RESUMEN
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
Asunto(s)
Amidas , Proteínas de Unión Periplasmáticas , Amidas/metabolismo , Amidas/química , Cristalografía por Rayos X , Proteínas de Unión Periplasmáticas/metabolismo , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Aminoácidos/metabolismo , Mesorhizobium/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Modelos Moleculares , Amidohidrolasas/metabolismo , Amidohidrolasas/química , Calcio/metabolismo , Unión ProteicaRESUMEN
WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by sas16 (P450Sas) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450Sas-mediated α,ß-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate (Z)-2-pent-1'-enyl-cinnamoyl-Thr-N-Me-Tyr. We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS, and further that P450Sas appears to be specific for substrates containing the (Z)-2-pent-1'-enyl-cinnamoyl group. A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates, including the substitution of the canonical active site alcohol residue with a phenylalanine (F250), which in turn is critical to P450Sas activity and WS9326A biosynthesis. Together, our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate, thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.
RESUMEN
Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.
Asunto(s)
Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas , Péptido Sintasas/química , Dominio Catalítico , Péptidos/metabolismo , PanteteínaRESUMEN
The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.
Asunto(s)
Burkholderiales , Hidrolasas , Hidrolasas/química , Burkholderiales/genética , Burkholderiales/metabolismo , Dominio Catalítico , Mutación , Tereftalatos Polietilenos/metabolismoRESUMEN
The emergence of oligomers is common during the evolution and diversification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head-to-head interfaces (e.g., in symmetrical dimers) or heterologous head-to-tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer-interfaces into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants and that trimerization corresponds with a reduction in the KM value of the enzyme from a promiscuous level to the physiologically relevant range. In addition, we find that removal of a C-terminal extension present in PaCDT leads to a variant with reduced catalytic activity, indicating that the C-terminal region has a role in tuning enzymatic activity. We show that these observations can be rationalized at the structural and dynamic levels, with trimerization and C-terminal extension leading to reduced sampling of non-catalytic conformational substates in molecular dynamics simulations. Overall, this work provides insight into how neutral sampling of distinct oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.
Asunto(s)
Simulación de Dinámica Molecular , Prefenato Deshidratasa , Prefenato Deshidratasa/química , Prefenato Deshidratasa/genética , Prefenato Deshidratasa/metabolismo , Pseudomonas aeruginosa , Conformación Molecular , Multimerización de ProteínaRESUMEN
Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera's activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen-deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.
Asunto(s)
Regulación Alostérica , Calmodulina , Glucosa Deshidrogenasas , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión , Calmodulina/química , Calmodulina/genética , Glucosa Deshidrogenasas/química , Glucosa Deshidrogenasas/genética , Ligandos , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , TermodinámicaRESUMEN
In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families. As ASR becomes more widely adopted as a protein engineering approach, it is important to understand the applications, limitations, and recent developments of this technique. This review highlights recent exemplifications of ASR, as well as technical aspects of the reconstruction process that are relevant to protein engineering.
Asunto(s)
Evolución Molecular , Proteínas , Evolución Biológica , Humanos , Filogenia , Ingeniería de Proteínas , Proteínas/genéticaRESUMEN
Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Asunto(s)
Aminoácidos/química , Dominio Catalítico , Péptido Sintasas/química , Péptidos/química , Sideróforos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Coenzima A/química , Cristalografía por Rayos X , Expresión Génica , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Dominios Proteicos , Estructura Terciaria de Proteína , Alineación de Secuencia , Sideróforos/biosíntesis , Especificidad por Sustrato , Thermobifida/química , Thermobifida/metabolismoRESUMEN
The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.
Asunto(s)
Factor XIIa/antagonistas & inhibidores , Péptidos Cíclicos/química , ARN Mensajero/metabolismo , Inhibidores de Serina Proteinasa/química , Sitios de Unión , Factor XIIa/metabolismo , Biblioteca de Genes , Código Genético , Humanos , Concentración 50 Inhibidora , Calicreínas/química , Calicreínas/metabolismo , Simulación de Dinámica Molecular , Tiempo de Tromboplastina Parcial , Péptidos Cíclicos/metabolismo , Estabilidad Proteica , Tiempo de Protrombina , Puromicina/química , ARN Mensajero/química , Inhibidores de Serina Proteinasa/metabolismo , Relación Estructura-ActividadRESUMEN
Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSPRepeat) can protect against malaria. However, it has also been suggested that the CSPRepeat is a decoy that prevents the immune system from mounting responses against other domains of CSP. Here, we show that, following parasite immunization, B cell responses to the CSPRepeat are immunodominant over responses to other CSP domains despite the presence of similar numbers of naive B cells able to bind these regions. We find that this immunodominance is driven by avid binding of the CSPRepeat to cognate B cells that are able to expand at the expense of B cells with other specificities. We further show that mice immunized with repeat-truncated CSP molecules develop responses to subdominant epitopes and are protected against malaria. These data demonstrate that the CSPRepeat functions as a decoy, but truncated CSP molecules may be an approach for malaria vaccination.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Inmunización/métodos , Vacunas contra la Malaria/administración & dosificación , Malaria/prevención & control , Péptidos/administración & dosificación , Plasmodium berghei/efectos de los fármacos , Proteínas Protozoarias/genética , Animales , Anopheles/parasitología , Anticuerpos Neutralizantes/biosíntesis , Linfocitos B/inmunología , Linfocitos B/parasitología , Femenino , Expresión Génica , Malaria/inmunología , Malaria/parasitología , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Péptidos/inmunología , Plasmodium berghei/inmunología , Plasmodium berghei/patogenicidad , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/inmunología , Plasmodium falciparum/patogenicidad , Unión Proteica , Proteínas Protozoarias/inmunología , Esporozoítos/inmunología , Esporozoítos/efectos de la radiación , Transgenes , Vacunas AtenuadasRESUMEN
Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Evolución Molecular , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catálisis , Dominio Catalítico , Enzimas/genética , Modelos Moleculares , Mutación , Filogenia , Prefenato Deshidratasa/química , Prefenato Deshidratasa/genética , Prefenato Deshidratasa/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP, and ATP with micromolar-range affinities. X-ray crystal structures of apo and nucleotide-bound SbtB7001 revealed that while AMP, ADP, and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop, and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased 10-fold in the presence of Ca2+, and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bicarbonatos/metabolismo , Cianobacterias , Nucleótidos de Adenina/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.
Asunto(s)
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Vías Biosintéticas/genética , Glicopéptidos/biosíntesis , Péptidos/metabolismo , Actinobacteria/genética , Antibacterianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Ciclización/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Glicopéptidos/química , Familia de Multigenes , Péptidos/químicaRESUMEN
Biosensors that selectively report on the presence of specific small molecule analytes have applications in many fields of research, medicine and biotechnology. Here, we review recent advances and emerging approaches in the design and optimisation of genetically encoded fluorescence-based small molecule biosensors. We discuss how natural sensory proteins can be exploited to produce novel biosensors and the strategies for optimizing ligand specificity and fluorescence readout. Finally, we provide insight into high-throughput sensor optimisation and discuss the challenges that are faced when designing novel biosensors.
Asunto(s)
Técnicas Biosensibles/métodos , Evolución Molecular Dirigida/métodos , Fluorescencia , Bibliotecas de Moléculas Pequeñas/análisis , Ingeniería de ProteínasRESUMEN
The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with ß-alanine. Ebony is a rare example of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine-N-acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic example of an alternative type of NRPS condensation domain; they also illustrate how the catalytic components of such assembly lines are significantly more diverse than a minimal set of conserved functional domains.
Asunto(s)
N-Acetiltransferasa de Arilalquilamina/química , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Péptido Sintasas/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Drosophila melanogaster/química , Dominios Proteicos , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme-substrate complementarity and transition-state stabilization and minimized sampling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent examples of computational enzyme design and directed evolution.
Asunto(s)
Escherichia coli/enzimología , Prefenato Deshidratasa/química , Prefenato Deshidratasa/genética , Proteínas Portadoras , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Análisis Mutacional de ADN , Evolución Molecular , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis , Mutación , Oligonucleótidos/genética , Filogenia , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Linfocitos B/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T/inmunología , Animales , Afinidad de Anticuerpos , Cristalografía por Rayos X , Femenino , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plasmodium falciparum/química , Plasmodium falciparum/genética , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Eukaryotic voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in electrically excitable cells, and are important pharmaceutical targets for the treatment of neurological disorders such as epilepsy, cardiac arrhythmias, and chronic pain. Evidence suggests that small, hydrophobic, VGSC-blocking drugs can gain access to binding residues within the central cavity of these channels by passing through lateral, lipid-filled "fenestrations" which run between the exterior of the protein and its central pore. Here, we use molecular dynamics simulations to investigate how the size and shape of fenestrations change over time in several bacterial VGSC models and a homology model of Nav1.4. We show that over the course of the simulations, the size of the fenestrations is primarily influenced by rapid protein motions, such as amino acid side-chain rotation, and highlight that differences between fenestration bottleneck-contributing residues are the primary cause of variations in fenestration size between the 6 bacterial models. In the eukaryotic channel model, 2 fenestrations are wide, but 2 are narrow due to differences in the amino acid sequence in the 4 domains. Lipid molecules are found to influence the size of the fenestrations by protruding acylchains into the fenestrations and displacing amino acid side-chains. Together, the results suggest that fenestrations provide viable pathways for small, flexible, hydrophobic drugs.