PI(4,5)P2 binding sites in the Ebola virus matrix protein VP40 modulate assembly and budding.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P2. The PI(4,5)P2-induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P2. CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs.

Simulation-Guided Molecular Modeling of Nisin and Lipid II Assembly and Membrane Pore Formation.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming ß-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Distinctive Features of the XBB.1.5 and XBB.1.16 Spike Protein Receptor-Binding Domains and Their Roles in Conformational Changes and Angiotensin-Converting Enzyme 2 Binding.

The emergence and the high transmissibility of the XBB.1.5 and XBB.1.16 subvariants of the SARS-CoV-2 omicron has reignited concerns over the potential impact on vaccine efficacy for these and future variants. We investigated the roles of the XBB.1.5 and XBB.1.16 mutations on the structure of the spike protein's receptor-binding domain (RBD) and its interactions with the host cell receptor ACE2. To bind to ACE2, the RBD must transition from the closed-form to the open-form configuration. We found that the XBB variants have less stable closed-form structures that may make the transition to the open-form easier. We found that the mutations enhance the RBD-ACE2 interactions in XBB.1.16 compared to XBB.1.5. We observed significant structural changes in the loop and motif regions of the RBD, altering well-known antibody-binding sites and potentially rendering primary RBD-specific antibodies ineffective. Our findings elucidate how subtle structural changes and interactions contribute to the subvariants' fitness over their predecessors.

Lipid-specific oligomerization of the Marburg virus matrix protein VP40 is regulated by two distinct interfaces for virion assembly.

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein-protein or lipid-protein interactions to inhibit virus budding.

Ebola virus protein VP40 binding to Sec24c for transport to the plasma membrane.

Outbreaks of the Ebola virus (EBOV) continue to occur and while a vaccine and treatment are now available, there remains a dearth of options for those who become sick with EBOV disease. An understanding at the atomic and molecular level of the various steps in the EBOV replication cycle can provide molecular targets for disrupting the virus. An important step in the EBOV replication cycle is the transport of EBOV structural matrix VP40 protein molecules to the plasma membrane inner leaflet, which involves VP40 binding to the host cell's Sec24c protein. Though some VP40 residues involved in the binding are known, the molecular details of VP40-Sec24c binding are not known. We use various molecular computational techniques to investigate the molecular details of how EBOV VP40 binds with the Sec24c complex of the ESCRT-I pathway. We employed different docking programs to identify the VP40-binding site on Sec24c and then performed molecular dynamics simulations to determine the atomic details and binding interactions of the complex. We also investigated how the inter-protein interactions of the complex are affected upon mutations of VP40 amino acids in the Sec24c-binding region. Our results provide a molecular basis for understanding previous coimmunoprecipitation experimental studies. In addition, we found that VP40 can bind to a site on Sec24c that can also bind Sec23 and suggests that VP40 may use the COPII transport mechanism in a manner that may not need the Sec23 protein in order for VP40 to be transported to the plasma membrane.

Significance of the RBD mutations in the SARS-CoV-2 omicron: from spike opening to antibody escape and cell attachment.

We computationally investigated the role of the omicron RBD mutations on its structure and interactions with the surrounding domains in the spike trimer as well as with ACE2. Our results suggest that, compared to WT and delta, the mutations in the omicron RBD facilitate a more efficient RBD "down" to "up" conformation as well as ACE2 attachment. These effects, combined with antibody evasion, may have contributed to its dominance over delta.

Mutation-induced changes in the receptor-binding interface of the SARS-CoV-2 Delta variant B.1.617.2 and implications for immune evasion.

Following the initial surges of the Alpha (B.1.1.7) and the Beta (B.1.351) variants, a more infectious Delta variant (B.1.617.2) is now surging, further deepening the health crises caused by the pandemic. The sharp rise in cases attributed to the Delta variant has made it especially disturbing and is a variant of concern. Fortunately, current vaccines offer protection against known variants of concern, including the Delta variant. However, the Delta variant has exhibited some ability to dodge the immune system as it is found that neutralizing antibodies from prior infections or vaccines are less receptive to binding with the Delta spike protein. Here, we investigated the structural changes caused by the mutations in the Delta variant's receptor-binding interface and explored the effects on binding with the ACE2 receptor as well as with neutralizing antibodies. We find that the receptor-binding ß-loop-ß motif adopts an altered but stable conformation causing separation in some of the antibody binding epitopes. Our study shows reduced binding of neutralizing antibodies and provides a possible mechanism for the immune evasion exhibited by the Delta variant.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine.

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys224, 225 and Lys274, 275). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes.

Membrane pore formation and ion selectivity of the Ebola virus delta peptide.

The Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV encodes a glycoprotein that when cleaved, produces the delta peptide. Experimental evidence suggests that the delta peptide functions as a viroporin that enhances virus particle release through the host cell membrane. However, the viroporin forming mechanism of the delta peptide is still not well understood. Guided by experimental information, we have computationally investigated the pore formation by different oligomers of the delta peptide. We have performed all-atom molecular dynamics (MD) simulations in an explicit membrane environment to investigate the pore-forming mechanism and stability of the pores. Our results suggest that the delta peptide forms stable pentameric pores. In addition, the pore is selective with respect to chloride ions, and the disulfide bond formed between Cys-29 and Cys-38 in the C-terminal of the peptide is essential for the pore stabilization and ion permeation. Our study provides helpful information on the pore-forming mechanism of filovirus delta peptides and such structural information can be important in designing and developing molecular modulators that target the delta peptide pore and disrupt the pathology of the Ebola virus.

Molecular mechanisms of pore formation and membrane disruption by the antimicrobial lantibiotic peptide Mutacin 1140.

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle.

Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments.

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.

Cooperative structural transitions in amyloid-like aggregation.

Amyloid fibril aggregation is associated with several horrific diseases such as Alzheimer's, Creutzfeld-Jacob, diabetes, Parkinson's, and others. Although proteins that undergo aggregation vary widely in their primary structure, they all produce a cross-ß motif with the proteins in ß-strand conformations perpendicular to the fibril axis. The process of amyloid aggregation involves forming myriad different metastable intermediate aggregates. To better understand the molecular basis of the protein structural transitions and aggregation, we report on molecular dynamics (MD) computational studies on the formation of amyloid protofibrillar structures in the small model protein ccß, which undergoes many of the structural transitions of the larger, naturally occurring amyloid forming proteins. Two different structural transition processes involving hydrogen bonds are observed for aggregation into fibrils: the breaking of intrachain hydrogen bonds to allow ß-hairpin proteins to straighten, and the subsequent formation of interchain H-bonds during aggregation into amyloid fibrils. For our MD simulations, we found that the temperature dependence of these two different structural transition processes results in the existence of a temperature window that the ccß protein experiences during the process of forming protofibrillar structures. This temperature dependence allows us to investigate the dynamics on a molecular level. We report on the thermodynamics and cooperativity of the transformations. The structural transitions that occurred in a specific temperature window for ccß in our investigations may also occur in other amyloid forming proteins but with biochemical parameters controlling the dynamics rather than temperature.

The Ebola virus protein VP40 hexamer enhances the clustering of PI(4,5)P2 lipids in the plasma membrane.

The Ebola virus is a lipid-enveloped virus that obtains its lipid coat from the plasma membrane of the host cell it infects during the budding process. The Ebola virus protein VP40 localizes to the inner leaflet of the plasma membrane and forms the viral matrix, which provides the major structure for the Ebola virus particles. VP40 is initially a dimer that rearranges to a hexameric structure that mediates budding. VP40 hexamers and larger filaments have been shown to be stabilized by PI(4,5)P2 in the plasma membrane inner leaflet. Reduction in the plasma membrane levels of PI(4,5)P2 significantly reduce formation of VP40 oligomers and virus-like particles. We investigated the lipid-protein interactions in VP40 hexamers at the plasma membrane. We quantified lipid-lipid self-clustering by calculating the fractional interaction matrix and found that the VP40 hexamer significantly enhances the PI(4,5)P2 clustering. The radial pair distribution functions suggest a strong interaction between PI(4,5)P2 and the VP40 hexamer. The cationic Lys side chains are found to mediate the PIP2 clustering around the protein, with cholesterol filling the space between the interacting PIP2 molecules. These computational studies support recent experimental data and provide new insights into the mechanisms by which VP40 assembles at the plasma membrane inner leaflet, alters membrane curvature, and forms new virus-like particles.

Role of phosphatidic acid lipids on plasma membrane association of the Ebola virus matrix protein VP40.

The Ebola virus matrix protein VP40 is responsible for the formation of the viral matrix by localizing at the inner leaflet of the human plasma membrane (PM). Various lipid types, including PI(4,5)P2 (i.e. PIP2) and phosphatidylserine (PS), play active roles in this process. Specifically, the negatively charged headgroups of both PIP2 and PS interact with the basic residues of VP40 and stabilize it at the membrane surface, allowing for eventual egress. Phosphatidic acid (PA), resulting from the enzyme phospholipase D (PLD), is also known to play an active role in viral development. In this work, we performed a biophysical and computational analysis to investigate the effects of the presence of PA on the membrane localization and association of VP40. We used coarse-grained molecular dynamics simulations to quantify VP40 hexamer interactions with the inner leaflet of the PM. Analysis of the local distribution of lipids shows enhanced lipid clustering when PA is abundant in the membrane. We observed that PA lipids have a similar role to that of PS lipids in VP40 association due to the geometry and charge. Complementary experiments performed in cell culture demonstrate competition between VP40 and a canonical PA-binding protein for the PM. Also, inhibition of PA synthesis reduced the detectable budding of virus-like particles. These computational and experimental results provide new insights into the early stages of Ebola virus budding and the role that PA lipids have on the VP40-PM association.

Ebola Virus Matrix Protein VP40 Single Mutations G198R and G201R Significantly Enhance Plasma Membrane Localization.

Viral proteins frequently undergo single or multiple amino acid mutations during replication, which can significantly alter their functionality. The Ebola virus matrix protein VP40 is multifunctional but primarily responsible for creating the viral envelope by binding to the inner leaflet of the host cell plasma membrane (PM). Changes to the VP40 surface cationic charge via mutations can influence PM interactions, resulting in altered viral assembly and budding. A recent mutagenesis study evaluated the effects of several mutations and found that mutations G198R and G201R enhanced VP40 assembly at the PM and virus-like particle budding. These two mutations lie in the loop region of the C-terminal domain (CTD), which directly interacts with the PM. To understand the role of these mutations in PM localization at the molecular level, we performed both all-atom and coarse-grained molecular dynamics simulations using a dimer-dimer configuration of VP40, which contains the CTD-CTD interface. Our studies indicate that the location of mutations on the outer surface of the CTD regions can lead to changes in membrane binding orientation and degree of membrane penetration. Direct PI(4,5)P2 interactions with the mutated residues seem to further stabilize and pull VP40 into the PM, thereby enhancing interactions with numerous amino acids that were otherwise infrequently or completely inaccessible. These multiscale computational studies provide new insights at the atomic and molecular level as to how VP40-PM interactions are altered through single amino acid mutations. Given the high case fatality rates associated with Ebola virus disease in humans, it is essential to explore the mechanisms of viral assembly in the presence of mutations to mitigate the severity of the disease and understand the potential of future outbreaks.

Kinetics of peptide secondary structure conversion during amyloid ß-protein fibrillogenesis.

Amyloid fibrils are a common component in many debilitating human neurological diseases such as Alzheimer's (AD), Parkinson's, and Creutzfeldt-Jakob, and in animal diseases such as BSE. The role of fibrillar Αß proteins in AD has stimulated interest in the kinetics of Αß fibril formation. Kinetic models that include reaction pathways and rate parameters for the various stages of the process can be helpful towards understanding the dynamics on a molecular level. Based upon experimental data, we have developed a mathematical model for the reaction pathways and determined rate parameters for peptide secondary structural conversion and aggregation during the entire fibrillogenesis process from random coil to mature fibrils, including the molecular species that accelerate the conversions. The model and the rate parameters include different molecular structural stages in the nucleation and polymerization processes and the numerical solutions yield graphs of concentrations of different molecular species versus time that are in close agreement with experimental results. The model also allows for the calculation of the time-dependent increase in aggregate size. The calculated results agree well with experimental results, and allow differences in experimental conditions to be included in the calculations. The specific steps of the model and the rate constants that are determined by fitting to experimental data provide insight on the molecular species involved in the fibril formation process.

Lattice model simulations of the effects of the position of a peptide trigger segment on helix folding and dimerization.

The folding and dimerization of proteins is greatly facilitated by the presence of a trigger site, a segment of amino acids that has a higher propensity for forming α-helix structure as compared to the rest of the chain. In addition to the helical propensity of each chain, dimerization can also be facilitated by interhelical interactions such as saltbridges, and interfacial contacts of different strengths. In this work, we are interested in understanding the interplay of these interactions in a model peptide system. We investigate how these different interactions influence the kinetics of dimer formation and the stability of the fully formed dimer. We use lattice model computer simulations to investigate how the effectiveness of the trigger segment and its saltbridges depends on the location along the protein primary sequence. For different positions of the trigger segment, heat capacity and free energy of unfolded and folded configurations are calculated to study the thermodynamics of folding and dimerization. The kinetics of the process is investigated by calculating characteristic folding times. The thermodynamic and kinetic data from the simulations combine to show that the dimerization process of the model system is faster when the segment with high helical propensity is located near either end of the peptide, as compared to the middle of the chain. The dependence of the stability of the dimer on the trigger segment's position is also studied. The stability can play a role in the ability of the dimer to perform a biological function that involves partial unzipping. The results on folding and dimer stability provide important insights for designing proteins that involve trigger sites.

Detecting Individual Proteins and Their Surface Charge Variations in Solution by the Potentiometric Nanoimpact Method.

Label-free detection and analysis of proteins in their natural form and their dynamic interactions with substrates at the single-molecule level are important for both fundamental studies and various applications. Herein, we demonstrate a simple potentiometric method to achieve this goal by detecting the native charge of protein in solution by utilizing the principle of single-entity electrochemistry techniques. When a charged protein moves near the vicinity of a floating carbon nanoelectrode connected to a high-impedance voltage meter, the distinct local electrostatic potential changes induced by the transient collision event of protein, also called the "nanoimpact" event, can be captured by the nanoelectrode as a potential probe. This potentiometric method is highly sensitive for charged proteins, and low-molecular-weight proteins less than 10 kDa can be detected in low-salt-concentration electrolytes. By analyzing the shape and magnitude of the recorded time-resolved potential change and its time derivative, we can reveal the charge and motion of the protein in the nonspecific protein-surface interaction event. The charge polarity variations of the proteins at different pH values were also successfully probed. Compared with synthetic spherical nanoparticles, the statistical analysis of many single-molecule nanoimpact events revealed a large variation in the recorded transient potential signals, which may be attributed to the intrinsic protein dynamics and surface charge heterogeneity, as suggested by the finite element method and molecular dynamic simulations.