Biology subjects

Lamb, D. C.

Publications and source records attributed to Lamb, D. C..

16 recordsLinked to original sources

bioRxiv2026

Developing a fluorescent derivative of GagPol as a tool for live-cell imaging of HIV-1 assembly

The HIV-1 assembly process is driven by the structural polyprotein Gag, which forms small cytosolic oligomers that traffic to the plasma membrane. While Gag alone is sufficient to drive particle formation, the incorporation of GagPol, a polyprotein comprising Gag and viral enzymes, is essential for productive infection. Maintenance of the proper Gag:GagPol ratio is crucial for virion infectivity. Yet, the mechanisms regulating their cytosolic interactions remain incompletely understood, in part because most studies rely on ensemble biochemical assays that hide cell-to-cell heterogeneity and lack spatial and temporal resolution. To overcome these limitations and investigate the dynamics of GagPol at the molecule level, we systematically tested different approaches to fluorescently label it. We successfully produced two functional fluorescent GagPol variants: one single-labeled GagPol, and a second double-labeled variant with a fluorescent protein added within Gag, that allows concurrent visualization of Gag and GagPol. These labeled versions, in combination with the use of raster image (cross-) correlation spectroscopy, enabled the quantification of Gag and GagPol relative concentrations and intermolecular interactions at the single-cell level. Overall, these variants set the stage for in-depth investigations of GagPol during HIV assembly providing insights into cytoplasmic trafficking, particle assembly, and the kinetics of these processes. ImportanceThe participation of Gag and GagPol in HIV-1 assembly is central to the generation of infectious virions. While the structural role of Gag and the enzymatic functions of GagPol are well described, little is known regarding the mechanisms underlying their cytosolic interactions, spatial organization, and relative incorporation into assembling particles. Here, we successfully developed two stable fluorescently labeled variants of GagPol; a single labeled variant with a label in the Pol domain and double-labeled construct in which the Gag domain is also tagged. Using these constructs, we can quantitatively investigate Gag and GagPol interactions on the single-cell level. Hence, they provide a framework for a comprehensive study of the oligomerization properties and trafficking of these polyproteins at different stages of HIV-1 assembly, including membrane recruitment and incorporation into nascent virions.

biophysics↗

bioRxiv2024

Resolving the Dynamic Interplay of the Conformational States in the TPR Domain of Human AMPylase FicD

The human Fic-enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum (ER): FicD reversibly modulates BiPs activity through attaching an adenosine monophosphate (AMP) to the substrate binding domain. This reduces BIPs chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, ATP-bound, and BiP-bound states revealed significant conformational variability in the tetratricopeptide repeat (TPR) motifs. In this study, we investigate the conformational dynamics of FicDs TPR motifs using two-color single-molecule Forster resonance energy transfer (smFRET). We demonstrate that the TPR motifs exhibit conformational dynamics between the TPR-out and TPR-in conformations on timescales ranging from milliseconds to microseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis (PDA) for the FRET constructs and propose a conformational landscape model for FicD where the TPR-in/out states exist in an equilibrium that is altered due to the presence of ATP and BiP.

biophysics↗

bioRxiv2024

Triple labeling resolves a GPCR intermediate state by 3-color single molecule FRET

The correlation of individual conformational changes in dynamic protein complexes remains challenging as most structural methods rely on averaged information over large numbers molecules. Single molecule FRET is a powerful tool for monitoring such conformational changes. When performed using three distinct probes, it enables the correlation of domain movements by providing up to three simultaneous distance measurements with high temporal resolution. Nevertheless, a major challenge lies in the site-specific attachment of three probes to unique positions within the target protein. Here, we propose an orthogonal triple-labeling strategy that is not compromised by native, reactive amino acid functionalities. It combines genetic code expansion and biorthogonal labeling of two different non-canonical amino-acids with an enzymatic self-labeling SNAP tag. We demonstrate its application by establishment of a 3-color sensor on the human metabotropic glutamate receptor 2, a dimeric, multidomain G protein-coupled neuroreceptor, and describe a previously unknown conformational intermediate state using 3-color single molecule FRET.

biophysics↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2023

Deep-Learning Assisted, Single-molecule Imaging analysis (Deep-LASI) of multi-color DNA Origami structures

Single-molecule experiments have changed the way we investigate the physical world but data analysis is typically time-consuming and prone to human bias. Here, we present Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software package consisting of an ensemble of deep neural networks to rapidly analyze single-, two- and three-color single-molecule data, in particular from single-molecule Forster Resonance Energy Transfer (FRET) experiments. Deep-LASI automatically sorts single molecule traces, determines FRET correction factors and classifies the state transitions of dynamic traces, all in ~20-100 ms per trajectory. We thoroughly benchmarked Deep-LASI using ground truth simulations as well as experimental data analyzed manually by an expert user and compared the results with a conventional Hidden Markov Model analysis. We illustrate the capabilities of the technique using a highly tunable L-shaped DNA origami structure and use Deep-LASI to perform titrations, analyze protein conformational dynamics and demonstrate its versatility for analyzing both total internal reflection fluorescence microscopy and confocal smFRET data.

biophysics↗

bioRxiv2022

mtHsp70 converts mitochondrial proteostasis distress into impaired protein import

Functional mitochondria are essential for cell viability and depend on protein import from the cytosol. Impaired protein import initiates various well-characterized cellular programs that rescue or remove dysfunctional mitochondria. However, the molecular mechanism that underlies the initial reduction of protein import into defective mitochondria remained unknown. Here, we found that the redistribution of mtHsp70, mitochondrial chaperone that is involved in both protein import and protein folding, regulates the efficiency of protein import. During early mitochondrial stress, before rescue programs are initiated and membrane potential is affected, mtHsp70-dependent import was specifically impaired and association of mtHsp70 with the import complex reduced. Even under non-stress conditions, the majority of mtHsp70 is found in a substrate-bound state. We propose that the availability of free mtHsp70 limits protein import into mitochondria during stress.

biochemistry↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗

bioRxiv2021

Nanoscale organization of the endogenous ASC speck

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to the formation of a supramolecular assembly of the inflammasome adaptor protein ASC, commonly referred to as the "ASC speck". Different models of the overall structure of the ASC speck, as well as the entire NLRP3 inflammasome, have been reported in the literature. While many experiments involve overexpression or in vitro reconstitution of recombinant ASC, the cytoplasmic endogenous ASC speck remains difficult to study due to its relatively small size and structural variability. Here, we use a combination of fluorescence imaging techniques including dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT) to visualize the endogenous ASC speck following NLRP3 inflammasome activation. We observe that the complex varies in diameter between [~]800 and 1000 nm and is composed of a dense core from which filaments extend into the periphery. We used a combination of anti-ASC antibodies as well as much smaller nanobodies for labeling, and find that the larger antibodies reliably label the lower-density periphery whereas the nanobody, which has a lower binding affinity, is more efficient in labeling the dense core. Imaging whole cells using dSTORM, furthermore, allowed us to sort the imaged structures into a pseudo-time sequence suggesting that the endogenous ASC speck becomes denser but not much larger during its formation. The reported results add an important piece of information towards a comprehensive understanding of the supramolecular structure of the endogenous inflammasome complex. SignificanceThe assembly of ASC into a supramolecular complex referred to as the ASC speck is a critical step in the NLRP3 inflammasome response. Despite significance research, the organization and formation of the endogenous ASC speck remain elusive. We used a combination of differently sized ASC labels with super-resolution microscopy to address the organization of the ASC speck at the nanoscale. Our results allow inference of the supramolecular structure of the unperturbed, endogenous speck and characterize how it changes during ASC recruitment. We find that the complex varies in size and is composed of a dense core from which filaments extend into the periphery. Notably, our results further indicate that during inflammasome formation, ASC specks become denser but only slightly larger.

immunology↗

bioRxiv2021

Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

pharmacology and toxicology↗

bioRxiv2020

Structural basis of rotavirus RNA chaperone displacement and RNA annealing

Rotavirus genomes are distributed between eleven distinct RNA molecules, all of which must be selectively co-packaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 auto-regulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, whilst accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch poised towards RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation whilst promoting inter-molecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

biophysics↗

bioRxiv2020

Tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: Is it still a virus?

Since the discovery of Acanthamoeba polyphaga Mimivirus, the first giant virus of amoeba, the historical hallmarks defining a virus have been challenged. Giant virion sizes can reach up to 2.3 {micro}m, making them visible by optical microscopy. They have large genomes of up to 2.5 Mb that encode proteins involved in the translation apparatus. Herein, we investigated possible energy production in Pandoravirus massiliensis, the largest of our giant virus collection. MitoTracker and TMRM mitochondrial membrane markers allowed for the detection of a membrane potential in virions that could be abolished by the use of the depolarizing agent CCCP. An attempt to identify enzymes involved in energy metabolism revealed that 8 predicted proteins of P. massiliensis exhibited low sequence identities with defined proteins involved in the universal tricarboxylic acid cycle (acetyl Co-A synthase; citrate synthase; aconitase; isocitrate dehydrogenase; -ketoglutarate decarboxylase; succinate dehydrogenase; fumarase). All 8 viral predicted ORFs were transcribed together during viral replication, mainly at the end of the replication cycle. Two of these proteins were detected in mature viral particles by proteomics. The product of the ORF132, a predicted protein of P. massiliensis, cloned and expressed in Escherichia coli, provided a functional isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle, which converts isocitrate to -ketoglutarate. We observed that membrane potential was enhanced by low concentrations of Acetyl-CoA, a regulator of the tricarboxylic acid cycle. Our findings show for the first time that energy production can occur in viruses, namely, pandoraviruses, and the involved enzymes are related to tricarboxylic acid cycle enzymes. The presence of a proton gradient in P. massiliensis coupled with the observation of genes of the tricarboxylic acid cycle make this virus a form a life for which it is legitimate to question what is a virus?.

microbiology↗

bioRxiv2020

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. MFS proteins are known to adopt different conformational states, yet details on the interconversion rates are crucially needed to understand or target their transport mechanism. Here, we studied the proton/multidrug antiporter LmrP as a model system for antibiotic resistance development in bacteria. The conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, yet the role of the transported substrate in these transitions has been puzzling. To measure LmrP structure in real-time, we performed solution-based single-molecule Forster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein interconverted between two structures at low rate (>>10 ms dwell time) at the cytosolic side while it interconverts dynamically between the 3 states (< 10 ms dwell time) at the extracellular side. When the Hoechst 33342 model substrate is added, inward conformational interconversions are greatly accelerated, coupled to an overall outward conformational halting, consistently with efficient proton exchange with the extracellular environment. Roxithromycin substrate binding did not halt but shift conformational interconversions from one pair of states to another. Substrate dependent structural heterogeneity is indicative of a general mechanism by which MFS transporters can efficiently transport a variety of substrates, and advocates for combined structure/dynamics-based drug design when targeting MDR transporters. BRIEF SUMMARYWe studied the conformational cycle of LmrP, a model for multidrug efflux pumps, using single-molecule Forster resonance energy transfer (smFRET). By following changes in FRET signal between different sets of positions, we specifically investigated how substrate binding modulates structural conversions between inward-open and outward-open states. Using a newly developed probabilistic analysis for describing sequential interconversion kinetics, we show that the apo protein slowly interconverts between defined states at the cytosolic and at the extracellular sides. Binding of the model substrate Hoechst33342 leads to an increase in conformational interconversions at the intracellular side while the extracellular side shows a drastic decrease in conversion, indicating a kinetic uncoupling between both sides. Remarkably, binding of roxithromycin, while also increasing interconversion on the intracellular side, did not slow the extracellular conversions. This indicates that multidrug pumps have evolved substrate-dependent transport mechanisms than enable transport of structurally diverse collection of substrates.

biophysics↗

bioRxiv2018

Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using Forster resonance energy transfer to study the conformational dynamics of a bimodular tandem cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. Our data reveal the existence of compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the inter-cohesin linker does not significantly alter the structural dynamics. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed but the cohesin-cohesin interactions persist. This suggests that the dockerin-binding interfaces are not involved in cohesin-cohesin interactions. The formation of cohesin-cohesin interactions is also observed in all-atom molecular dynamics simulations of the system. From the simulations, we identify possible inter-cohesin binding modes, none of which show obstruction of the cohesin-dockerin binding interfaces. Our results go beyond the view of scaffoldin as \"beads on a string\". We propose that both the flexibility and cohesin-cohesin interactions are important factors for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies. Hence, the flexibility of the linker region and cohesin-cohesin interactions should be considered when designing cellulosomes for industrial applications.

biophysics↗

bioRxiv2018

Stability of local secondary structure determines selectivity of viral RNA chaperones

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus NSP2 and avian reovirus {sigma}NS. Here, we compared the abilities of NSP2 and {sigma}NS to mediate sequence-specific interactions between rotavirus genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while {sigma}NS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric {sigma}NS oligomerises into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

biophysics↗

bioRxiv2018

Dimer arrangement and monomer flattening determine actin filament formation

Actin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

biophysics↗