20-Flag-SVG28 showed a configuration similar to native TOM20 32 , which has a transmembrane region ranging from residue 1 to 24 ( Fig. 1b ). The TOM20-Flag-SVG28 pore structure was maintained after a 200 ns simulation within 1,2-dioleoyl-sn-glycero-3-phosphocholine:cholesterol (DOPC:Chol, 2:1 molar ratio) bilayer lipid membrane ( Fig. 1c ). While the SVG28 region maintained stable pore structures, the transmembrane TOM20 α-helix modestly fluctuated ( Supplementary Fig. 1a , b ). Thus, addition of the TOM20 targeting motif does not appear to impact the overall structural integrity of the SVG28-formed pore. Lastly, the Flag tag was exposed to the water layer as expected ( Fig. 1c ). Stability of pore-forming peptides synthesized in vitro To assess the pore-forming capability of TOM-Flag-SVG28 on membrane, we performed electrophysiological measurement across a planar lipid bilayer composed of DOPC:Chol. The molecular mass of the synthesized construct was analyzed by MALDI-TOF mass spectrometry and found to match the expected value ( Supplementary Fig. 1c ), confirming the molecular identity. Upon electrophysiological measurement of TOM20-Flag-SVG28, only five current events were observed during 8 h of recording, indicating that the events occurred infrequently. This low frequency was likely attributed to the poor expression of SVG28, likely due to its highly hydrophobic nature. Therefore, we employed a more hydrophilic variant, SVG28D2 29 . TOM20-Flag-SVG28D2 was confirmed to be stable in MD simulation, and was successfully produced using cell-free synthesis followed by MALDI-TOF/MS confirmation ( Supplementary Fig. 2a-c ). TOM20-Flag-SVG28D2 produced approximately a five-fold higher event frequency than TOM20-Flag-SVG28. Based on these findings, subsequent analyses were conducted using both TOM20-Flag-SVG28 and TOM20-Flag-SVG28D2. Pore properties of SVG28 peptides in lipid membranes Initial trials of electrophysiological measurements using lipid bilayers did not indicate stable pore formation, despite the detection of interactions between these molecules and the lipid membranes ( Supplementary Fig. 3 ). Suspecting that the presence of transmembrane TOM20 increased unwanted aggregation of SVG28 peptides, we added DOPC:DOPG liposomes to a cell-free protein synthesis solution for efficient membrane insertion of SVG28 peptides with proper folding. As a result, clear pore formation was observed for both TOM20-Flag-SVG28 and TOM20-Flag-SVG28D2 ( Fig. 2a ). To quantitatively evaluate pore-forming ability, the observed current signals were classified into stable or unstable pore formation based on their waveforms. Both constructs exhibited stable pore formation ( Fig. 2b ), with TOM20-Flag-SVG28 displaying a more stable pore formation compared to TOM20-Flag-SVG28D2 ( Fig. 2b ). This is consistent with the observation that SVG28D2, in the absence of TOM20, exhibited less stable pore formation than SVG28. Although direct comparison of the pore-forming stability between these constructs is complicated by factors such as synthesis yield and the extent of liposome incorporation, these results demonstrate that SVG28 peptides are capable of self-assembling into stable pores, even with TOM20 modification. Download figure Open in new tab Figure 2. TOM20-Flag-SVG28 and TOM20-Flag-SVG28D2 nanopore characterization (a) The typical current-time traces of TOM20-Flag-SVG28 and TOM20-Flag-SVG28D2 (b) The ratio of duration of stable pore formation and unstable pore formation of SVG28, SVG28D2, TOM20-Flag-SVG28, and TOM20-Flag-SVG28D2. For the data of SVG28, the raw data reported in S. Fujita et al . (unpublished work) was reanalyzed. (c-d) Histograms of the current conductance of TOM20-Flag-SVG28 (c) and TOM20-Flag-SVG28D2 (d) . Peak-fitting curves are plotted for peaks that can be fitted with a Gaussian function (e-f) I-V curves of TOM20-Flag-SVG28 (e) and TOM20-Flag-SVG28D2 (f) To further characterize these pore structures, we analyzed their single-channel properties. TOM20-Flag-SVG28 showed an ionic conductance ( I/V ) of 0.3 nS, while TOM20-Flag-SVG28D2 showed 0.4 nS, with a subpopulation of 1.1 nS in 1 M KCl ( Fig. 2c-d ). Based on the relationship between pore size and pore conductance in natural β-barrel proteins 30 , SVG28-Flag-TOM20 is estimated to form 5-mer pores composed of 10 β-strands with a pore diameter of 1.0 nm. Similarly, TOM20-Flag-SVG28D2 is estimated to form approximately 5-mer pores composed of 10.6 β-strands with a 1.1 nm pore diameter, while the 1.1 nS subpopulation corresponds to approximately 7-mer pores comprising 13.6 β-strands and a pore diameter of 1.7 nm, having sufficient pore sizes to transport protons. Furthermore, current-voltage ( I-V) curves exhibited an asymmetry with a higher current intensity at negative voltages ( Fig. 2e-f ). This is due to charge asymmetry in the SVG28 pore, wherein one terminus is positively charged and the other is negatively charged 30 . This demonstrates that TOM20-Flag-SVG28 and TOM20-Flag-SVG28D2 retain the defining structural and biophysical characteristics of the native SVG28 pore. Localizing pore-forming synthetic peptides to mitochondria To test whether the SVG28 peptides could manipulate organelle properties by forming a pore at a targeted location, DNA constructs encoding TOM20-Flag-SVG28, TOM20-Flag-SVG28D2, or TOM20-Flag as a control were generated ( Fig. 3a ). When each of these was transfected in HeLa cells, fluorescence imaging of these cells after immunostaining using a Flag antibody indicated all three constructs were successfully targeted to mitochondria ( Fig. 3b ). Strikingly, cells expressing Tom20-SVG28 or Tom20-SVG28D2, but not a control Tom20-Flag, exhibited altered mitochondrial morphology ( Fig. 3b ); while mitochondria in this cell type generally take tubular shape, SVG28- or SVG28D2-expressing cells showed more spherical mitochondria. ( Fig. 3b-c ). This result suggests that the pore-forming peptides SVG28 and SVG28D2 are capable of altering mitochondrial morphology in living cells when expressed on the mitochondrial outer membranes. Download figure Open in new tab Figure 3. SVG28 and SVG28D2 located in mitochondrial membrane (a) Illustration of the domains of the proteins (b) Representative images of mitochondria in HeLa cells labelled with Flag, Flag-SVG28, and Flag-SVG28D2. Scale bars, 10 μm (c) Graphs of the percentage of mitochondrial fragmentation. The means ± SD are shown (n=3). The P-values were obtained using a two-tailed paired Student’s t-test. Significance values are **p<0.01 Functional remodeling of mitochondria To evaluate the impact of mitochondrial targeting of SVG28 and SVG28D2 on mitochondrial properties, we constructed TOM20-Flag-SVG28, TOM20-Flag-SVG28D2, or TOM20-Flag, each of which was fused to a Halo tag via internal ribosomal entry site (IRES) ( Fig. 4a ). After expressing these constructs, we measured two major functions of mitochondria: membrane potential and ATP production using their fluorescence indicators ( Fig. 4b ). Download figure Open in new tab Figure 4. Effect of pore membrane on functional aspects of mitochondria (a) Illustration of the domains of the proteins (b) Illustration of mitochondrial potential and ATP synthesis (c) Fluorescence images of TMRE in HeLa cells expressing Flag, Flag-SVG28, and Flag-SVG28D2 at 0 min, 9 mins before CCCP treatment, and after CCCP treatment of 14 mins. Scale bars, 10 μm (d) Graphs of TMRE mean intensity at mitochondria. The P-values were obtained using one-way ANOVA with Tukey’s post hoc analysis. Significance values are *p<0.05 and ****p<0.0001 (e) Representative FRET images of miAT1.03 along with TOM20-Flag, TOM20-Flag-SVG28, and TOM20-Flag-SVG28D2. Scale bars, 10 μm (f) Graphs of FRET/CFP ratio value. The P-values were obtained using one-way ANOVA with Tukey’s post hoc analysis. Significance values are ***p<0.001 and ****p<0.0001 We first assessed mitochondrial membrane potential in HeLa cells expressing either SVG28, SVG28D2, or Flag control. To measure membrane potential, we employed tetramethyl rhodamine methyl ester (TMRE). Cells were incubated with TMRE for 30 minutes before live-cell imaging. TMRE fluorescence intensity was lower in cells expressing SVG28 and SVG28D2 than in those expressing a control construct. carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment resulted in complete loss of TMRE signal in all conditions ( Fig. 4c ). To evaluate the membrane potential, we quantified TMRE fluorescence intensity in mitochondria of expressing versus non-expressing cells. The result showed that the mitochondrial membrane potential was decreased by roughly 30% with the expression of poreforming membrane peptides SVG28 and SVG28D2 ( Fig. 4d ). We next measured mitochondrial ATP levels using mitAT1.03, an organelle-targeted Forster resonance energy transfer (FRET)-based biosensor of ATP 33 . Methods for measurement and analysis of CFP-to-YFP FRET were described previously 34 - 37 . FRET efficiency was determined by calculating the FRET signal normalized to CFP intensity. Expression of SVG28 and SVG28D2 resulted in a decrease in the FRET signal by roughly 10%, compared to the expression of the control construct ( Fig. 4e-f ). As a control, 2-deoxy-D-glucose was used to inhibit glycolysis and reduce ATP production. The 2DG treatment caused a strong decrease in the FRET signal in all three cases. This finding suggests that expression of pore-forming peptides leads to a reduction in ATP synthesis, likely due to the waning membrane potential. Mitochondrial inner membrane integrity Given the observed reductions in mitochondrial membrane potential and ATP synthesis, we hypothesized that these effects might result from the disintegration of inner membranes due to compromise to their outer membrane. If this is the case, then this should lead to the release of matrix-resident proteins ( Fig. 5a ). To test this possibility, we co-expressed mitochondria matrix protein Subunit 9 (Su9) of mitochondrial ATPase, that was fused to mCherry with either TOM20-Flag-SVG28, TOM20-Flag-SVG28D2, or TOM20-Flag. Fluorescence imaging of Su9-mCherry revealed no significant difference in the intensity or subcellular distribution between the peptide-expressing and control cells ( Fig. 5b-c ). These results indicate that the peptides did not cause any detectable leakage of soluble matrix proteins into the cytosol. Combined with the characterization in vitro , it is highly likely that SVG28 peptides formed a pore on the outer membranes, which permeated protons in the intermembrane space of mitochondria, thereby dissipating the proton gradient across the inner membranes. Download figure Open in new tab Figure 5. Mitochondrial matrix protein did not release to cytosol (a) Illustration of the presence of SVG28 or SVG28D2 (SVG pore) at the outer mitochondrial membrane, with Su9-mCherry which localized to the mitochondrial matrix, under condition of intact mitochondria or rupture of mitochondrial membrane (b) Representative images of Su9-mCherry showing Su9-mCherry localization to matrix of mitochondria co-expressed with TOM20-Flag, TOM20-Flag-SVG28, or TOM20-Flag-SVG28D2. Scale bars, 10 μm (c) Plots illustrating the relative mitochondrial-to-cytosolic fluorescence intensity ratio. No statistically significant differences were observed (p> 0.05). Discussion In this study, we demonstrate a unique strategy for precise manipulation of mitochondrial morphology and function by targeting engineered pore-forming peptides, SVG28 and SVG28D2, to the mitochondrial outer membrane via fusion with TOM20. Our results show that these peptides localize to mitochondria in HeLa cells, inducing mitochondrial fragmentation accompanied by decreased membrane potential and ATP production, while maintaining matrix compartmentalization. This highlights selective modulation of mitochondrial membrane integrity without catastrophic membrane rupture. Interestingly, the fusion of SVG28 to the transmembrane domain of TOM20 appears to enhance the stability of SVG28. While TOM20 is conventionally used as a targeting sequence for the outer membrane localization of exogenous proteins 38 - 40 , its demonstrated structural compatibility with SVG28 may also characterize TOM20 as a stabilizing scaffold that reduces proteolytic degradation and/or maintains membrane insertion competence. Channelrhodopsins are light-gated cation channels composed of single-chain, seven-transmembrane helix proteins, typically 40–77 kDa in size, and have been targeted to mitochondria to achieve temporal control of ion flux and membrane potential 41 - 43 . However, their relatively large and complex architecture may limit broader applicability within the mitochondria and also in other organelles. These challenges underscore the need for smaller, more stable, mitochondria-adapted tools to reliably modulate mitochondrial physiology without the drawbacks associated with native channelrhodopsin constructs 41 ; 42 By contrast, SVG28 pores are composed of ∼3 kDa monomers, each forming two transmembrane domains, whose minimal architecture may facilitate more efficient integration into organelle membranes. In the future, strategies could be developed to regulate the gating of SVG28 pores, such as engineering peptide variants responsive to chemical inducers, small molecules, or light. For example, light-controllable SVG28 pores could be achieved by incorporating photo-switchable amino acids or optogenetic modules. In conclusion, we present a synthetic biology approach to directly manipulate mitochondrial integrity and function by leveraging 28–amino acid peptides rationally designed de novo to self-assemble into nanoscale pores within biological membranes. This platform provides a versatile foundation for advancing applications in cell biology and nanotechnology. Because these synthetic molecular tools are genetically encoded, they can be introduced readily into target cells, tissues, and organisms. Together, these unique features open opportunities for organelle-targeted therapies and the creation of synthetically engineered organelles. Methods Plasmid construction Plasmids of TOM20-Flag, TOM20-Flag-SVG28, and TOM20-Flag-SVG28D2 were cloned into the C1 vector using Gibson assembly. The gene encoding IRES-Halo was subcloned into the C1 vector of TOM20-Flag, TOM20-Flag-SVG28, and TOM20-Flag-SVG28D2. All plasmids were cloned or subcloned using Gibson assembly, transformed into DH5α, and confirmed by DNA sequencing. Cell culture HeLa cells (ATCC CCL-2) were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Corning, 10-013-CV) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, F6178), and 1% penicillin-streptomycin (Pen-Strep; Thermo Fisher Scientific, 15140163). Cells were cultured at 37°C in a humidified incubator under 5% CO□. MD simulations The 7-mer structure of TOM20-Flag-SVG28D2 was modeled using the AlphaFold3 31 server with 40 oleic acid molecules to mimic membranous environment. Due to the undesirable predicted topology for TOM20-Flag-SVG28, its 7-mer structure was modeled using homology modeling 44 with TOM20-Flag-SVG28D2. Each structure was embedded in DOPC:Chol. (2:1 mol:mol) membranes with 1 M KCl using the CHARMM-GUI membrane builder 45 . The systems were initially equilibrated using the protocol provided by the CHARMM-GUI, followed by an additional 500 ps of equilibration, and subsequently simulated for 200 ns with the CHARMM36m force field. All simulations were executed with GROMACS-2024.1 46 . Cell-free synthesis DNA templates for CFPS were synthetic genes provided by Integrated DNA Technologies, USA. Purefrex2.0 (GeneFrontier, Japan) 47 was used for CFPS. Synthesis was performed at 37LJ for 6 hours. A reaction mixture containing 1 ng/µL DNA, 20 µL of solution I, 2 µL of solution II, 4 µL of solution III, and 10 µL of nuclease-free water was prepared. After the translation, the peptides were desalted and purified with GL-Tip SDB (GL Sciences, Japan). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was performed using the Autoflex Speed MALDI-TOF instrument (Bruker Daltonics Inc., Billerica, MA, USA) in linear positive mode. PEG6000 was used for external mass calibration. Lyophilized peptides obtained from 10 μL of the reaction mixture were dissolved in 3 μL of TA30 solution (acetonitrile:0.1% trifluoroacetic acid = 3:7, v/v). A matrix solution was prepared by dissolving 100 μg of α-cyano-4-hydroxycinnamic acid (CHCA) in 100 μL of TA30. The peptide sample and matrix were mixed at a 1:2 (v/v) ratio, and 1 μL of the mixture was spotted onto the MALDI target plate. Liposome-added cell-free synthesis Liposomes were prepared using the gentle hydration method. First, DOPC and DOPG dissolved in chloroform at 1-to-1 molar ratio were placed in a glass microtube and evaporated. The lipid films were hydrated with 500 µL of 0.5 M glucose for 3 hours at 40LJ. After the hydration, 200 µL of 0.5 M glucose and 100 mM HEPES (pH 7.4) were added to 200 µL of liposome suspension. The liposome solution was extruded using an extruder (Avanti Polar Lipids, USA) equipped with a 1.0 µm-pored membrane (Whatman, UK). A 20 µL of the CFPS reaction mixture described above was prepared with the addition of 10 µL of 10 mg/ml liposome solution, for a total volume of 30 µL. After the CFPS, the reaction mixture was left at 4LJ for approximately one week. Then, 150 µL of 50 mM HEPES was added to the PURE reaction solution, which was subsequently centrifuged at 20,000 xg for 5 min. The supernatant was carefully discarded after centrifugation. Another 150 µL of 50 mM HEPES was added to the solution, and then it was centrifuged again. The supernatant was completely removed, and the pellet was resuspended in 10 µL of 50 mM HEPES. A mixture of 10 µL of the liposome solution and 190 µL of 500 mM glucose and 50 mM HEPES was extruded using an extruder equipped with a 0.1 µm-pored membrane (Whatman, UK). Channel current measurement As shown in our previous reports, we prepared planar bilayer lipid membranes (BLMs) using the droplet contact method within a microdevice, with two 4.0 mm-diameter wells and a 100-µm-diameter pore for the droplet contact site in a 5 µm-thick parylene C (polychloro- p -xylylene, Parylene Japan, Japan) film ( Supplementary Fig. 4 ). Each well had a through-hole at the bottom containing Ag/AgCl electrodes. Two lipid monolayers containing the aqueous droplets came into contact through the small hole in the parylene film, forming lipid bilayers. First, 2.5 µL of 10 mg/ml DOPC:Cholesterol= 2:1 (mol:mol) in n -decane was added to both chambers. Then, 23 µL of solution containing 1 mg/ml liposome, 250 mM glucose, 25 mM HEPES, 1 M KCl, and 10 mM MOPS was added to the voltage-applied chamber, and 23 µL of solution containing 1 M KCl and 10 mM MOPS was added to the grounded chamber. The channel current was monitored using a JET patch-clamp amplifier (Tecella, USA). Signals were detected with a 4 kHz low-pass filter at a sampling frequency of 20 kHz. A constant voltage of +100 mV was applied to the recording chamber, and the ground side chamber was grounded. Data were analyzed using Clampfit 11.3 (Molecular Devices, USA), Excel (Microsoft, USA), and OriginPro 2022 (OriginLab Corporation, USA). To quantify pore-forming ability, current signals were classified into stable or unstable pore-formation signals 30 ; 48 . Stable pore formation is characterized by a sharp increase in signal rise in less than 10 ms and a 95% confidence interval of the pore current value that is 10 times less than the baseline, or by a sharp increase in signal rise in less than 10 ms with staircase-like current-time traces and a 95% confidence interval of the pore current value that is 10 times less than the baseline for each step ( Supplementary Fig. 5a ). Unstable pore formation is characterized by a sharp increase in signal rise in less than 10 ms and a 95% confidence interval of the pore current value that is 10 times greater than the baseline, or by a blunt increase in signal rise in more than 10 ms ( Supplementary Fig. 5b ). Signals longer than 5 s and greater than 0.2 nS were used for analysis. Signal distributions were calculated using n = 20 data for one measurement device. Mitochondrial Localization Assay HeLa cells per well were seeded per well into poly-D-lysin-coated 8-well glass chamber slides (Sigma, P6407-5MG, Cellvis, C8-1-N). Cells were transfected the following day with plasmids of TOM20-Flag, TOM20-Flag-SVG28, and TOM20-Flag-SVG28D2 using FUGENE HD (Promega, E2311), according to the manufacturer’s protocol. After 16 hours of transfection, cells were fixed and immunostained using primary antibodies against Flag (mouse, Millipore Sigma, F1804) and TOM20 (rabbit, Cell Signaling, 42406S). For imaging, an Eclipse Ti inverted fluorescence microscope (Nikon) equipped with a x60 oil-immersion objective lens was used. Mitochondrial membrane potential measurement To assess mitochondrial membrane potential, HeLa cells were seeded and transfected as described above. After 16 hours, cells were incubated with tetramethyl rhodamine ethyl ester (TMRE; Thermo Fisher) at a final concentration of 7 nM for 30 minutes at 37°C before live imaging. TMRE fluorescence was used as a measurement of mitochondrial inner membrane potential. As a positive control for membrane potential dissipation, cells were treated with the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at a final concentration of 10 μM. Live-cell imaging was performed for 10 minutes following TMRE staining, followed by an additional 15 minutes of imaging during CCCP treatment using an Eclipse Ti inverted fluorescence microscope equipped with a x60 oil-immersion objective. Mitochondrial ATP synthesis measurement To evaluate ATP production in mitochondria, a mitochondria-targeted FRET-based ATP biosensor, mitAT1.03 was used with CFP excitation light and collecting emission with YFP emission filter at 37°C. HeLa cells were seeded in 8-well glass-bottom chambers coated with poly-D-lysin and then were co-transfected with mitAT1.03 and either TOM20-Flag-IRES-Halo, TOM20-Flag-SVG28-IRES-Halo, or TOM20-Flag-SVG28D2-IRES-Halo using FuGene-HD. After transfection, live-cell imaging was performed at 37°C using excitation at the CFP wavelength and capturing emission through a YFP filter to measure FRET signal. For a control condition, cells were treated with 25 mM 2-deoxy-D-glucose (2-DG) to inhibit glycolysis and reduce ATP production. Fluorescence intensities from the CFP and FRET channels were quantified using FIJI (ImageJ), and the FRET/CFP ratio was calculated for each condition. Data represent the average of at least three biologically independent experiments. Su-9 at matrix mitochondria release To determine the function of pore membrane for protein release, Su9-mCherry was co-transfected with TOM20-Flag-IRES-Halo, TOM20-Flag-SVG28-IRES-Halo, or TOM20-Flag-SVG28D2-IRES-Halo in HeLa cells using FuGene-HD after cells seeded in 8-well glass-bottom chambers coated with poly-D-lysine. After transfection of 16 hours, live-cell imaging was performed at 37°C with 5% COL□. Fluorescence intensities of Su9-mCherry at mitochondria and cytosol were quantified using FIJI (ImageJ), and the Mitochondria/Cytosol ratio (Mito/Cyto ratio) was calculated for each condition. Data represent the average of at least three biologically independent experiments. Statistical analyses All data are mean ± SD, as indicated in the legends. Data for each condition were obtained from at least three independent biological replicate experiments. The two-tailed paired Student’s t-test was used to measure differences between the two groups. For multiple-group comparisons, one-way ANOVA was applied under the assumption of normal distribution. Detailed statistical methods are described in the individual figure legends. These statistical analyses were performed by using GraphPad Prism 8. A value of p <0.05 was considered significant. Funding JSPS KAKENHI Grant Number 21H05229, 25H00416, and JST-CREST JPMJCR21B2 for RK. NT is grateful to the Global Innovation Research Young Researcher International Travel Grant (FY2024), Institute of Global Innovation Research, Tokyo University of Agriculture and Technology (TUAT). Author Contributions TI and RK conceived the idea. NTHN initiated the project. NTHN, RK, and TI designed the experiments. NTHN performed the experiments and analyzed the data under the supervision of TI. SF and NT performed experiments and prepared the draft of the sections on MD simulations and electrophysiological measurements under the supervision of RK. NTHN wrote all the remaining manuscript in consultation with RK and TI. All the authors contributed to the final version of the manuscript. Competing Interests The authors declare no competing interests. Data Availability The datasets generated during this study are available from the corresponding author upon request. Source data are provided with this paper. Acknowledgement We thank members of the Inoue and Kawano laboratories for technical assistance and valuable discussions. In addition, we are grateful for the initial support from the Global Innovation Research (GIR) Program organized by the Tokyo University of Agriculture and Technology. Funder Information Declared Japan Science and Technology Agency, https://ror.org/00097mb19 , JPMJCR21B2 Japan Society for the Promotion of Science , 21H05229 , 25H00416 Reference 1. ↵ Wang Q , Yuan Y , Liu J , Li C , Jiang X. 2024 . The role of mitochondria in aging, cell death, and tumor immunity . Frontiers in Immunology . 15 : 1520072 . OpenUrl PubMed 2. Nguyen TT , Wei S , Nguyen TH , Jo Y , Zhang Y , Park W , Gariani K , Oh C-M , Kim HH , Ha K-T. 2023 . Mitochondria-associated programmed cell death as a therapeutic target for age-related disease . Experimental & Molecular Medicine . 55 ( 8 ): 1595 – 1619 . OpenUrl PubMed 3. Spinelli JB , Haigis MC . 2018 . The multifaceted contributions of mitochondria to cellular metabolism . Nature cell biology . 20 ( 7 ): 745 – 754 . OpenUrl CrossRef PubMed 4. Brand M , Orr A , Perevoshchikova I , Quinlan C. 2013 . The role of mitochondrial function and cellular bioenergetics in ageing and disease . British Journal of Dermatology . 169 ( 2):1-8 . 5. ↵ Ahola S. 2024 . Mitochondrial bioenergetics and metabolism: Implication for human health and disease . Frontiers in Molecular Biosciences . 11 : 1468758 . OpenUrl PubMed 6. ↵ Romanello V , Sandri M. 2021 . The connection between the dynamic remodeling of the mitochondrial network and the regulation of muscle mass . Cellular and Molecular Life Sciences . 78 ( 4 ): 1305 – 1328 . OpenUrl CrossRef PubMed 7. Yang Z , Wang L , Yang C , Pu S , Guo Z , Wu Q , Zhou Z , Zhao H. 2022 . Mitochondrial membrane remodeling . Frontiers in Bioengineering and Biotechnology . 9 : 786806 . OpenUrl 8. ↵ Quintana-Cabrera R , Scorrano L. 2023 . Determinants and outcomes of mitochondrial dynamics . Molecular cell . 83 ( 6 ): 857 – 876 . OpenUrl CrossRef PubMed 9. ↵ Khan T , Waseem R , Zehra Z , Aiman A , Bhardwaj P , Ansari J , Hassan MI , Islam A. 2022 . Mitochondrial dysfunction: Pathophysiology and mitochondria-targeted drug delivery approaches . Pharmaceutics . 14 ( 12 ): 2657 . OpenUrl PubMed 10. Wang W , Zhao F , Ma X , Perry G , Zhu X. 2020 . Mitochondria dysfunction in the pathogenesis of alzheimer’s disease: Recent advances . Molecular Neurodegeneration . 15 ( 1 ): 30 . OpenUrl PubMed 11. ↵ Wen H , Deng H , Li B , Chen J , Zhu J , Zhang X , Yoshida S , Zhou Y. 2025 . Mitochondrial diseases: From molecular mechanisms to therapeutic advances . Signal Transduction and Targeted Therapy . 10 ( 1 ): 9 . OpenUrl 12. ↵ Flores-Romero H , Dadsena S , García-Sáez AJ . 2023 . Mitochondrial pores at the crossroad between cell death and inflammatory signaling . Molecular Cell . 83 ( 6 ): 843 – 856 . OpenUrl CrossRef PubMed 13. Flores-Romero H , Ros U , Garcia-Saez AJ . 2020 . Pore formation in regulated cell death . The EMBO Journal . 39 ( 23 ): e105753 . OpenUrl CrossRef PubMed 14. ↵ Iranpour S , Arif M , Szegezdi E. 2025 . Disrupting membranes, controlling cell fate: The role of poreforming proteins in cell death and therapy . Apoptosis.1-28 . 15. ↵ Koneru JK , Prakashchand DD , Dube N , Ghosh P , Mondal J. 2021 . Spontaneous transmembrane pore formation by short-chain synthetic peptide . Biophysical Journal . 120 ( 20 ): 4557 – 4574 . OpenUrl PubMed 16. Tuerkova A , Kabelka I , Králová T , Sukeník L , Pokorná Š , Hof M , Vácha R. 2020 . Effect of helical kink in antimicrobial peptides on membrane pore formation . eLife . 9 : e47946 . OpenUrl PubMed 17. Lipkin R , Lazaridis T. 2017 . Computational studies of peptide-induced membrane pore formation . Philosophical Transactions of the Royal Society B: Biological Sciences . 372 ( 1726 ): 20160219 . OpenUrl CrossRef PubMed 18. ↵ Gilbert RJC . 2016 . Protein–lipid interactions and non-lamellar lipidic structures in membrane pore formation and membrane fusion . Biochimica et Biophysica Acta (BBA) -Biomembranes . 1858 ( 3 ): 487 – 499 . OpenUrl PubMed 19. ↵ Sánchez-Hernández R , Benítez-Angeles M , Hernández-Vega AM , Rosenbaum T. 2024 . Recent advances on the structure and the function relationships of the trpv4 ion channel . Channels . 18 ( 1 ): 2313323 . OpenUrl PubMed 20. Kwon DH , Zhang F , McCray BA , Feng S , Kumar M , Sullivan JM , Im W , Sumner CJ , Lee S-Y. 2023 . Trpv4rho gtpase complex structures reveal mechanisms of gating and disease . Nature Communications . 14 ( 1 ): 3732 . OpenUrl PubMed 21. Nadezhdin KD , Talyzina IA , Parthasarathy A , Neuberger A , Zhang DX , Sobolevsky AI . 2023 . Structure of human trpv4 in complex with gtpase rhoa . Nature Communications . 14 ( 1 ): 3733 . OpenUrl PubMed 22. ↵ Yu Z , Li J , Zhu J , Zhu M , Jiang F , Zhang J , Li Z , Zhong M , Kaye JB , D. J. 2014 . A synthetic transmembrane segment derived from trpv4 channel self-assembles into potassium-like channels to regulate vascular smooth muscle cell membrane potential . Journal of Materials Chemistry B . 2 ( 24 ): 3809 – 3818 . OpenUrl PubMed 23. Allolio C , Magarkar A , Jurkiewicz P , Baxová K , Javanainen M , Mason PE , Š achl R , Cebecauer M , Hof M , Horinek D. 2018 . Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore . Proceedings of the National Academy of Sciences . 115 ( 47 ): 11923 – 11928 . OpenUrl Abstract / FREE Full Text 24. Lunde PK , Manfra O , Støle TP , Lunde M , Martinsen M , Carlson CR , Louch WE . 2023 . Polyarginine cell-penetrating peptides bind and inhibit serca2 . Cells . 12 ( 19 ): 2358 . OpenUrl 25. ↵ Tomasello F , Messina A , Lartigue L , Schembri L , Medina C , Reina S , Thoraval D , Crouzet M , Ichas F , De Pinto V et al. 2009 . Outer membrane vdac1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis . Cell Research . 19 ( 12 ): 1363 – 1376 . OpenUrl CrossRef PubMed Web of Science 26. Hodge T , Colombini M. 1997 . Regulation of metabolite flux through voltage-gating of vdac channels . The Journal of membrane biology . 157 ( 3 ): 271 – 279 . OpenUrl CrossRef PubMed Web of Science 27. Ravi B , Kanwar P , Sanyal SK , Bheri M , Pandey GK . 2021 . Vdacs: An outlook on biochemical regulation and function in animal and plant systems . Frontiers in Physiology . 12 : 683920 . OpenUrl PubMed 28. ↵ Varughese JT , Buchanan SK , Pitt AS . 2021 . The role of voltage-dependent anion channel in mitochondrial dysfunction and human disease . Cells . 10 ( 7 ): 1737 . OpenUrl 29. ↵ Fujita S , Kawamura I , Kawano R. 2023 . Cell-free expression of de novo designed peptides that form β-barrel nanopores . ACS nano . 17 ( 4 ): 3358 – 3367 . OpenUrl PubMed 30. ↵ Shimizu K , Mijiddorj B , Usami M , Mizoguchi I , Yoshida S , Akayama S , Hamada Y , Ohyama A , Usui K , Kawamura I et al. 2022 . De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide . Nature Nanotechnology . 17 ( 1 ): 67 – 75 . OpenUrl PubMed 31. ↵ Abramson J , Adler J , Dunger J , Evans R , Green T , Pritzel A , Ronneberger O , Willmore L , Ballard AJ , Bambrick J. 2024 . Accurate structure prediction of biomolecular interactions with alphafold 3 . Nature . 630 ( 8016 ): 493 – 500 . OpenUrl CrossRef PubMed 32. ↵ Callegari S , Kirk NS , Gan ZY , Dite T , Cobbold SA , Leis A , Dagley LF , Glukhova A , Komander D. 2025 . Structure of human pink1 at a mitochondrial tom-vdac array . Science . 388 ( 6744 ): 303 – 310 . OpenUrl PubMed 33. ↵ Imamura H , Huynh Nhat KP , Togawa H , Saito K , Iino R , Kato-Yamada Y , Nagai T , Noji H. 2009 . Visualization of atp levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators . Proceedings of the National Academy of Sciences . 106 ( 37 ): 15651 – 15656 . OpenUrl Abstract / FREE Full Text 34. ↵ Miyamoto T , Uosaki H , Mizunoe Y , Han S-I , Goto S , Yamanaka D , Masuda M , Yoneyama Y , Nakamura H , Hattori N. 2021 . Rapid manipulation of mitochondrial morphology in a living cell with icmm . Cell Reports Methods . 1 ( 4 ). 35. Watanabe S , Nihongaki Y , Itoh K , Uyama T , Toda S , Watanabe S , Inoue T. 2022 . Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase a/acyltransferases . Nature communications . 13 ( 1 ): 4413 . OpenUrl PubMed 36. Nakamura H , Rho E , Lee CT , Itoh K , Deng D , Watanabe S , Razavi S , Matsubayashi HT , Zhu C , Jung E. 2023 . Actuator, a listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization . Cell reports . 42 ( 10 ). 37. ↵ Miyamoto T , Rho E , Kim A , Inoue T. 2018 . Cellular application of genetically encoded sensors and impeders of ampk . Ampk: Methods and protocols . Springer . p. 255 – 272 . 38. ↵ Han X , Maita N , Shimada A , Kohda D. 2022 . Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of tom20 . Protein Science . 31 ( 10 ): e4433 . OpenUrl PubMed 39. Yamamoto H , Itoh N , Kawano S , Yatsukawa Y-i , Momose T , Makio T , Matsunaga M , Yokota M , Esaki M , Shodai T. 2011 . Dual role of the receptor tom20 in specificity and efficiency of protein import into mitochondria . Proceedings of the National Academy of Sciences . 108 ( 1 ): 91 – 96 . OpenUrl Abstract / FREE Full Text 40. ↵ Kanaji S , Iwahashi J , Kida Y , Sakaguchi M , Mihara K. 2000 . Characterization of the signal that directs tom20 to the mitochondrial outer membrane . The Journal of cell biology . 151 ( 2 ): 277 – 288 . OpenUrl Abstract / FREE Full Text 41. ↵ Ernst P , Xu N , Qu J , Chen H , Goldberg MS , Darley-Usmar V , Zhang JJ , O’Rourke B , Liu X , Zhou L. 2019 . Precisely control mitochondria with light to manipulate cell fate decision . Biophysical journal . 117 ( 4 ): 631 – 645 . OpenUrl PubMed 42. ↵ Tkatch T , Greotti E , Baranauskas G , Pendin D , Roy S , Nita LI , Wettmarshausen J , Prigge M , Yizhar O , Shirihai OS . 2017 . Optogenetic control of mitochondrial metabolism and ca2+ signaling by mitochondria-targeted opsins . Proceedings of the National Academy of Sciences . 114 ( 26 ): E5167 – E5176 . OpenUrl Abstract / FREE Full Text 43. ↵ Deisseroth K , Hegemann P. 2017 . The form and function of channelrhodopsin . Science . 357 ( 6356 ): eaan5544 . OpenUrl Abstract / FREE Full Text 44. ↵ Waterhouse A , Bertoni M , Bienert S , Studer G , Tauriello G , Gumienny R , Heer FT , de Beer TAP , Rempfer C , Bordoli L. 2018 . Swiss-model: Homology modelling of protein structures and complexes . Nucleic acids research . 46 ( W1 ): W296 - W303 . OpenUrl CrossRef PubMed 45. ↵ Wu EL , Cheng X , Jo S , Rui H , Song KC , Dávila-Contreras EM , Qi Y , Lee J , Monje-Galvan V , Venable RM . 2014 . Charmm-gui membrane builder toward realistic biological membrane simulations . Wiley Online Library . 46. ↵ Van Der Spoel D , Lindahl E , Hess B , Groenhof G , Mark AE , Berendsen HJ . 2005 . Gromacs: Fast, flexible, and free . Journal of computational chemistry . 26 ( 16 ): 1701 – 1718 . OpenUrl CrossRef PubMed Web of Science 47. ↵ Shimizu Y , Inoue A , Tomari Y , Suzuki T , Yokogawa T , Nishikawa K , Ueda T. 2001 . Cell-free translation reconstituted with purified components . Nature biotechnology . 19 ( 8 ): 751 – 755 . OpenUrl CrossRef PubMed Web of Science 48. ↵ Sekiya Y , Sakashita S , Shimizu K , Usui K , Kawano R. 2018 . Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides . Analyst . 143 ( 15 ): 3540 – 3543 . OpenUrl View the discussion thread. Back to top Previous Next Posted August 27, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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Share Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation Nhung Thi Hong Nguyen , Shoko Fujita , Nanami Takeuchi , Ryuji Kawano , Takanari Inoue bioRxiv 2025.08.26.672490; doi: https://doi.org/10.1101/2025.08.26.672490 Share This Article: Copy Citation Tools Self-Assembling Synthetic Peptides Remodel Mitochondrial Properties via Nanopore Formation Nhung Thi Hong Nguyen , Shoko Fujita , Nanami Takeuchi , Ryuji Kawano , Takanari Inoue bioRxiv 2025.08.26.672490; doi: https://doi.org/10.1101/2025.08.26.672490 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Synthetic Biology Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25423) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24286) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88429) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)