Biology subjects

Sharp, J. S.

Publications and source records attributed to Sharp, J. S..

16 recordsLinked to original sources

bioRxiv2025

Hydroxyl and Trifluoromethyl Radical Carbohydrate Footprinting for Probing Protein Binding Components of Oligosaccharides

Carbohydrates are found in various forms in living organisms, both as free-standing glycans as well as glycoconjugates including glycoproteins, glycolipids, and glycosaminoglycans. These structures play crucial roles in many biological processes, often mediated or influenced by interactions of carbohydrates with other biomolecules. However, studying these interactions is particularly challenging due to the structural complexity of carbohydrates, their dynamic conformational behavior, and the low binding affinities often involved. To address these challenges, we are developing a novel method that leverages mass spectrometry-based radical footprinting of carbohydrates (RFC). We monitored changes in the solvent accessibility of specific regions within oligosaccharides by measuring variations in the apparent rate of hydroxyl radical and trifluoromethyl radical-mediated oxidation. In our studies, a collection of trisaccharide isomers and N, N',N''-triacetylchitotriose (NAG3) shows no significant change in modification in non-binding protein solutions. However, in the presence of two proteins that bind NAG3 specifically, NAG3 oxidation is reduced. We find that the free reducing end is the primary site of hydroxyl radical oxidation under covalent labeling conditions, allowing it to distinguish interactions at the glycan reducing end. Trifluoromethyl radicals, conversely, label broadly across the trisaccharide by substitution into a C-H bond. Overall, this approach offers a powerful new approach for identifying glycan-protein interactions and mapping the binding interface of glycans.

biochemistry↗

bioRxiv2024

Radical Protein Footprinting in Mammalian Whole Blood

Hydroxyl Radical Protein Footprinting (HRPF) is a powerful tool to probe protein higher-order structure, as well as protein-protein and protein-carbohydrate interactions. It is mostly performed in vitro, but recent advances have extended its use to live cells, nematodes, and 3D cultures. However, application in living mammalian tissues has not been accomplished. Here, we present the first successful use of radical protein footprinting (RPF) in mammalian whole blood from wild-type (WT) and type 2 diabetes mellitus (T2DM) BKS. Cg Dock7m +/+ Leprdb/J mice. Using persulfate photoactivated with the FOX Photolysis System, we achieved effective protein labeling without significant disruption to blood cell morphology. An optimized quenching protocol eliminated background labeling. We report oxidative modifications in 11 selected proteins, revealing disease-associated conformational changes in multiple proteins. These findings demonstrate the feasibility of RPF in mammalian blood and open new opportunities for structural proteomics in preclinical models and clinical samples.

biochemistry↗

bioRxiv2023

Exploring the Residue-Level Interactions between the R2ab Protein and Polystyrene Nanoparticles

In biological systems, proteins can bind to nanoparticles to form a "corona" of adsorbed molecules. The nanoparticle corona is of high interest because it impacts the organisms response to the nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. Ultimately, a residue-level mapping of protein behavior on nanoparticle surfaces is needed, but this mapping is difficult to obtain with traditional approaches. Here, we have investigated the interaction between R2ab and polystyrene nanoparticles (PSNPs) at the level of individual residues. R2ab is a bacterial surface protein from Staphylococcus epidermidis and is known to interact strongly with polystyrene, leading to biofilm formation. We have used mass spectrometry after lysine methylation and hydrogen-deuterium exchange (HDX) NMR spectroscopy to understand how the R2ab protein interacts with PSNPs of different sizes. Through lysine methylation, we observe subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX measurements reveal that certain regions of the R2ab protein undergo faster exchange rates in the presence of PSNPs, suggesting conformational changes upon binding. Both results support a recently proposed "adsorbotope" model, wherein adsorbed proteins consist of unfolded anchor points interspersed with regions of partial structure. Our data also highlight the challenges of characterizing complex protein-nanoparticle interactions using these techniques, such as fast exchange rates. While providing insights into how proteins respond to nanoparticle surfaces, this research emphasizes the need for advanced methods to comprehend these intricate interactions fully at the residue level. TOC Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/554951v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19b8ac5org.highwire.dtl.DTLVardef@b3948aorg.highwire.dtl.DTLVardef@1dd2ef5org.highwire.dtl.DTLVardef@35e938_HPS_FORMAT_FIGEXP M_FIG C_FIG Lysine methylation and hydrogen-deuterium exchange can reveal useful structural details about protein adsorption to nanoparticle surfaces.

biophysics↗

bioRxiv2022

Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography

Protein post-translational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic post-translational modifications are very challenging to structurally characterize using modern techniques, including covalent labeling methods, due to the presence of multiple proteoforms and conformers together in solution. Here, we have coupled ion exchange HPLC with a flash oxidation system (IEX LC-FOX) to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase (AP). Real-time dosimetry indicates no difference in effective radical dose between peaks or across the peak, demonstrating both the lack of scavenging of the NaCl gradient and the lack of a concentration effect on radical dose between peaks of different intensities. The use of IEX LC-FOX allows us to structurally probe each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system reintroduce heterogeneity. We found significant differences in residue-level oxidation between the hydroxyl radical footprint of non-phosphorylated, mono-phosphorylated and di-phosphorylated ovalbumin. Not only were our data consistent with the previously reported stabilization of ovalbumin structure by phosphorylation, but local structural changes were also consistent with the measured order of dephosphorylation of Ser344 being removed first. These results demonstrate the utility of IEX LC-FOX for measuring the structural effects of PTMs, even in dynamic systems.

biochemistry↗

bioRxiv2021

Validated Determination of NRG1 Ig-like Domain Structure by Mass Spectrometry Coupled with Computational Modeling

High resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry-based method that measures the solvent exposure of multiple amino acids in a single experiment, offering constraints for experimentally-informed computational modeling. HR-HRPF-based modeling has previously been used to accurately model the structure of proteins of known structure, but the technique has never been used to determine the structure of a protein of unknown structure leaving questions of unintentional bias and applicability to unknown structures unresolved. Here, we present the use of HR-HRPF-based modeling to determine the structure of the Ig-like domain of NRG1, a protein with no close homolog of known structure. Independent determination of the protein structure by both HR-HRPF-based modeling and heteronuclear NMR was carried out, with results compared only after both processes were complete. The HR-HRPF-based model was highly similar to the lowest energy NMR model, with a backbone RMSD of 1.6 [A]. To our knowledge, this is the first use of HR-HRPF-based modeling to determine a previously uncharacterized protein structure.

biophysics↗

bioRxiv2021

Differential response to prey quorum signals indicates predatory range of myxobacteria

A potential keystone taxa, myxobacteria contribute to the microbial food web as generalist predators. However, the extent of myxobacterial impact on microbial community structure remains unknown. The chemical ecology of these predator-prey interactions provides insight into myxobacterial production of biologically active specialized metabolites used to benefit consumption of prey as well as the perception of quorum signals secreted by prey. Using comparative transcriptomics and metabolomics, we compared how the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus respond to structurally distinct exogenous quorum signaling molecules. Investigating acylhomoserine lactone (AHL) and quinolone type quorum signals used by the clinical pathogen Pseudomonas aeruginosa, we identified a general response to AHL signals from both myxobacteria as well as a unique response from C. ferrugineus when exposed to the quinolone signal 4-hydroxy-2-heptylquinolone (HHQ). Oxidative detoxification of HHQ in C. ferrugineus was not observed from M. xanthus. Subsequent predation assays indicated P. aeruginosa to be more susceptible to C. ferrugineus predation. These data indicate that as generalist predators myxobacteria demonstrate a common response to the ubiquitous AHL quorum signal class, and we suggest this response likely involves recognition of the homoserine lactone moiety of AHLs. We also suggest that oxidation of HHQ and superior predation of P. aeruginosa observed from C. ferrugineus provides an example of how prey signaling molecules impact predatory specialization of myxobacteria by influencing prey range. SummaryMultiomic analysis of transcriptional and metabolic responses from the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus exposed to prey signaling molecules of the acylhomoserine lactone and quinolone quorum signaling classes provided insight into myxobacterial specialization associated with predatory eavesdropping. We suggest that the general response observed from both myxobacteria exposed to acylhomoserine lactone quorum signals is likely due to the generalist predator lifestyles of myxobacteria and ubiquity of acylhomoserine lactone signals. We also provide data that indicates the core homoserine lactone moiety included in all acylhomoserine lactone scaffolds to be sufficient to induce this general response. Comparing both myxobacteria, unique transcriptional and metabolic responses were observed from Cystobacter ferrugineus exposed to the quinolone signal 4-hydroxy-2-heptylquinoline (HHQ) natively produced by Pseudomonas aeruginosa. We suggest that this unique response and ability to metabolize quinolone signals contribute to the superior predation of P. aeruginosa observed from C. ferrugineus. These results further demonstrate myxobacterial eavesdropping on prey signaling molecules and provide insight into how responses to exogenous signals might correlate with prey range of myxobacteria. Originality-Significance StatementThis manuscript provides the first multiomic analysis of how predatory myxobacteria respond to exogenous prey signaling molecules and details the differences observed by comparing responses from two myxobacteria.

microbiology↗

bioRxiv2020

Self-Organized Amphiphiles are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments

The analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. Here, we investigated the effect of bulk hydroxyl radical scavenging in FPOP using both isolated cellular membranes as well as detergent micelles. We found no significant change in radical scavenging activity upon the addition of disrupted cellular membranes with the membrane concentration in the range of 0-25600 cell/L using an inline radical dosimeter. We confirmed the non-scavenging nature of the membrane with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration acts as a potent hydroxyl radical scavenger as expected, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. These results suggest that any scavenging effect of membranes or organized amphiphilic membrane mimetics in FPOP experiments are not due to bulk hydroxyl radical scavenging, but may be due to a localized scavenging phenomenon.

biochemistry↗

bioRxiv2020

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 {micro}g/L, 1.08 mg/L, 1.77 {micro}g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

microbiology↗

bioRxiv2019

Rapid Quantification of Peptide Oxidation Isomers from Complex Mixtures

Hydroxyl radical protein footprinting (HRPF) is a powerful technique for probing changes in protein topography, based on quantifying the amount of oxidation of different regions of a protein. While quantification of HRPF oxidation at the peptide level is relatively common, quantification at the residue level is challenging due to the influence of oxidation on MS/MS fragmentation and the large number of complex and only partially chromatographically resolved isomeric peptide oxidation products. HRPF quantification of isomeric peptide oxidation products (where the peptide sequence is the same but isomeric oxidation products are formed at different sites) at the residue level by electron transfer dissociation tandem mass spectrometry (ETD MS/MS) has been demonstrated in both model peptides and HRPF products, but the method is hampered by the partial separation of oxidation isomers by reversed phase chromatography. This requires custom MS/MS methods to equally sample all isomeric oxidation products across their elution window, greatly increasing method development time and reducing the oxidation products quantified in a single LC-MS/MS run. Here we present a zwitterionic hydrophilic interaction capillary chromatography (ZIC-HILIC) method to ideally co-elute all isomeric peptide oxidation products while separating different peptides. This allows us to relatively quantify peptide oxidation isomers using an ETD MS/MS spectrum acquired at any point across the single peptide oxidation isomer peak, greatly simplifying data acquisition and data analysis at both the peptide and amino acid level.

biochemistry↗

bioRxiv2019

Solo acylhomoserine lactone synthase from predatory myxobacterium suggests beneficial participation in interspecies cross talk

Considered a key taxon in microbial communities, myxobacteria exist as coordinated swarms that utilize an excreted combination of lytic enzymes and specialized metabolites to facilitate predation of numerous microbial phyla. This capacity to produce biologically active metabolites and the associated abundance of natural product biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all the biosynthetic gene clusters associated with myxobacteria deposited in the antiSMASH database ([~]1,000 total), only one putative acylhomoserine lactone synthase, agpI, was observed in genome data from the myxobacterium Archangium gephyra. Without an acylhomoserine lactone (AHL) receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was the first example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second myxobacterial AHL synthase from Vitiosangium sp. strain GDMCC 1.1324. During axenic cultivation conditions, no detectible AHL metabolites were observed in A. gephyra extracts. However, heterologous expression of each synthase in Escherichia coli provided detectible quantities of 3 AHL signals including 2 known AHLs, C8-AHL and C9-AHL. These results suggest that A. gephyra AHL production is dormant during axenic cultivation conditions and requires an unknown external cue for activation. The orphaned AHL synthase, AgpI, is the first to be reported from a predatory myxobacterium, and predator production of prey quorum signals provides unique insight into interspecies crosstalk within polymicrobial communities. ImportanceThe presence of orphaned quorum signal receptors and associated recognition and response to exogenous acylhomoserine lactone quorum signals observed in microbial communities provides evidence for small molecule-mediated interspecies interactions. While the high occurrence of orphaned AHL receptors from bacteria that do not produce cognate AHL signals suggests the involvement of AHL signals as a shared chemical resource in polymicrobial communities, no orphaned AHL synthases have been determined to be functional in a species without an associated AHL receptor. An orphan signal synthase from a predatory myxobacterium provides an alternative perspective on the evolution and benefits of quorum signaling systems within these communities.

microbiology↗

bioRxiv2019

Intrinsic Buffer Hydroxyl Radical Dosimetry Using Tris(Hydroxymethyl)Aminomethane

Fast Photochemical Oxidation of Proteins (FPOP) is a powerful covalent labeling tool that uses hydroxyl radicals generated by laser flash photolysis of hydrogen peroxide to footprint protein surfaces. Because radical production varies with many experimental parameters, hydroxyl radical dosimeters have been introduced to track the effective radical dosage experienced by the protein analyte. FPOP experiments performed using adenine optical radical dosimetry containing protein in Tris buffer demonstrated unusual dosimetry behavior. We have investigated the behavior of Tris under oxidative conditions in detail. We find that Tris can act as a novel gain-of-signal optical hydroxyl radical dosimeter in FPOP experiments. This new dosimeter is also amenable to inline real-time monitoring thereby allowing real-time adjustments to compensate for differences in samples for their quenching ability.

biochemistry↗

bioRxiv2019

Online Size Exclusion Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

Structural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline fast photochemical oxidation of proteins (FPOP). Using a controlled synthetic mixture of holomyoglobin and apomyoglobin, we demonstrate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to off-line FPOP of each pure conformer. We then applied LC-FPOP to analyze the adalimumab heat-shock aggregation process. We found that the LC-FPOP footprint of unaggregated adalimumab was consistent with a previously published footprint of the native IgG. The LC-FPOP footprint of the aggregation product indicated that heat shock aggregation primarily protected the hinge region, suggesting this region is involved with the heat shock aggregation process of this molecule. LC-FPOP offers a new method to probe dynamic conformationally heterogeneous mixtures such as biopharmaceutical aggregates, and obtain accurate information on the topography of each conformer.

biochemistry↗

bioRxiv2019

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

biochemistry↗

bioRxiv2018

Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g. ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultra-fast benchtop method for performing HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond timescale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g. ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be normalized on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

biochemistry↗

bioRxiv2017

Mapping of the Fondaparinux Binding Site of JR-FL gp120 by High Resolution Hydroxyl Radical Protein Footprinting and Computational Docking

The adhesion of HIV gp120 antigen to human cells is modulated in part by interactions with heparan sulfate. The HXB2 strain of gp120 has been shown to interact with heparin primarily through the V3 loop, although other domains including the C-terminal domain were also implicated. However, the JR-FL strain (representative of CCR5-interacting strains that make up newest infections) was shown to have a drastically lowered affinity to heparin due to the loss of several basic residues in the V3 loop, and deletion of the V3 loop in JR-FL gp120 was shown to abrogate some, but not all, heparin binding. Here, we use high resolution hydroxyl radical protein footprinting to measure the changes in protein surface oxidation levels that result from the binding of a model heparin fragment (fondaparinux). Protection in both the V3 loop and the N-terminus of JR-FP gp120 is observed. The well-defined composition of fondaparinux allowed us to perform docking simulations, which showed two clusters of fondaparinux binding: the V3 loop, and a domain consisting of the N- and C-termini. Together, the experimental and theoretical results indicate the heparin/heparan sulfate binding sites on JR-FL gp120 and the efficient interaction of fondaparinux, a widely exploited therapeutic carbohydrate, on gp120.

biochemistry↗

bioRxiv2017

Quantitative Protein Topography Measurements By High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

We report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area ( ) of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use measurements from HR-HRPF to differentiate molecular models of high accuracy (< 3[A] backbone RMSD) from models of lower accuracy (> 4[A] backbone RMSD). The ability of data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

biochemistry↗