Biology subjects

Gardner, K. H.

Publications and source records attributed to Gardner, K. H..

15 recordsLinked to original sources

bioRxiv2026

Conformational changes induced in ubiquitin by circular protein-DNA chimeras

Proteins are dynamic molecular machines that change shape in response to physical and chemical perturbations. Although single-molecule force spectroscopy provides precise information about the stretching of proteins in response to tunable forces, it does so without structural detail. Circular protein-DNA chimeras, with DNA attached to pairs of surface sites, have been introduced as an alternative way to tunably apply forces to proteins. Intriguingly, these chimeras should be tractable for atomic-level study by nuclear magnetic resonance (NMR) spectroscopy and other structural methods. Here, we describe the NMR-scale synthesis of circular chimeras of single-and double-stranded DNA with ubiquitin, an essential component of many cellular pathways. We designed these chimeras to probe a two-residue retraction of ubiquitins C-terminal {beta}5 strand, normally triggered by phosphorylation of serine 65 during initiation of mitophagy. We probed the resulting conformational changes by NMR and found that the attachment of a single strand of DNA suffices to alter this conformational equilibrium. A control bearing two separate short single DNA strands recapitulated much of the circular chimeras NMR properties, supporting a dominant role for local protein-DNA interactions rather than spring-like action by single- or double-stranded DNA. These results provide a necessary benchmark for future studies using DNA springs to probe the functional dynamics of proteins. Significance statementLigands, post-translational modifications, and mechanical inputs reshape proteins through forces that propagate across their structures. The underlying mechanical properties of proteins mediating these changes are rarely accessible with atomic-level detail. By producing NMR-scale circular protein-DNA chimeras, we provide a route to examining how defined physical perturbations alter protein conformational land-scapes. The results establish both the promise of this strategy and the need to account for local DNA-protein interactions.

biophysics↗

bioRxiv2026

Small Molecule Regulation of CLOCK:BMAL1 DNA Binding Activity

CLOCK:BMAL1 is a bHLH-PAS transcription factor complex that utilizes its bHLH (basic helix-loop-helix) domains to bind E-box motifs in DNA and tandem PAS (PER-ARNT-SIM) domains to heterodimerize and interact with regulatory proteins to generate circadian rhythms. PAS domains are evolutionarily conserved modules that frequently bind small molecule ligands within buried cavities to perform sensory and signal transduction functions. CLOCK and BMAL1 PAS domains have cavities that could be leveraged to regulate the transcription factor, and consequently, the circadian clock. Using NMR spectroscopy, we identified small molecules that bind within a cavity inside the PAS-A domain of CLOCK and its paralog NPAS2, which sits at an important flexible junction in the structured core of the heterodimer. We identified a gatekeeping mutant in the core of CLOCK PAS-A that significantly decreased ligand binding affinity. High-pressure NMR studies showed that ligand binding or the gatekeeping mutant significantly stabilized the domain. Finally, we showed that ligands induced dose-dependent displacement of CLOCK:BMAL1 from DNA in vitro. Together, these data demonstrate that small molecules can regulate DNA binding by the circadian transcription factor CLOCK:BMAL1 through occupancy of a PAS domain cavity.

biochemistry↗

bioRxiv2026

Structural Basis of a Novel Heme Binding Bacterial One-Component Switch

One-component systems (OCSs) integrate sensory and effector functions within a single protein, enabling rapid gene expression changes in response to environmental cues. Here, we characterized a novel heme binding OCS protein, FG214, from Fimbriimonas ginsengisoli, a redox-regulated helix-turn-helix transcription factor in which heme iron ligand state controls a monomer-to-dimer switch. Data supporting this included our observation of the FG214 PAS domain binding a hexacoordinate heme b in oxidized conditions and undergoing a slate of redox and ligand-dependent conformational changes, transitioning from a monomer to a homodimer. Spectroscopic and structural data revealed that oxidation stabilizes the likely HTH-PAS intramolecular domain interface, while reduction of the heme iron dissociates the HTH, freeing previously-sequestered homodimerization surfaces. Similar effects were seen by addition of a small molecule ferric heme ligand, as directly visualized with a 1.47 [A] crystal structure of an imidazole-bound truncated construct. Using in vitro DNA-binding assays, we identified an artificial promoter sequence and demonstrated ligand-enhanced protein-DNA binding. Finally, we performed in vivo proof of concept experiments establishing FG214 as a redox-sensitive scaffold for biosensor engineering. Together, these findings define FG214 as a novel heme-binding PAS DNA binding protein, complementing known heme-PAS two-component signaling switches.

biochemistry↗

bioRxiv2025

A Pipeline for Screening Small Molecule-Enhanced Protein Stability in A Bacterial Orphan Receptor

Bacterial one-component signaling proteins integrate sensory and gene regulation functions within the same polypeptide, creating powerful natural sensors of environmental conditions which can also be adapted into powerful tools for synthetic biology and biotechnology. A key sensor motif within many of these proteins is the Per- ARNT-Sim (PAS) domain, known for its conserved fold yet highly divergent sequences, allowing for a broad range of ligands to control PAS protein function by changes in small molecule binding occupancy or configuration. This diversity introduces a challenging step - identification of ligands for "orphan" PAS proteins which show signatures of ligand binding but no copurifying high-affinity bound small molecules - into characterization and engineering of such proteins. In this study, we characterized CU228, a putative PAS-HTH transcription factor from Candidatus Solibacter usitatus, as a novel model system for searching for novel ligands by small molecule stabilization. Bioinformatics and structural analyses predicted a PAS domain with an Trp-rich internal cavity, suggesting potential small molecule interactions. Using a [~]760 compound fragment library, differential scanning fluorimetry identified three ligands (KG-96, KG- 408, and KG-484) that substantially increased CU228 thermal stability with {Delta}Tm values up to +10{degrees}C. Microfluidic modulation spectroscopy (MMS) revealed ligand-induced preservation of -helical and {beta}-sheet integrity under thermal stress. Saturation transfer difference NMR confirmed direct binding of all three ligands and enabled estimation of micromolar-range dissociation constants, consistent with expected fragment-level affinity. Our findings expand the analytical toolbox for probing protein-ligand interactions in flexible, signal-responsive systems, laying the groundwork for designing synthetic chemogenetic variants of one-component transcription factors.

biochemistry↗

bioRxiv2025

Hydraulic Activation of the AsLOV2 photoreceptor

How proteins transduce light into mechanical energy remains a central question in biology. This study tests the hypothesis that blue light activation of the LOV2 (light, oxygen, voltage sensitive) domain of Avena sativa phototropin 1 (AsLOV2), gives rise to concerted water movement that induces protein conformational extensions. Using electron and nuclear magnetic resonance spectroscopy, along with molecular dynamics simulations at high pressure, we find AsLOV2 activation can be initiated by blue light or high pressure, followed by selective and concerted expulsion of low-entropy, tetrahedrally coordinated "wrap" water from the protein hydration shell. These findings suggest that interfacial water serves as constituents to reshape the proteins free energy landscape during activation. Our study highlights hydration water as an active hydraulic fluid that can drive long-range conformational changes underlying protein mechanics upon light activation and offers a new concept for engineering externally controllable protein actuators. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/660617v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1a35f9dorg.highwire.dtl.DTLVardef@da4991org.highwire.dtl.DTLVardef@113e9b2org.highwire.dtl.DTLVardef@53d9fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Context-Dependent Variability Of HIF Heterodimers Influences Interactions With Macromolecular And Small Molecule Partners

Hypoxia inducible factors (HIFs) are transcription factors that coordinate cellular responses to low oxygen levels, functioning as an /{beta} heterodimer which binds a short hypoxia response element (HRE) DNA sequence. Prior studies suggest HIF/HRE complexes are augmented by the binding of additional factors nearby, but those interactions are not well understood. Here, we integrated structural and biochemical approaches to investigate several functionally relevant HIF assemblies with other protein, small molecule, and DNA partners. First, we used cryo-electron microscopy (cryo-EM) to establish HIF-1 and HIF-2 self-assemble to form novel "dimer-of-heterodimers" (DoHD) complexes on extended human EPO enhancer sequences, with one heterodimer bound at a canonical HRE site and the second binding in an inverted fashion to an HRE-adjacent sequence (HAS) 8 bp away. Consistent with ARNT PAS-B domains predominating interactions within a DoHD, we found HIF-1 and HIF-2 co-assemble mixed DoHD complexes on the same DNA. Second, we saw that despite the increased complexities of the larger complexes, ligands for the isolated ARNT or HIF-2 PAS-B domains are still capable of binding and disrupting both the heterodimer and DoHD complexes, albeit with variable potencies depending on the ligand. Finally, we combined cryo-EM and hydrogen- deuterium exchange by mass spectrometry (HDX-MS) to show how HIF-1 and HIF-2 heterodimers engage the transforming acidic coiled-coil containing protein 3 (TACC3) coactivator via both ARNT and HIF- subunits, though this was unseen in the larger DoHD. Our findings highlight the importance of both molecular context and dynamics in biomolecular complex formation, adding to the complexities of potential regulation. Significance StatementHypoxia inducible factors (HIFs) are transcription factors that regulate oxygen-dependent cellular processes with implications in certain types of cancers. Current molecular structures of HIFs bound to short DNA fragments provide insights into their function, but leave open questions about how they bind longer natural DNA fragments and interact with small molecules and protein coactivators. Integrating structural and biochemical techniques, we discovered a novel assembly in which two HIFs bind together on a single extended DNA fragment, forming a "dimer-of-heterodimers", which exhibits some differences in ligand and coactivator binding than heterodimers or isolated PAS domains. Our studies highlight how functional contexts can shift structural paradigms and provide greater insight into the mechanisms by which HIFs and similar bHLH-PAS transcription factors operate.

biochemistry↗

bioRxiv2025

Pulsatory response of the BcLOV4 photoreceptor through intramolecular feed-forward regulation

Proteins are information processors, but their computations are typically considered at steady state. Here we find that individual proteins can dynamically encode information about their environment and that such response dynamics have been conserved throughout evolution. The fungal protein BcLOV4 exhibits pulsatory light responses shaped by the magnitude of environmental light and temperature. Response adaptation resulted from competitive interactions between domains that sensed either light or temperature. Temperature-sensing was encoded in a modular domain and could be tuned by mutations within co-evolved loops. Photo-thermal response dynamics were conserved in homologues from fungi that diverged >300 million years ago, and the characteristic temperature of pulsatory responses had adapted to match the ecological niche of the hosts, ranging from Antarctica to thermal ponds. These findings uncover a class of dynamic proteins, determine molecular principles of time-varying protein activation, and suggest functional importance for light- and temperature-conditioned protein activity pulses. One-Sentence SummaryIndividual proteins can dynamically encode information through interactions between their component domains, revealing principles for complex signal processing in natural and engineered proteins.

systems biology↗

bioRxiv2024

Variations in kinase and effector signaling logic in a two component signaling network

The general stress response (GSR) protects bacteria from a wide range of stressors. In Alphaproteobacteria, GSR activation is coordinated by HWE/HisKA2 family histidine kinases (HKs), which can exhibit non-canonical structure and function. For example, while most light-oxygen-voltage sensor-containing HKs are light activated dimers, the Rubellimicrobium thermophilum RT-HK has inverted "dark on, light off" signaling logic with a tunable monomer/dimer equilibrium. Here, we further investigate these atypical behaviors of RT-HK and characterize its downstream signaling network. Using hydrogen-deuterium exchange mass spectrometry, we find that RT-HK uses a signal transduction mechanism similar to light-activated systems, despite its inverted logic. Mutagenesis reveals that RT-HK autophosphorylates in trans, with changes to the J helix linking sensor and kinase domains affecting autophosphorylation levels. Exploring downstream effects of RT-HK, we identified two GSR genetic regions, each encoding a copy of the central regulator PhyR. In vitro measurements of phosphotransfer from RT-HK to the two putative PhyRs revealed that RT-HK signals only to one, and does so at an increased intensity in the dark, consistent with its reversed logic. X-ray crystal structures of both PhyRs revealed a substantial shift within the receiver domain of one, suggesting a basis for RT-HK specificity. We probed further down the pathway using nuclear magnetic resonance to determine that the single NepR homolog interacts with both unphosphorylated PhyRs, and this interaction is decoupled from activation in one PhyR. This work expands our understanding of HWE/HisKA2 family signal transduction, revealing marked variations from signaling mechanisms previously identified in other GSR networks.

biochemistry↗

bioRxiv2024

Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here, we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionalities they can deliver and biological questions that can be probed.

molecular biology↗

bioRxiv2023

Identification of Small Molecule Ligand Binding Sites On and In the ARNT PAS-B Domain

Transcription factors are generally challenging to target with small molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions include several naturally ligand-regulated transcription factors, including our prior work with the heterodimeric HIF-2 transcription factor which showed that small molecule binding within an internal pocket of the HIF-2 PAS-B domain can disrupt its interactions with its dimerization partner, ARNT. Here, we explore the feasibility of similarly targeting small molecules to the analogous ARNT PAS-B domain itself, potentially opening a promising route to simultaneously modulate several ARNT-mediated signaling pathways. Using solution NMR screening of an in-house fragment library, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the TACC3 transcriptional coactivator. However, these ligands have only modest binding affinities, complicating characterization of their binding sites. We address this challenge by combining NMR, MD simulations, and ensemble docking to identify ligand-binding hotspots on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/TACC3 inhibitors, KG-548 and KG-655, bind to a {beta}-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, while KG-548 binds exclusively to the {beta}-sheet surface, KG-655 can additionally bind within a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. All three ligands promoted ARNT PAS-B homodimerization, albeit to varying degrees. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

biochemistry↗

bioRxiv2023

Signal-regulated unmasking of the nuclear localization motif in the PAS domain regulates the nuclear translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

biochemistry↗

bioRxiv2020

TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. Here, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

developmental biology↗

bioRxiv2020

Use of High Pressure NMR Spectroscopy to Rapidly Identify Proteins with Internal Ligand-Binding Voids

Small molecule binding within internal cavities provides a way to control protein function and structure, as exhibited in numerous natural and artificial settings. Unfortunately, most ways to identify suitable cavities require high-resolution structures a priori and may miss potential sites. Here we address this limitation via high-pressure solution NMR spectroscopy, taking advantage of the distinctive nonlinear pressure-induced chemical shift changes observed in proteins containing internal cavities and voids. We developed a method to rapidly characterize such nonlinearity among backbone 1H and 15N amide signals without needing to have sequence-specific chemical shift assignments, taking advantage of routinely available 15N-labeled samples, instrumentation, and 2D 1H/15N HSQC experiments. From such data, we find a strong correlation in the site-to-site variability in such nonlinearity with the total void volume within proteins, providing insights useful for prioritizing domains for ligand binding and indicating mode-of-action among such protein/ligand systems. We suggest that this experimental approach is a rapid and useful probe of otherwise hidden dynamic architectures of proteins, providing novel insights and opportunities into ligand binding and control. Significance StatementMany proteins can be regulated by internally binding small molecule ligands, but it is often not clear a priori which proteins are controllable in such a way. Here we describe a rapid method to address this challenge, using solution NMR spectroscopy to monitor the response of proteins to the application of high pressure. While the locations of NMR signals from many proteins respond to high pressure with linear chemical shift changes, proteins containing internal cavities that can bind small molecule ligands respond with easily identified non-linear changes. We demonstrate this approach on several proteins and protein/ligand complexes, suggesting that it has general utility.

biophysics↗

bioRxiv2020

High-Pressure NMR Reveals Volume and Compressibility Differences Between Two Stably Folded Protein Conformations

Proteins often interconvert between different conformations in ways critical to their function. While manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic beta-strand slip as it switches between two stably-folded conformations. Coupling high pressure and biomolecular NMR, we obtained the first quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wildtype equivalent, and the interconversion proceeds through a chiefly-unfolded intermediate state. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

biophysics↗

bioRxiv2020

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (J-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to J helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 s, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between J helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

biophysics↗