Biology subjects

Oliveira, A. S. F.

Publications and source records attributed to Oliveira, A. S. F..

16 recordsLinked to original sources

bioRxiv2026

Cancer-associated mutations reconfigure dynamical responses in EGFR kinase

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays a fundamental role in regulating cellular proliferation, survival, and differentiation. EGFR's kinase domain catalyses the autophosphorylation that drives downstream signalling and is a known hotspot for cancer-associated mutations. Such substitutions often enhance receptor activation, driving oncogenesis and therapeutic resistance, with major implications for prognosis and treatment choice; consequently, a detailed understanding of this domain's functional dynamics, and how mutations reshape them, is essential. Here, we apply dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to probe how changes at the ATP-binding site are transmitted throughout the kinase domain, ultimately shaping the behaviour of distal regions in the wild-type protein and in two of the most clinically observed non-small cell lung cancer variants, L858R and L858R+T790M. D-NEMD reveals previously uncharacterised communication networks linking the ATP pocket to key functional motifs, including the P-loop, C-helix, hinge region, activation loop, and F-helix, showing the intricate allosteric connectivity within this domain. Notably, numerous cancer-related mutation sites, despite being distant from the ATP site, map onto or lie close to these communication pathways, indicating that D-NEMD simulations can identify functionally relevant allosteric positions and potential mutation sites in EGFR. Our simulations further reveal that the two variants rewire EGFR's internal signal propagation network in distinct ways: L858R diminishes the transmission of structural changes to distal regions of the C-lobe, whereas introducing T790M in addition to L858R partially restores wild-type-like behaviour through compensatory rerouting of early-stage dynamical pathways.

biochemistry↗

bioRxiv2025

A dynamical-nonequilibrium molecular dynamics (D-NEMD) alanine scanning approach for identifying allosteric positions in proteins

Allosteric effects are widespread in proteins, but predicting the impact of sequence substitutions at positions distant from ligand-binding or enzyme active sites remains challenging, as their effects are mediated through complex dynamical networks. Pinpointing such positions experimentally is labour-intensive and low throughput. Equilibrium molecular dynamics (MD) simulations are useful: analysis methods can identify functional distal sites and networks, while free energy simulations can predict effects on stability or binding, given sufficient sampling; such simulations are typically time-consuming, requiring extensive simulation of each individual mutant. Here, we introduce a dynamical-nonequilibrium MD (D-NEMD) protocol using alanine substitutions as targeted perturbations to identify distal positions that influence enzyme function. Using the well characterised class A {beta}-lactamase KPC-2 as a test system, we show that D-NEMD distinguishes functional from non-functional sites based on whether substitutions generate structured, long-range responses that reach the active site. KPC-2 is clinically important due to its broad substrate spectrum and the emergence of resistance-associated point mutations, many of which act through non-local effects on catalytic residues. Alanine substitutions at positions 179 and 164, which disrupt the {Omega}-loop salt bridge, trigger persistent structural responses propagating through the protein and reach both catalytic residues and the oxyanion-hole backbone. Likewise, alanine substitution at position 220 elicits pronounced responses extending to the active-site {beta}-sheet and key loops, consistent with its known role in substrate specificity. In contrast, substitution at position 276--mutations of which have negligible kinetic impact--produces only local displacements with minimal propagation and no effect on catalytically relevant regions. These differential response patterns align with experimentally observed resistance phenotypes. D-NEMD therefore provides a fast, generalisable, and predictive approach for identifying allosterically connected distal positions. The protocol complements equilibrium MD, is straightforward to implement, and offers a tractable route for prioritising candidate sites for mechanistic study or future mutational scanning efforts. Statement of significanceWe introduce a dynamical-nonequilibrium MD (D-NEMD) protocol that uses alanine substitutions as the perturbation to identify residues, particularly those distal to an active or binding site, that modulate biological activity. We test this approach on the clinically important {beta}-lactamase KPC-2: it distinguishes positions that elicit long-range responses that reach catalytic residues from another with only local effects, in agreement with experiment. The D-NEMD approach allows for tests of statistical robustness of responses to mutation, enabling prioritisation of candidate residues for experimental mutation. This should aid in identifying distal sites that may influence enzyme activity or serve other allosteric roles. The approach is generalisable to other proteins of biomedical and biotechnological interest.

biochemistry↗

bioRxiv2025

pH-induced structural changes in SARS-CoV-2 spike variants

pH critically influences SARS-CoV-2 infectivity and stability by, for example, triggering pH-dependent conformational changes in the spike glycoprotein that can facilitate membrane fusion and viral entry into host cells. Using the emerging dynamical nonequilibrium molecular dynamics (D-NEMD) simulations approach, we investigated how biologically relevant pH shifts affect the functional dynamics of the fully glycosylated spike from the ancestral, Delta, and Omicron BA.1 variants of concern. For this, over 1100 nonequilibrium simulations were conducted to capture the pH-induced structural and dynamic changes that occur following transitions from physiological to acidic and alkaline conditions, with the former mimicking the low pH environment within endosomes, and the latter the high pH conditions accessible to nascent exhaled aerosols. D-NEMD reveals that pH changes trigger distinct, variant-specific conformational responses in key regions of the spike, including the receptor binding domain (RBD), fusion peptide proximal region (FPPR), and C-terminal domain (CTD). The ancestral spike shows broad pH sensitivity, characterised by directional motions and region-specific structural rearrangements that depend on the pH conditions. The spike of the Delta variant displays increased reactivity to alkaline pH, potentially explaining its reduced stability in alkaline aerosols. The spike of Omicron BA.1, in contrast, responds strongly to acidic conditions with spontaneous RBD opening and pronounced structural rearrangements in the FPPR and CTD. This behaviour aligns with this variants preference for endosomal entry and reduced reliance on TMPRSS2-mediated fusion. The Omicron BA.1 spike also shows increased resilience to alkaline pH, suggesting greater environmental stability. Our findings further emphasise the key role of glycans in spike activation, with glycan N234 stabilising the RBD "up" conformation during pH-induced transitions in Omicron under acidic conditions. These insights, together, highlight pH as a potential evolutionary pressure for SARS-CoV-2 and underscore the importance of glycosylation and environmental pH variability in shaping the behaviour of viral fusion proteins.

biochemistry↗

bioRxiv2025

How Varenicline Works: Identifying Critical Receptor and Ligand-based Interactions

Approved by the FDA in 2006, varenicline became the first nicotinic-based therapeutic for smoking cessation and has since been used by tens of millions of smokers worldwide. Varenicline works by targeting the 4{beta}2 nicotinic acetylcholine receptor (nAChR), the primary focus for nicotine addiction, where ligand recognition by the receptor triggers ion channel opening. While widely recognized that vareniclines development was rooted in the well-established pharmacology of cytisine, the two compounds display notably different profiles, not only at nAChRs, but also at key off-target sites such as the 5-HT3 serotonin receptor. Despite vareniclines widespread use and proven efficacy as a smoking cessation aid, our knowledge of the precise molecular mechanism underlying its action, particularly the specific receptor-ligand interactions that underpin its functional specificity, remains incomplete. Through a multidisciplinary approach that integrates complementary fields of research, this study reveals the critical receptor-ligand interactions that distinguish varenicline from related nAChR agonists, such as cytisine and nicotine. Our findings reveal previously unrecognized, critical hydrogen bonding interactions within the 4{beta}2 binding sites, specifically involving 4T139, 4T183, and {beta}2S133, that are uniquely and selectively engaged by varenicline. Of these, {beta}2S133 emerged as the pivotal determinant of vareniclines function, with substitution by valine significantly impairing the ligand efficacy. Furthermore, the design and synthesis of novel varenicline analogues shed new light into the functional importance of the ligands quinoxaline moiety, revealing that not just the presence but also the precise positioning of this hydrogen bond acceptor are critical for receptor activation by varenicline. Together, these findings uncover a previously uncharacterized interaction network essential for vareniclines function at 4{beta}2, offering a deeper and more comprehensive framework for understanding its distinct pharmacological profile while expanding our broader understanding of how ligand binding is translated into function in these receptors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/659675v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@a77e39org.highwire.dtl.DTLVardef@4fec37org.highwire.dtl.DTLVardef@11d26a2org.highwire.dtl.DTLVardef@d35640_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

Dynamical Responses Predict a Distal Site that Modulates Activity in an Antibiotic Resistance Enzyme

{beta}-Lactamases, which hydrolyse {beta}-lactam antibiotics, are key determinants of antibiotic resistance. Predicting the sites and effects of distal mutations in enzymes is challenging. For {beta}-lactamases, the ability to make such predictions would contribute to understanding activity against, and development of, antibiotics and inhibitors to combat resistance. Here, using dynamical non-equilibrium molecular dynamics (D-NEMD) simulations combined with experiments, we demonstrate that intramolecular communication networks differ in three class A SulpHydryl Variant (SHV)-type {beta}-lactamases). Differences in network architecture and correlated motions link to catalytic efficiency and {beta}-lactam substrate spectrum. Further, the simulations identify a distal residue 89 in the clinically important Klebsiella pneumoniae carbapenemase 2 (KPC-2), as a participant in similar networks, suggesting that mutation at this position would modulate enzyme activity. Experimental kinetics, biophysical and structural characterisation of the naturally occurring, but previously biochemically uncharacterised, KPC-2G89D mutant with several antibiotics and inhibitors reveals significant changes in hydrolytic spectrum, specifically reducing activity towards carbapenems without effecting major structural or stability changes. These results show that D-NEMD simulations can predict distal sites where mutation affects enzyme activity. This approach could have broad application in understanding enzyme evolution, and in engineering of natural and de novo enzymes.

biochemistry↗

bioRxiv2024

Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to unpick the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

biochemistry↗

bioRxiv2023

Substrate recognition and selectivity in SARS-CoV-2 main protease: Unveiling the role of subsite interactions through dynamical nonequilibrium molecular dynamics simulations

The main protease (Mpro) of the SARS-CoV-2 coronavirus employs a cysteine-histidine dyad in its active site to catalyse hydrolysis of the viral polyproteins. It is well established that binding of the substrate P1-Gln in the S1 subsite of Mpro active site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mpro dynamically recognises and responds to substrate binding remains difficult to probe by experimental methods. We thus employed the dynamical nonequilibrium molecular dynamics (D-NEMD) approach to probe the response of Mpro to systematic substrate variations. The results emphasise the importance of P1-Gln for initiating a productive enzymatic reaction. Specifically, substituting P1-Gln with alanine disrupts the conformations of the Cys145 and His41 dyad, causing Cys145 to transition from the productive gauche conformation to the non-productive trans conformation. Importantly, our findings indicate that Mpro exhibits dynamic responses to substrate binding and likely to substrate-mimicking inhibitors within each of the S4-S2' subsites. The results inform on the substrate selectivity requirements and shed light on the observed variations in hydrolytic efficiencies of Mpro towards different substrates. Some interactions between substrate residues and enzyme subsites involve more induced fit than others, implying that differences in functional group flexibility may optimise the binding of a substrate or inhibitor in a particular subsite.

biochemistry↗

bioRxiv2023

Allosteric modulation by the fatty acid site in the glycosylated SARS-CoV-2 spike

The trimeric spike protein plays an essential role in the SARS-CoV-2 virus lifecycle, facilitating virus entry through binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating viral-host membrane fusion. The SARS-CoV-2 spike contains a fatty acid (FA) binding site at the interface between two neighbouring receptor-binding domains. This site, also found in some other coronaviruses, binds free fatty acids such as linoleic acid. Binding at this site locks the spike in a non-infectious, closed conformation. This site is coupled to functionally important regions, but the effects of glycans on these allosteric effects have not been investigated. Understanding allostery and how this site modulates the behaviour of the spike protein could potentiate the development of promising alternative strategies for new coronavirus therapies. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to investigate allosteric effects of the FA site in the fully glycosylated spike of the original SARS-CoV-2 ancestral variant. The results show allosteric networks that connect the FA site to important functional regions of the protein, including some more than 40 [A] away, including the receptor binding motif, an antigenic supersite in the N-terminal domain, the furin cleavage site, regions surrounding the fusion peptide, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected connection between different allosteric sites. Notably, 65% of amino acid substitutions, deletions and insertions in the Alpha, Beta, Delta, Gamma and Omicron variants map onto or close to the identified allosteric pathways. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spikes revealed that the presence of glycans does not qualitatively change the internal allosteric pathways within the protein, with some glycans facilitating the transmission of the structural changes within and between subunits. Significance statementThe spike protein is crucial for the SARS-CoV-2 virus, enabling the fusion of the viral and host cell membranes. This protein contains several allosteric sites, including a fatty acid binding site at the interface between every two neighbouring receptor-binding domains. This site modulates the behaviour of the protein, with the binding of various free fatty acids and other small molecules influencing the spikes structure. In particular, the binding of linoleic acid, an essential fatty acid molecule, stabilizes the protein in a non-infectious locked conformation, thus making it inaccessible for binding to human receptors. Here, we investigate how the fatty acid site modulates the structural and dynamical behaviour of the fully glycosylated protein. Our work reveals complex patterns of communication between the fatty acid site and functionally important regions of the spike (including the receptor binding motif, the antigenic supersite in the N-terminal domain, the heme/biliverdin site, furin cleavage site and the fusion-peptide surrounding regions) and shed new light on the roles of glycans in this protein.

biochemistry↗

bioRxiv2022

Human cellular model systems of β-thalassemia enablein-depth analysis of disease phenotype

{beta}-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we created human disease cellular model systems for {beta}-thalassemia, which accurately recapitulate the phenotype of patient erythroid cells. We also developed a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilised the assay to demonstrate positive response of lines to verified reagents, providing validation for such applications. TMT-based comparative proteomics confirmed the same profile of proteins previously reported, whilst providing new insights into the altered molecular mechanisms in {beta}-thalassemia erythroid cells, with upregulation of a wide range of biological pathways and processes observed. Overall, the lines provide a sustainable supply of disease cells as novel research tools, for identifying new therapeutic targets, and as screening platforms for novel drugs and therapeutic reagents.

cell biology↗

bioRxiv2022

SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid

The SARS-CoV-2 spike protein contains a fatty acid binding site, also found in some other coronaviruses (e.g. SARS-CoV), which binds linoleic acid and is functionally important. When occupied by linoleic acid, it reduces infectivity, by locking the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. These simulations show that the fatty acid site is coupled to functional regions of the protein, some of them far from the site (e.g. in the receptor-binding motif, N-terminal domain, the furin cleavage site located in position 679-685 and the fusion peptide-surrounding regions) and identify the allosteric networks involved in these connections. Comparison of the response of the original ( Wuhan) spike with four variants: Alpha, Delta, Delta plus and Omicron BA.1 show that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the fatty acid site on Alpha are generally similar to the original protein, except for the receptor-binding motif and S71-R78 region which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629 and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, e.g. in differences in transmissibility and virulence. Experimental comparison of the effects of linoleic acid on different variants is warranted.

biochemistry↗

bioRxiv2022

Structural and temporal basis for agonism in the α4β2 nicotinic acetylcholine receptor

Despite decades of study, the structural mechanisms underpinning agonist efficacy in pentameric ligand-gated ion channels remain poorly understood. Here, a combination of extensive equilibrium and dynamical-nonequilibrium molecular dynamics simulations was used to obtain a detailed description of the structural and dynamic changes induced within the human 4{beta}2 nicotinic acetylcholine receptor by a full and a partial agonist, namely acetylcholine and nicotine, and map how these rearrangements propagate within this receptor. These simulations reveal how the agonists modulate the patterns associated with intra and inter-domain communication and the evolution of the agonist-specific structural rearrangements. For the first time, we show that full and partial agonists, although generally using similar routes for through-receptor signal transmission, induce different amplitudes of conformational rearrangements in key functional motifs, thus impacting the rates of signal propagation within the protein. The largest agonist-induced conformational differences are located in the Cys loop, loops C and 1-{beta}1 in the 4 subunit, loops F and {beta}1-{beta}2 in the {beta}2 subunit and in the extracellular selectivity filter.

biochemistry↗

bioRxiv2021

Identification and Validation of Novel Microtubule Suppressors with an Imidazopyridine Scaffold through Structure Based Virtual Screening and Docking.

Targeting the colchicine binding site of /{beta} tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, the development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure-based pharmacophore approach and docking using three programs MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-Hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b] pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6.1 {+/-}0.1 M), inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.

cancer biology↗

bioRxiv2021

Structure and mechanism of SARS-CoV-2 Spike N679-V687 deletion variant elucidate cell-type specific evolution of viral fitness

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriS{Delta} variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

biochemistry↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗

bioRxiv2019

Exploration of the role of CHRNA5-A3-B4 genotype in smoking behaviours

Genome-wide association studies have identified associations between variation at rs16969968/rs1051730 in the CHRNA5-A3-B4 gene cluster and smoking related outcomes. Experiments in rodents have described the nicotinic acetylcholine receptors (nAChRs) subunits encoded by this gene cluster and showed a lack of nicotine aversion in nAChRs deficient animal models. We conducted a nicotine challenge and a smoking topography study in humans, hypothesising that: 1. responses to a nicotine challenge would differ according to the rs16969968/rs1051730 genotype and 2. genotype may influence nicotine intake via smoking topography.\n\nWe used linear regressions to examine associations between rs16969968/rs1051730 genotype and subjective (questionnaires) and objective (physiological parameters) responses following acute nicotine exposure in never smokers (hypothesis 1) or cigarette smoking in current smokers (hypothesis 2). There was evidence to suggest nicotine exposure increases blood pressure and heart rate, and negatively affects mood, but insufficient evidence that these effects differ by genotype. Carriers of the minor allele following smoking one cigarette, exhibited reduced cravings (b=-2.46, 95% CI -4.87 to - 0.06, p=0.04) and inhaled less smoke per cigarette (b=-0.24, 95% CI - 0.43 to - 0.06, p=0.01) and per puff (b=-0.18, 95% CI -0.32 to -0.01, p=0.02). These results suggest that we need to carefully consider the translational value of the findings of aversion behaviour in nAChRs rodent models, and that deeper inhalation does not explain the strong association between rs16969968/rs1051730 genotype and objective biomarkers of tobacco exposure.

neuroscience↗

bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗