Biology subjects

Craggs, T. D.

Publications and source records attributed to Craggs, T. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Fluorescence cross-correlation spectroscopy quantifies affinity, cooperativity, and kinetic stability in ternary protein complexes

Many biological processes and emerging therapeutic modalities rely on higher-order protein complexes whose properties cannot be predicted from their constituent binary interactions. However, methods for directly quantifying affinity, cooperativity, and kinetic stability within such assemblies remain limited. Here, we establish fluorescence cross-correlation spectroscopy (FCCS) as a solution-phase approach for characterizing multicomponent protein interactions and apply it to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab. Using fluorescently labelled HER2, trastuzumab, and pertuzumab, we quantified binary binding affinities, directly measured ternary complex formation, and characterized the dissociation kinetics of binary and ternary complexes. FCCS measurements revealed positive cooperativity in the formation of the HER2-trastuzumab-pertuzumab ternary complex, while dissociation experiments demonstrated that the ternary complex is kinetically more stable than the corresponding binary interactions. Together, these findings provide direct solution-phase evidence that cooperative interactions stabilize the HER2-trastuzumab-pertuzumab complex, offering a molecular explanation for the enhanced efficacy of dual HER2 targeting in cancer therapy. More broadly, this work demonstrates that FCCS can robustly quantify affinity, cooperativity, and kinetic stability of multicomponent protein complexes using a commercially available platform. We provide a broadly accessible framework for studying higher-order protein interactions and supporting the development of next-generation multispecific and combination therapeutics. Significance StatementMany proteins function as part of multicomponent complexes, yet most experimental methods characterize interactions only one pair at a time, or indirectly through secondary reporters, or necessitating saturation of binary interactions first. We show that fluorescence cross-correlation spectroscopy can directly quantify how multiple binding partners interact simultaneously by measuring affinity, cooperativity, and kinetic stability in solution. Applying this approach to the clinically important HER2-targeting antibodies trastuzumab and pertuzumab reveals cooperative stabilization of their ternary complex. Because the measurements are performed on a commercially available instrument and require no surface immobilization, this broadly accessible method should facilitate mechanistic studies of complex biomolecular interactions and aid the development of multispecific and combination therapeutics.

biophysics↗

bioRxiv2026

DNA damage induces long range changes to duplex structure - a non-protein start to damage detection?

DNA-binding proteins must quickly locate specific sites on DNA to enable replication, repair, and transcription. While sequence-specific recognition is well understood, the physical basis of structure-specific recognition remains unclear, limiting our understanding of DNA damage repair. Proteins must distinguish damaged sites within largely undamaged DNA; however, studying this is challenging due to DNAs dynamic nature. We hypothesised that DNA damage causes changes in DNA structure, signalling protein recruitment. Using confocal single-molecule FRET, we analysed seven DNA duplexes containing modifications such as ribonucleotide, 8-oxoguanine (8-oxoG), abasic sites, nicks, and gaps, which are all involved in the base excision repair (BER) pathway. Each construct was measured with nine dye pairs in triplicate to capture changes in bending, twisting, and stretching. An automated analysis pipeline processed 162 measurements, enabling rigorous statistical comparisons. All modifications altered FRET efficiencies compared to undamaged DNA, including the subtlest change: a single oxygen difference (ribo-vs deoxyribonucleotide). Abasic sites, nicks, and gaps had the greatest effects. These findings provide direct evidence that DNA damage affects duplex structure and dynamics beyond the lesion site, suggesting DNA flexibility changes may act as an early signal for repair protein recruitment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/709887v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@a85839org.highwire.dtl.DTLVardef@3813dborg.highwire.dtl.DTLVardef@19fa06aorg.highwire.dtl.DTLVardef@dc9729_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

A universal protein ladder for standardisation of diverse FRET assays

Fluorescence resonance energy transfer (FRET) is the highly distance dependent (3-10 nm) transfer of energy from a donor to an acceptor fluorophore, with transfer efficiency inversely proportional to the distance between the fluorophores. Consequently FRET serves as a powerful spectroscopic ruler for probing molecular interactions. Whilst cell based FRET assays report bulk relative changes in FRET efficiency in a population, single molecule FRET (smFRET) is capable of deconvoluting these population averages into distinct structural states. However, the lack of universal benchmarks prevents the direct translation of in vitro distance measurements to the intracellular environment and vice versa. Here, we present a modular protein ladder designed to harmonize FRET data across diverse platforms. Using an engineered repeating TPR motif and self-labeling enzymes, we demonstrate that our standards yield consistent FRET efficiencies across expression systems (mammalian and bacterial) and labelling strategies (self labelling enzymes and click chemistry with non-canonical amino acids). By providing a predictable calibration curve, the ladder enables interpolation between different experimental FRET modalities, including confocal smFRET, flow cytometry based-FRET and Fluorescence Lifetime Imaging Microscopy FRET (FLIM-FRET). This is the necessary infrastructure to relate molecular distances from the test tube to the cell.

biophysics↗

bioRxiv2026

RNA i-Motif Formation at Neutral pH

i-Motifs are non-canonical nucleic acid secondary structures formed in sequences rich in cytosine. Previous work has shown that DNA i-motifs may form at neutral pH and provided evidence to suggest they influence biological functions. RNA i-motifs are less stable than DNA i-motifs, so more questions surround the biological relevance of these structures in RNA. Using biophysical methods, we found that increasing cytosine tract lengths resulted in increased thermal stability but not pH stability. In ensemble solution experiments RNA i-motifs appear globally unfolded at neutral pH. However, single molecule experiments revealed that 1% of RNA i-motifs remain folded in solution. This has implications for the potential of formation of RNA i-motifs in cells.

biophysics↗

bioRxiv2025

The interplay between supercoiling and DNA modifying enzymes at the single-molecule level

DNA supercoiling refers to the overwinding or underwinding of the double helix, forming plait-like structures called plectonemes. Supercoiling is involved in all biological processes that occur on DNA. Despite this involvement, single-molecule in vitro studies on these processes largely work with relaxed DNA, overlooking the role supercoiling is playing in these processes. We have constructed 18-kb linear DNA substrates that can be tethered to a microscope coverslip at multiple sites, allowing the template to be topologically constrained. We can then use an intercalating dye to induce positive or negative supercoiling, which we can observe at the single-molecule level using TIRF microscopy. We directly visualise plectonemes diffusing along the DNA. However, in the presence of the restriction enzyme EcoRV, plectonemes remain stationary at the EcoRV binding site. We show that EcoRV has an increased cleavage efficiency in the presence of supercoiling, even though its binding efficiency is unaffected. These findings reveal a relationship between DNA topology and the activity of DNA modifying enzymes. We hypothesise that supercoiling makes cleavage more energetically favourable for restriction enzymes that bend DNA. This work reveals the kinetic interplay between supercoiling and enzyme activity, highlighting how DNA itself can modulate the processes that act upon it. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/680164v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a9890forg.highwire.dtl.DTLVardef@1653ecaorg.highwire.dtl.DTLVardef@1fd7bfcorg.highwire.dtl.DTLVardef@d70357_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A DNA scaffold approach facilitates 5' labelling of the SARS-CoV-2 RNA pseudoknot for smFRET investigation

Single-molecule Forster resonance energy transfer (smFRET) studies of highly structured RNA molecules are often frustrated by issues with efficient dye conjugation. Here, we develop a DNA scaffold-based labelling strategy, and apply it to the frameshift-stimulating RNA pseudoknot from the SARS-CoV-2 genome. We prepare FRET-active reporters containing both Cy3 (donor) and Cy5 (acceptor) molecules and conduct measurements on freely diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. We identify that freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl-dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X-ray and cryo-EM structures. This work is a proof-of-principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches towards the study of RNA conformational dynamics in pseudoknots, riboswitches and other structured RNA elements.

biophysics↗

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2025

Structural basis of multitasking by the apicoplast DNA polymerase from Plasmodium falciparum

Plasmodium falciparum is a unicellular eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, which is replicated by a dedicated apicoplast replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol) for copying the apicoplast DNA. Being the only DNA polymerase in the apicoplast, apPol is expected to multitask, catalysing both replicative and lesion bypass synthesis. Replicative synthesis typically relies on a restrictive active site for high accuracy while lesion bypass requires an open active site. This raises the question how does apPol combine the structural features of multiple DNA polymerases in a single protein. Using single particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its DNA and nucleotide substrates in five pre-chemistry conformational states, allowing us to describe the events leading up to nucleotide incorporation and answer how apPol incorporates features of multiple polymerases. We found that, unlike most replicative polymerases, apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers open state we identified a nascent base pair checkpoint that can preferentially select a Watson-Crick base pair, an essential requirement for replicative synthesis. Taken together these structural features explain how apPol may balance replicative and lesion bypass synthesis.

biochemistry↗

bioRxiv2024

Kinesin-1 is highly flexible and adopts an open conformation in the absence of cargo

Kinesin-1 is an essential anterograde microtubule motor protein. The core kinesin motor is a homodimer of two heavy chains; N-terminal motor domains hydrolyse ATP and walk along microtubules, whilst a long elongated coiled-coil stalk and an intrinsically disordered C-terminal tail region bind cargos. Kinesin autoinhibition is key to preventing futile ATP consumption and occurs, at least in part, through direct interactions between N-terminal motor domains and C-terminal inhibitory motifs. Despite significant advances in our understanding of kinesin walking, little is known about the kinesin-1 conformational landscape of the stalk and tail domains. Here we apply solution based biophysical analysis tools to study conformational changes in kinesin-1, with full rotational freedom, and in response to changes in ionic strength, mutations, and the presence of microtubules. This has allowed us to uncover the inherent flexibility in kinesin-1 which gives insights into autoinhibition and the regulation of intracellular transport.

biophysics↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

bioRxiv2024

Cooperation between Intrinsically Disordered Regions regulates CBP condensate behaviour.

Intrinsically disordered regions (IDRs) have emerged as crucial regulators of protein function, allowing proteins to sense and respond to their environment. Creb binding protein (CBP) and EP300 (p300) are transcription coactivators that regulate gene expression in multicellular organisms, following their recruitment to cis-regulatory elements. CBP and p300 contain large IDRs, however little is known about how these different IDRs work together to regulate CBP function. Here, we show that CBP-IDRs cooperate to control different aspects of CBP behaviour in the nucleus, by regulating the properties of fluid-like condensates formed by endogenous CBP. We show how IDRs with different sequence properties make unique contributions to CBP behaviour by establishing a balance between positive and negative regulation of CBP condensates. These conflicting interactions are functionally important, shaping CBPs response to factors such as lysine acetylation, that influence condensate formation. When disrupted, regulatory CBP-IDRs change how CBP interacts with chromatin, alter patterns of CBP-dependent histone acetylation and change gene expression. Together, our work highlights how IDRs with different sequences, spatially segregated in the same protein, can cooperate to shape protein function.

molecular biology↗

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↗

bioRxiv2022

An Open-source, Cost-efficient Excitation Module for Single-molecule Microscopy

In fluorescence microscopy, scientific-grade laser diodes are key components and contribute a considerable expense to the total cost of the microscope setup. Existing open-source laser excitation modules are mainly designed for super-resolution imaging and have not been fully characterised to meet the requirements of single-molecule spectroscopy measurements. In this paper we introduce an open-source, cost-efficient excitation module that can be used for confocal, accurate single-molecule FRET, fluorescence correlation spectroscopy (FCS) and low-power super-resolution techniques. The module delivers two diode lasers (520 nm and 638 nm) via a single-mode fibre, with stable power output (<1% deviation), which can be modulated on the microsecond timescale. Here, we benchmark these lasers against smFRET standards, and recover consistent diffusion coefficients from FCS measurements, thereby demonstrating their suitability for a range of single-molecule spectroscopic experiments, whilst reducing the overall costs of our open-source smFRET instrument (the smfBox) by ~50%, making these types of experiments even more accessible to the widest possible userbase.

biophysics↗

bioRxiv2021

(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.

microbiology↗

bioRxiv2020

Multi-Modal Single-Molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. The ever-growing need to acquire high-throughput, dynamic data from multicolor species is driving the development of optical schemes that optimize the achievable spectral, temporal, and spatial resolution needed in order to follow biological, chemical and physical processes. Here we introduce Continuously Controlled Spectral-resolution (CoCoS) microscopy, an imaging scheme that encodes color into spatial read-out in the image plane, with continuous control over the spectral resolution. The concept enables single-frame acquisition of multiple color channels, allowing simultaneous, single-molecule colocalization for barcoding and Forster resonance energy transfer (FRET) experiments. The simple control over the spectral dispersion allows switching between imaging modalities at a click of a button. We demonstrate the utility of CoCoS for multicolor localization microscopy of microRNA barcodes in clinical samples, single-molecule FRET measurements, and single-molecule spectroscopy. CoCoS may be integrated as a simple add-on to existing microscopes and will find use in applications that aim to record dynamic, multicolor localization events such as in multiplex FRET and tracking of multi-component, interacting complexes.

biophysics↗

bioRxiv2019

The smfBox: an open-source platform for single-molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRETs broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

biophysics↗

bioRxiv2018

DNA replication initiation in Bacillus subtilis; Structural and functional characterisation of the essential DnaA-DnaD interaction

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we show that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail by NMR. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

biochemistry↗

bioRxiv2018

Substrate conformational dynamics drive structure-specific recognition of gapped DNA by DNA polymerase

DNA-binding proteins utilise different recognition mechanisms to locate their DNA targets. Some proteins recognise specific nucleotide sequences, while many DNA repair proteins interact with specific (often bent) DNA structures. While sequence-specific DNA binding mechanisms have been studied extensively, structure-specific mechanisms remain unclear. Here, we study structure-specific DNA recognition by examining the structure and dynamics of DNA polymerase I (Pol) substrates both alone and in Pol-DNA complexes. Using a rigid-body docking approach based on a network of 73 distance restraints collected using single-molecule FRET, we determined a novel solution structure of the singlenucleotide-gapped DNA-Pol binary complex. The structure was highly consistent with previous crystal structures with regards to the downstream primer-template DNA substrate; further, our structure showed a previously unobserved sharp bend (~120{degrees}) in the DNA substrate; we also showed that this pronounced bending of the substrate is present in living bacteria. All-atom molecular dynamics simulations and single-molecule quenching assays revealed that 4-5 nt of downstream gap-proximal DNA are unwound in the binary complex. Coarsegrained simulations on free gapped substrates reproduced our experimental FRET values with remarkable accuracy (<{Delta}FRET> = -0.0025 across 34 independent distances) and revealed that the one-nucleotide-gapped DNA frequently adopted highly bent conformations similar to those in the Pol-bound state ({Delta}G < 4 kT); such conformations were much less accessible to nicked (> 7 kT) or duplex (>> 10 kT) DNA. Our results suggest a mechanism by which Pol and other structure-specific DNA-binding proteins locate their DNA targets through sensing of the conformational dynamics of DNA substrates.\n\nSignificance StatementMost genetic processes, including DNA replication, repair and transcription, rely on DNA-binding proteins locating specific sites on DNA; some sites contain a specific sequence, whereas others present a specific structure. While sequence-specific recognition has a clear physical basis, structure-specific recognition mechanisms remain obscure. Here, we use single-molecule FRET and computer simulations to show that the conformational dynamics of an important repair intermediate (1nt-gapped DNA) act as central recognition signals for structure-specific binding by DNA polymerase I (Pol). Our conclusion is strongly supported by a novel solution structure of the Pol-DNA complex wherein the gapped-DNA is significantly bent. Our iterative approach combining precise single-molecule measurements with molecular modelling is general and can elucidate the structure and dynamics for many large biomachines.

biophysics↗