Biology subjects

Pollack, L.

Publications and source records attributed to Pollack, L..

11 recordsLinked to original sources

bioRxiv2025

Predicting lead hotspots in urban stormwater ponds across the Twin Cities Metro

Stormwater ponds play an important role in urban food webs. Critically these same ponds could also serve as pollution hotspots since stormwater ponds can act as local concentrators of urban runoff. One such contaminant of concern is lead, which remains a significant issue for human and ecosystem health in the United States despite regulatory bans on its use in paint and gasoline imposed in the 1970s. Despite high levels of lead in some urban stormwater, little is known about the distribution of lead in urban aquatic ecosystems. Therefore, we characterized lead within the sediment, water column, and surrounding soil of stormwater ponds across the Minneapolis-Saint Paul Metro Area. We hypothesized that lead would be highest in ponds that receive runoff from landscapes with older construction (i.e., legacy leaded paint and gasoline), have high traffic volume (i.e., legacy leaded gasoline), and areas with low impervious surface cover (i.e., increased mobilization of contaminated soil). Moreover, we hypothesized that stormwater ponds capture lead within sediments, with more dissolved lead at the bottom of the water column, where it would interact with lead containing sediments. Across ponds, we found that age of parcel development where the pond was located was the strongest predictor of surface sediment lead content. Within pond sediments, we found that lead concentrations increased with depth below the sediment surface, which is unsurprising since depth is likely correlated with time. The strongest predictor of surface water lead concentration was the strength of pond stratification, while the strongest predictor of bottom water lead concentration was pond duckweed cover and water conductivity. Water column oxygen concentrations varied across ponds yet were not important in determining dissolved lead within the water column. Importantly, lead within pond water remained quite low despite elevated sediment lead levels. These findings confirm that stormwater ponds can act as one source of environmental lead remediation by capturing lead within sediments under a wide range of environmental conditions. Our results suggest relatively low lead release from ponds to downstream areas, indicating that ponds generally serve as sinks, not sources within the urban lead cycle.

ecology↗

bioRxiv2025

Nucleotide-specific RNA conformations and dynamics within ribonucleoprotein condensates

Ribonucleoprotein (RNP) condensates have distinct physiological and pathological significance, but the structure of RNA within them is not well understood. Using contrast-variation solution X-ray scattering, which discerns only the RNA structures within protein-RNA complexes, alongside ensemble-based structural modeling we characterize the conformational changes of flexible poly-A, poly-U and poly-C single stranded RNA as it interacts with polybasic peptides, eventually forming condensed coacervate mixtures. At high salt concentrations, where macromolecular association is weak, we probe association events that precede the formation of liquid-like droplets. Structural changes occur in RNA that reflect charge screening by the peptides as well as{pi} -{pi} interactions of the bases with basic residues. At lower salt concentrations, where association is enhanced, poly-A RNA within phase separated RNP mixtures exhibit a broad scattering peak, suggesting subtle ordering. Coarse-grained molecular dynamics simulations are used to elucidate the nucleotide-specific dynamics within RNP condensates. While adenine-rich condensates behave like stable semidilute solutions, uracil-rich RNA condensates appear to be compositionally fluctuating. This approach helps understand how RNA sequence contributes to the molecular grammar of RNA-protein phase separation.

biophysics↗

bioRxiv2024

Scaling and Merging Time-Resolved Laue Data with Variational Inference

Time-resolved X-ray crystallography (TR-X) at synchrotrons and free electron lasers is a promising technique for recording dynamics of molecules at atomic resolution. While experimental methods for TR-X have proliferated and matured, data analysis is often difficult. Extracting small, time-dependent changes in signal is frequently a bottleneck for practitioners. Recent work demonstrated this challenge can be addressed when merging redundant observations by a statistical technique known as variational inference (VI). However, the variational approach to time-resolved data analysis requires identification of successful hyperparameters in order to optimally extract signal. In this case study, we present a successful application of VI to time-resolved changes in an enzyme, DJ-1, upon mixing with a substrate molecule, methylglyoxal. We present a strategy to extract high signal-to-noise changes in electron density from these data. Furthermore, we conduct an ablation study, in which we systematically remove one hyperparameter at a time to demonstrate the impact of each hyperparameter choice on the success of our model. We expect this case study will serve as a practical example for how others may deploy VI in order to analyze their time-resolved diffraction data.

biophysics↗

bioRxiv2024

Resolving DJ-1 Glyoxalase Catalysis Using Mix-and-Inject Serial Crystallography at a Synchrotron

DJ-1 (PARK7) is an intensively studied protein whose cytoprotective activities are dysregulated in multiple diseases. DJ-1 has been reported as having two distinct enzymatic activities in defense against reactive carbonyl species that are difficult to distinguish in conventional biochemical experiments. Here, we establish the mechanism of DJ-1 using a synchrotron-compatible version of mix-and-inject-serial crystallography (MISC), which was previously performed only at XFELs, to directly observe DJ-1 catalysis. We designed and used new diffusive mixers to collect time-resolved Laue diffraction data of DJ-1 catalysis at a pink beam synchrotron beamline. Analysis of structurally similar methylglyoxal-derived intermediates formed through the DJ-1 catalytic cycle shows that the enzyme catalyzes nearly two turnovers in the crystal and defines key aspects of its glyoxalase mechanism. In addition, DJ-1 shows allosteric communication between a distal site at the dimer interface and the active site that changes during catalysis. Our results rule out the widely cited deglycase mechanism for DJ-1 action and provide an explanation for how DJ-1 produces L-lactate with high chiral purity.

biochemistry↗

bioRxiv2024

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death

Transglutaminase 2 (TG2) is a GTP-binding/protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that maintain the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotide-bound TG2 adopts a monomeric closed conformation while calcium-bound TG2 assumes an open conformational state that can form higher order oligomers. SAXS analysis also suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time-resolved SAXS to show that LM11 increases the ability of calcium to drive TG2 to an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state. Significance StatementThe multi-functional protein transglutaminase 2 (TG2) undergoes large conformational changes in response to nucleotide and calcium binding, resulting in diverse cellular effects that can differentially promote either cancer cell survival or cell death. Previous biochemical and structural characterizations have revealed that TG2 primarily adopts two conformational states, a closed nucleotide-bound conformation, and an open calcium-bound conformation. In this study, we use advanced structural methods to describe the conformational changes associated with TG2 activation and inhibition and define the mechanism by which small molecule inhibitors maintain TG2 in a structural state that kill cancer cells.

biophysics↗

bioRxiv2023

Length-dependent Intramolecular Coil-to-Globule Transition in Poly(ADP-ribose) Induced by Cations

Poly(ADP-ribose) (PAR), a non-canonical nucleic acid, is essential for DNA/RNA metabolism and protein condensation, and its dysregulation is linked to cancer and neurodegeneration. However, key structural insights into PARs functions remain largely uncharacterized, hindered by the challenges in synthesizing and characterizing PAR, which are attributed to its length heterogeneity. A central issue is how PAR, comprised solely of ADP-ribose units, attains specificity in its binding and condensing proteins based on chain length. Here, we integrate molecular dynamics simulations with small-angle X-ray scattering to analyze PAR structures. We reveal the diverse structural ensembles of PAR and the factors influencing them, including a notable length-dependent compaction of PAR upon the addition of small amounts of Mg2+ ions. Unlike PAR15, PAR22 forms ADP-ribose bundles via local intramolecular coil-to-globule transitions. Understanding these length-dependent structural changes could be central to deciphering the specific biological functions of PAR.

biophysics↗

bioRxiv2023

RNA structures and dynamics with A resolution revealed by x-ray free electron lasers

RNA macromolecules, like proteins, fold to assume shapes that are intimately connected to their broadly recognized biological functions; however, because of their high charge and dynamic nature, RNA structures are far more challenging to determine. We introduce an approach that exploits the high brilliance of x-ray free electron laser sources to reveal the formation and ready identification of [A] scale features in structured and unstructured RNAs. New structural signatures of RNA secondary and tertiary structures are identified through wide angle solution scattering experiments. With millisecond time resolution, we observe an RNA fold from a dynamically varying single strand through a base paired intermediate to assume a triple helix conformation. While the backbone orchestrates the folding, the final structure is locked in by base stacking. In addition to understanding how RNA triplexes form and thereby function as dynamic signaling elements, this new method can vastly increase the rate of structure determination for these biologically essential, but mostly uncharacterized macromolecules.

biophysics↗

bioRxiv2022

Heterogeneity in the M. tuberculosis β-lactamase inhibition by Sulbactam

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC)1-5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals6. Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating7-9, cooperativity, induced fit10,11 and conformational selection11-13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans-enamine. This was made possible in part by the application of the singular value decomposition14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

molecular biology↗

bioRxiv2022

RNA triplex structures revealed by WAXS-driven MD simulations

RNA triple helices are commonly observed tertiary motifs that are increasingly associated with critical biological functions, including signal transduction. Because the recognition of their biological importance is relatively recent, their full range of structures and function has not yet been elucidated. The integration of solution wide-angle X-ray scattering (WAXS) with data-driven molecular dynamics (MD) simulations, described here, provides a new way to capture the structures of major-groove RNA triplexes that evade crystallographic characterization. This method yields excellent agreement between measured and computed WAXS profiles, and allows for an atomically detailed visualization of these motifs. Using correlation maps, the relationship between well-defined features in the scattering profiles and real space characteristics of RNA molecules is easily defined, including the subtle conformational variations in the double-stranded RNA upon the incorporation of a third strand by base-triples. This readily applicable approach provides unique insight into some of the interactions that stabilize RNA tertiary structure and enable function.

biophysics↗

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2017

Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis -lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.\n\nSynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

biophysics↗