Structure and Binding Properties to Blood Co-Factors of the Least Sulfated Galactan Found in the Cell Wall of the Red Alga Botryocladia occidentalis.

Three different populations of sulfated polysaccharides can be found in the cell wall of the red alga Botryocladia occidentalis. In a previous work, the structures of the two more sulfated polysaccharides were revised. In this work, NMR-based structural analysis was performed on the least sulfated polysaccharide and its chemically modified derivatives. Results have revealed the presence of both 4-linked α- and 3-linked ß-galactose units having the following chemical features: more than half of the total galactose units are not sulfated, the α-units occur primarily as 3,6-anhydrogalactose units either 2-O-methylated or 2-O-sulfated, and the ß-galactose units can be 4-O-sulfated or 2,4-O-disulfated. SPR-based results indicated weaker binding of the least sulfated galactan to thrombin, factor Xa, and antithrombin, but stronger binding to heparin cofactor II than unfractionated heparin. This report together with our previous publication completes the structural characterization of the three polysaccharides found in the cell wall of the red alga B. occidentalis and correlates the impact of their composing chemical groups with the levels of interaction with the blood co-factors.

Exploring Residue-Level Interactions between the Biofilm-Driving 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 significant interest because it impacts an organism's response to a nanomaterial. Understanding the corona requires knowledge of protein structure, orientation, and dynamics at the surface. 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. Lysine methylation experiments reveal subtle but statistically significant changes in methylation patterns in the presence of PSNPs, indicating altered protein surface accessibility. HDX rates become slower overall in the presence of PSNPs. However, some regions of the R2ab protein exhibit faster than average exchange rates in the presence of PSNPs, while others are slower than the average behavior, suggesting conformational changes upon binding. HDX rates and methylation ratios support a recently proposed "adsorbotope" model for PSNPs, wherein adsorbed proteins consist of unfolded anchor points interspersed with partially structured regions. 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 R2ab adsorbs onto PSNP surfaces, this research emphasizes the need for advanced methods to comprehend residue-level interactions in the nanoparticle corona.

Dimethylthiourea as a Quencher in Hydroxyl Radical Protein Footprinting Experiments.

Hydroxyl radical protein footprinting (HRPF) is a mass-spectrometry-based method for studying protein structures, interactions, conformations, and folding. This method is based on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. While catalase is commonly used as a quencher after the labeling of a protein by the hydroxyl radicals to efficiently remove the remaining hydrogen peroxide, it has some disadvantages. Catalase quenching adds a relatively high amount of protein to the sample, limiting the sensitivity of the method due to dynamic range issues and causing significant issues when dealing with more complex samples. We evaluated dimethylthiourea (DMTU) as a replacement for catalase in the quenching HRPF reactions. We observed that DMTU is highly effective at quenching HRPF oxidation. DMTU does not cause the background protein issues that catalase does, resulting in an increased number of protein identifications from complex mixtures. We recommend the replacement of catalase quenching with DMTU for all HRPF experiments.

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 organism's 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.

The Sea Cucumber Thyonella gemmata Contains a Low Anticoagulant Sulfated Fucan with High Anti-SARS-CoV-2 Actions against Wild-Type and Delta Variants.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-ß-N-acetylgalactosamine-(1→4)-ß-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

Structure, anti-SARS-CoV-2, and anticoagulant effects of two sulfated galactans from the red alga Botryocladia occidentalis.

The structure of the sulfated galactan from the red alga Botryocladia occidentalis (BoSG) was originally proposed as a simple repeating disaccharide of alternating 4-linked α-galactopyranose (Galp) and 3-linked ß-Galp units with variable sulfation pattern. Abundance was estimated only for the α-Galp units: one-third of 2,3-disulfation and one-third of 2-monosulfation. Here, we isolated again the same BoSG fractions from the anion-exchange chromatography, obtaining the same NMR profile of the first report. More careful NMR analysis led us to revise the structure. A more complex sulfation pattern was noted along with the occurrence of 4-linked α-3,6-anhydro-Galp (AnGalp) units. Interestingly, the more sulfated BoSG fraction showed slightly reduced in vitro anti-SARS-CoV-2 activities against both wild-type and delta variants, and significantly reduced anticoagulant activity. The BoSG fractions showed no cytotoxic effects. The reduction in both bioactivities is attributed to the presence of the AnGalp unit. Docking scores from computational simulations using BoSG disaccharide constructs on wild-type and delta S-proteins, and binding analysis through competitive SPR assays using blood (co)-factors (antithrombin, heparin cofactor II and thrombin) and four S-proteins (wild-type, delta, gamma, and omicron) strongly support the conclusion about the deleterious impact of the AnGalp unit.

Selective 2-desulfation of tetrasaccharide-repeating sulfated fucans during oligosaccharide production by mild acid hydrolysis.

Sulfated fucans (SFs) from echinoderms, such as sea cucumbers and sea urchins, present linear and regular sulfation patterns within defined oligosaccharide building blocks. The high molecular weights of these polymers pose a problem in advanced structure-activity relationship studies for which derived oligosaccharides are more appropriate tools for investigation. However, enzymes capable of specifically depolymerizing SFs, fucanases, are not very common. Scarce abundance and unknown catalytic activities are additional barriers to exploiting fucanases. Oligosaccharide production by controlled chemical reactions such as mild acid hydrolysis then becomes a convenient strategy. As a consequence, physicochemical studies are necessary to understand the structural modifications caused on SFs by this chemical hydrolysis. Hence, in this work, we subjected three tetrasaccharide-repeating SFs from sea cucumbers, Isostichopus badionotus (IbSF), Holothuria floridana (HfSF), and Lytechinus variegatus (LvSF) to mild acid hydrolysis for oligosaccharide production. Interestingly, selective 2-desulfation reaction was observed in all three SFs. Through our study, we indicate that selective 2-desulfation is a common and expected phenomenon in oligosaccharide production by mild acid hydrolysis of SFs, including those composed of tetrasaccharide-repeating units.

Structural characterization and biological activity of an α-glucan from the mollusk Marcia hiantina (Lamarck, 1818).

Marcia hiantina (Mollusca, Bivalvia) (Lamarck, 1818), is an edible clam mainly distributed along the tropical coastal regions. Recent researches have demonstrated that clams can possess compounds, including polysaccharides, with a wide range of biological actions including antioxidant, immunomodulatory and antitumor activities. Here an α-glucan was isolated from M. hiantina by hot water, purified by anion exchange chromatography, and its structure was characterized by a combination of multiple nuclear magnetic resonance (NMR) methods (1D 1H, 1H-1H COSY, 1H-1H TOCSY, 1H-1H NOESY, 1H-13C HSQC and 1H-13C HSQC-NOESY spectra), gas chromatography-mass spectrometry, and high performance size exclusion chromatography (HPSEC). The analysis from NMR, monosaccharide composition, methylation analyses and HPSEC combined with multi-angle light scattering (MALS) of M. hiantina-derived α-glycan confirmed a branched polysaccharide exclusively composed of glucose (Glc), mostly 4-linked in its backbone, branched occasionally at 6-positions, and having a molecular weight of ~ 570 kDa. The mollusk α-glucan was subjected to four cell-based assays: (i) viability of three cell lines (RAW264.7, HaCaT, and HT-29), (ii) activity on lipopolysaccharide (LPS)-induced prostaglandin production in RAW264.7 cells, (iii) inhibitory activities of in H2O2- and LPS-induced reactive oxygen species (ROS) production in HMC3 cells, and (iv) HaCaT cell proliferation. Results have indicated no cytotoxicity, potent inhibition of both H2O2- and LPS-induced ROS, and potent cell proliferative activity.

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

Protein posttranslational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic PTMs 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. We have coupled an ion exchange high-performance liquid chromatography separation with a flash oxidation system [ion exchange chromatography liquid chromatography-flash oxidation (IEX LC-FOX)] to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase. Real-time dosimetry indicates no difference in the 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 into each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system to reintroduce heterogeneity. We found significant differences in the residue-level oxidation between the hydroxyl radical footprint of nonphosphorylated, monophosphorylated, and diphosphorylated OVA. Not only were our data consistent with the previously reported stabilization of OVA 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.

Rational Design of a Cu Chelator That Mitigates Cu-Induced ROS Production by Amyloid Beta.

Alzheimer's disease severely perturbs transition metal homeostasis in the brain leading to the accumulation of excess metals in extracellular and intraneuronal locations. The amyloid beta protein binds these transition metals, ultimately causing severe oxidative stress in the brain. Metal chelation therapy is an approach to sequester metals from amyloid beta and relieve the oxidative stress. Here we have designed a mixed N/O donor Cu chelator inspired by the proposed ligand set of Cu in amyloid beta. We demonstrate that the chelator effectively removes Cu from amyloid beta and suppresses reactive oxygen species (ROS) production by redox silencing and radical scavenging both in vitro and in cellulo. The impact of ROS on the extent of oxidation of the different aggregated forms of the peptide is studied by mass spectrometry, which, along with other ROS assays, shows that the oligomers are pro-oxidants in nature. The aliphatic Leu34, which was previously unobserved, has been identified as a new oxidation site.

Differential response to prey quorum signals indicates predatory specialization of myxobacteria and ability to predate Pseudomonas aeruginosa.

Multiomic analysis of transcriptional and metabolic responses from the predatory myxobacteria Myxococcus xanthus and Cystobacter ferrugineus exposed to prey signalling molecules of the acylhomoserine lactone and quinolone quorum signalling classes provided insight into predatory specialization. Acylhomoserine lactone quorum signals elicited a general response from both myxobacteria. We suggest that this 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 2-heptylquinolin-4(1H)-one (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 signalling molecules and provide insight into how responses to exogenous signals might correlate with prey range of myxobacteria.

Laser-free Hydroxyl Radical Protein Footprinting to Perform Higher Order Structural Analysis of Proteins.

Hydroxyl Radical Protein Footprinting (HRPF) is an emerging and promising higher order structural analysis technique that provides information on changes in protein structure, protein-protein interactions, or protein-ligand interactions. HRPF utilizes hydroxyl radicals (▪OH) to irreversibly label a protein's solvent accessible surface. The inherent complexity, cost, and hazardous nature of performing HRPF have substantially limited broad-based adoption in biopharma. These factors include: 1) the use of complicated, dangerous, and expensive lasers that demand substantial safety precautions; and 2) the irreproducibility of HRPF caused by background scavenging of ▪OH that limit comparative studies. This publication provides a protocol for operation of a laser-free HRPF system. This laser-free HRPF system utilizes a high energy, high-pressure plasma light source flash oxidation technology with in-line radical dosimetry. The plasma light source is safer, easier to use, and more efficient in generating hydroxyl radicals than laser-based HRPF systems, and the in-line radical dosimeter increases the reproducibility of studies. Combined, the laser-free HRPF system addresses and surmounts the mentioned shortcomings and limitations of laser-based techniques.

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding.

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.

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

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is 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. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/µL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution 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 is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.

Flash Oxidation (FOX) System: A Novel Laser-Free Fast Photochemical Oxidation Protein Footprinting Platform.

Hydroxyl radical protein footprinting (HRPF) is a powerful and flexible technique for probing changes in protein topography. With the development of the fast photochemical oxidation of proteins (FPOP), it became possible for researchers to perform HRPF in their laboratory on a very short time scale. While FPOP has grown significantly in popularity since its inception, adoption remains limited due to technical and safety issues involved in the operation of a hazardous Class IV UV laser and irreproducibility often caused by improper laser operation and/or differential radical scavenging by various sample components. Here, we present a new integrated FOX (Flash OXidation) Protein Footprinting System. This platform delivers sample via flow injection to a facile and safe-to-use high-pressure flash lamp with a flash duration of 10 µs fwhm. Integrated optics collect the radiant light and focus it into the lumen of a capillary flow cell. An inline radical dosimeter measures the hydroxyl radical dose delivered and allows for real-time compensation for differential radical scavenging. A programmable fraction collector collects and quenches only the sample that received the desired effective hydroxyl radical dose, diverting the carrier liquid and improperly oxidized sample to waste. We demonstrate the utility of the FOX Protein Footprinting System by determining the epitope of TNFα recognized by adalimumab. We successfully identify the surface of the protein that serves as the epitope for adalimumab, identifying four of the five regions previously noted by X-ray crystallography while seeing no changes in peptides not involved in the epitope interface. The FOX Protein Footprinting System allows for FPOP-like experiments with real-time dosimetry in a safe, compact, and integrated benchtop platform.

Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins 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 validate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to offline 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 that 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 that can be separated by SEC such as biopharmaceutical aggregates and to obtain accurate information on the topography of each conformer.

Identification of a solo acylhomoserine lactone synthase from the myxobacterium Archangium gephyra.

Considered a key taxon in soil and marine microbial communities, myxobacteria exist as coordinated swarms that utilize a combination of lytic enzymes and specialized metabolites to facilitate predation of microbes. This capacity to produce specialized metabolites and the associated abundance of biosynthetic pathways contained within their genomes have motivated continued drug discovery efforts from myxobacteria. Of all myxobacterial biosynthetic gene clusters deposited in the antiSMASH database, only one putative acylhomoserine lactone (AHL) synthase, agpI, was observed, in genome data from Archangium gephyra. Without an AHL receptor also apparent in the genome of A. gephyra, we sought to determine if AgpI was an uncommon example of an orphaned AHL synthase. Herein we report the bioinformatic assessment of AgpI and discovery of a second AHL synthase from Vitiosangium sp. 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. The functional, orphaned AHL synthase, AgpI, is unique to A. gephyra, and its utility to the predatory myxobacterium remains unknown.

De Novo Design of a Self-Assembled Artificial Copper Peptide that Activates and Reduces Peroxide.

Copper-containing metalloenzymes constitute a major class of proteins which catalyze a myriad of reactions in nature. Inspired by the structural and functional characteristics of this unique class of metalloenzymes, we report the conception, design, characterization, and functional studies of a de novo artificial copper peptide (ArCuP) within a trimeric self-assembled polypeptide scaffold that activates and reduces peroxide. Using a first principles approach, the ArCuP was designed to coordinate one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallographic, UV-vis and EPR data demonstrate that Cu binds via the Nε atoms of His forming a T2Cu environment. When reacted with hydrogen peroxide, the putative copper-hydroperoxo species is formed where a reductive priming step accelerates the rate of its formation and reduction. Mass spectrometry was used to identify specific residues undergoing oxidative modification, which showed His oxidation only in the reduced state. The redox behavior of the ArCuP was elucidated by protein film voltammetry. Detailed characterization of the electrocatalytic behavior of the ArCuP led us to determine the catalytic parameters (KM, kcat), which established the peroxidase activity of the ArCuP. Combined spectroscopic and electrochemical data showed a pH-dependence on the reactivity, which was optimum at pH 7.5.

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area of proteins. This technique relies on the reaction of amino acid side chains with hydroxyl radicals freely diffusing in solution. FPOP generates these radicals in situ by laser photolysis of hydrogen peroxide, creating a burst of hydroxyl radicals that is depleted on the order of a microsecond. When these hydroxyl radicals react with a solvent-accessible amino acid side chain, the reaction products exhibit a mass shift that can be measured and quantified by mass spectrometry. Since the rate of reaction of an amino acid depends in part on the average solvent accessible surface of that amino acid, measured changes in the amount of oxidation of a given region of a protein can be directly correlated to changes in the solvent accessibility of that region between different conformations (e.g., ligand-bound versus ligand-free, monomer vs. aggregate, etc.) FPOP has been applied in a number of problems in biology, including protein-protein interactions, protein conformational changes, and protein-ligand binding. As the available concentration of hydroxyl radicals varies based on many experimental conditions in the FPOP experiment, it is important to monitor the effective radical dose to which the protein analyte is exposed. This monitoring is efficiently achieved by incorporating an inline dosimeter to measure the signal from the FPOP reaction, with laser fluence adjusted in real-time to achieve the desired amount of oxidation. With this compensation, changes in protein topography reflecting conformational changes, ligand-binding surfaces, and/or protein-protein interaction interfaces can be determined in heterogeneous samples using relatively low sample amounts.

Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor.

The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.