Loss Of Side Chain Research Articles

Entropy Hotspots for the Binding of Intrinsically Disordered Ligands to a Receptor Domain

Proline-rich motifs (PRMs) are widely used for mediating protein-protein interactions with weak binding affinities. Because they are intrinsically disordered when unbound, conformational entropy plays a significant role for the binding. However, residue-level differences of the entropic contribution in the binding of different ligands remain not well understood. We use all-atom molecular dynamics simulation and the maximal information spanning tree formalism to analyze conformational entropy associated with the binding of two PRMs, one from the Abl kinase and the other from the nonstructural protein 1 of the 1918 Spanish influenza A virus, to the N-terminal SH3 (nSH3) domain of the CrkII protein. Side chains of the stably folded nSH3 experience more entropy change upon ligand binding than the backbone, whereas PRMs involve comparable but heterogeneous entropy changes among the backbone and side chains. In nSH3, two conserved nonpolar residues forming contacts with the PRM experience the largest side-chain entropy loss. In contrast, the C-terminal charged residues of PRMs that form polar contacts with nSH3 experience the greatest side-chain entropy loss, although their “fuzzy” nature is attributable to the backbone that remains relatively flexible. Thus, residues that form high-occupancy contacts between nSH3 and PRM do not reciprocally contribute to entropy loss. Furthermore, certain surface residues of nSH3 distal to the interface with PRMs gain entropy, indicating a nonlocal effect of ligand binding. Comparing between the PRMs from cAbl and nonstructural protein 1, the latter involves a larger side-chain entropy loss and forms more contacts with nSH3. Consistent with experiments, this indicates stronger binding of the viral ligand at the expense of losing the flexibility of side chains, whereas the backbone experiences less entropy loss. The entropy “hotspots” as identified in this study will be important for tuning the binding affinity of various ligands to a receptor.

Embrittlement induced fracture behavior and mechanisms of perfluorosulfonic-acid membranes after chemical degradation

Chemical and mechanical degradations of perfluorosulfonic-acid membranes are two factors contributing to the reduced durability of fuel cells. While the mechanisms of isolated chemical or mechanical degradation are extensively investigated, the impact of chemical degradation on mechanical degradation is not fully understood. In this paper, the fracture behavior of Nafion 212 membranes with different chemical degradation levels are investigated. To characterize the degradation level and probe the molecular origins of chemical degradation, fluoride release, Fourier-transformer infrared spectra, conductivity and swelling behavior are measured. It is found that chemical degradation causes predominant loss of side-chains. A transition from ductile to brittle fracture is observed with increasing degradation level. In addition, the cross-sections are examined to link fracture behavior with microstructure changes. While the decreased fracture toughness is attributed to reduced mechanical properties such as Young's modulus and break strain, the decreased crack propagation resistance is ascribed to reduced plastic zone size ahead of crack tip, which reduces the plastic energy dissipation. These findings not only provide new mechanical dataset of degraded membranes for performance and durability modelling to take into account of chemical degradation effect but also improve the understanding of membrane durability.

Functional role of glycosaminoglycans in decellularized lung extracellular matrix

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFβ1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFβ1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. Statement of significanceDespite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.

Hydroxylation of Steroids by a Microbial Substrate-Promiscuous P450 Cytochrome (CYP105D7): Key Arginine Residues for Rational Design.

Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

The Role of Active-Site Residues Phe98, His239, and Arg243 in DNA Binding and in the Catalysis of Human Uracil-DNA Glycosylase SMUG1.

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme–DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme–DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.

Functional importance of αAsp-350 in the catalytic sites of Escherichia coli ATP synthase

Negatively charged residue αAsp-350 of the highly conserved VISIT-DG sequence is required for Pi binding and maintenance of the phosphate-binding subdomain in the catalytic sites of Escherichia coli F1Fo ATP synthase. αAsp-350 is situated in close proximity, 2.88 Å and 3.5 Å, to the conserved known phosphate-binding residues αR376 and βR182. αD350 is also in close proximity, 1.3 Å, to another functionally important residue αG351. Mutation of αAsp-350 to Ala, Gln, or Arg resulted in substantial loss of oxidative phosphorylation and reduction in ATPase activity by 6- to 16-fold. The loss of the acidic side chain in the form of αD350A, αD350Q, and αD350R caused loss of Pi binding. While removal of Arg in the form of αR376D resulted in the loss of Pi binding, the addition of Arg in the form of αG351R did not affect Pi binding. Our data demonstrates that αD350R helps in the proper orientation of αR376 and βR182 for Pi binding. Fluoroaluminate, fluoroscandium, and sodium azide caused almost complete inhibition of wild type enzyme and caused variable inhibition of αD350 mutant enzymes. NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole) caused complete inhibition of wild type enzyme while some residual activity was left in mutant enzymes. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites. Phosphate protected against NBD-Cl inhibition of wild type and αG351R mutant enzymes but not inhibition of αD350A, αD350Q, αD350R, or αR376D mutant enzymes. These results demonstrate that αAsp-350 is an essential residue required for phosphate binding, through its interaction with αR376 and βR182, for normal function of phosphate binding subdomain and for transition state stabilization in ATP synthase catalytic sites.

Cell wall polysaccharides degradation and ultrastructure modification of apricot during storage at a near freezing temperature

The effects of near freezing temperature (NFT) storage at −1.9 °C on cell wall degradation of ‘Shushanggan’ apricot was studied comparing to 0 °C and 5 °C storage. Our results indicated that NFT storage strongly inhibited the solubilization of Na2CO3-soluble pectin and cellulose, by the suppression of cell wall modifying enzymes (polygalacturonase, β-Galactosidase, pectin methyl esterase and cellulase) and related genes expressions. The loss of side chains was the main modification in CDTA (Cyclohexane-diamine-tetraacetic Acid)-soluble pectin during storage and made the main contribution to the softening of apricot, while the loss of side chain was suppressed by NFT storage. Microscopic observation showed that NFT storage delayed the degradation of pectin fraction and protected cell wall structure from loosing. This study proves that NFT storage is an effective technology to suppress the cell wall polysaccharides degradation and ultrastructure modification of apricot.

General Mechanism of Cα-C Peptide Backbone Bond Cleavage in Matrix-Assisted Laser Desorption/Ionization In-Source Decay Mediated by Hydrogen Abstraction.

Nitrogen-centered and β-carbon-centered hydrogen-deficient peptide radicals are considered to be intermediates in the matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD)-induced Cα-C bond cleavage of peptide backbones when using an oxidizing matrix. To understand the general mechanism of Cα-C bond cleavage by MALDI-ISD, I study the fragmentation of model peptides and investigate the fragment formation pathways using calculations with density functional theory and transition state theory. The calculations indicate that the nitrogen-centered radical immediately undergoes Cα-C bond cleavage, leading to the formation of an a•/x fragment pair. In contrast, the dissociation of the β-carbon-centered radical is kinetically feasible under MALDI-ISD conditions, leading to the formation of an a/x• fragment pair. To discriminate these processes, I focus on the yield of d fragments, which originate from a• radicals through radical-induced side-chain loss, not from a fragments. The Cα-C bond cleavage on the C-terminal side of the carbamidomethylated cysteine residue is found to produce d fragments instead of a fragments. According to the calculation of the rate constant, the corresponding fragmentation occurs within 1ns. The intense signal arising from d fragments and the lack of or weak signal from a fragments strongly suggest that the Cα-C bond cleavage occurs through a nitrogen-centered radical intermediate. In addition to the side-chain loss, the resulting a• radical undergoes hydrogen atom abstraction by the matrix. The results for a deuterium-labeled peptide indicate that the matrix abstracts a hydrogen atom from either the amide nitrogen or the β-carbon.

Highly Drug-Resistant HIV-1 Protease Mutant PRS17 Shows Enhanced Binding to Substrate Analogues.

We report the structural analysis of highly drug-resistant human immunodeficiency virus protease (PR) variant PRS17, rationally selected by machine learning, in complex with substrate analogues. Crystal structures were solved of inhibitor-free inactive PRS17-D25N, wild-type PR/CA-p2 complex, and PRS17 in complex with substrate analogues, CA-p2 and p2-NC. Peptide analogues p2-NC and CA-p2 exhibit inhibition constants of 514 and 22 nM, respectively, for PRS17 or approximately 3-fold better than for PR. CA-p2 is a better inhibitor of PRS17 than are clinical inhibitors (Ki = 50–8390 nM) except for amprenavir (Ki = 11 nM). G48V resistance mutation induces curled flap tips in PRS17-D25N structure. The inner P2–P2′ residues of substrate analogues in PRS17 complexes maintain similar conformations to those of wild-type complex, while significant conformational changes are observed in the peripheral residues P3, P4′ of CA-p2 and P3, P4, and P3′ of p2-NC. The loss of β-branched side chain by V82S mutation initiates a shift in 80’s loop and reshapes the S3/S3′ subsite, which enhances substrate binding with new hydrogen bonds and van der Waals interactions that are absent in the wild-type structures. The steric hindrance caused by G48V mutation in the flap of PRS17 contributes to altered binding interactions of P3 Arg, P4′ norleucine of CA-p2, and P4 and P3′ of p2-NC with the addition of new hydrogen bonds and van der Waals contacts. The enhanced interaction of PRS17 with substrate analogues agrees with their relative inhibition, suggesting that this mutant improves substrate binding while decreasing affinity for clinical inhibitors.

Development of Novel Free Radical Initiated Peptide Sequencing Reagent: Application to Identification and Characterization of Peptides by Mass Spectrometry.

By incorporating a high proton affinity moiety to the charge localized free radical-initiated peptide sequencing (CL-FRIPS) reagent, FRIPS-MS technique has extended the applicability to hydrophobic peptides and peptides without basic amino acid residues (lysine, arginine, and histidine). Herein, the CL-FRIPS reagent has three moieties: (1) pyridine acting as the basic site to locate the proton, (2) 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, a stable free radical) acting as the free radical precursor to generate the nascent free radical in the gas phase, and (3) N-hydroxysuccinimide (NHS) activated carboxylic acid acting as the coupling site to derivatize the N-terminus of peptides. The CL-FRIPS reagent allows for the characterization of peptides by generating sequencing ions, enzymatic cleavage-like radical-induced side chain losses, and the loss of TEMPO simultaneously via one-step collisional activation. Further collisional activation of enzymatic cleavage-like radical-induced side chain loss ions provides more information for the structure determination of peptides. The application of CL-FRIPS reagent to characterize peptides is proved by employing bovine insulin as the model peptide. Both scaffold structure of bovine insulin and sequencing information of each chain are achieved. Graphical Abstract.

W-Type ions formed by electron transfer dissociation of Cys-containing peptides investigated by infrared ion spectroscopy.

In mass spectrometry‐based peptide sequencing, electron transfer dissociation (ETD) and electron capture dissociation (ECD) have become well‐established fragmentation methods complementary to collision‐induced dissociation. The dominant fragmentation pathways during ETD and ECD primarily involve the formation of c‐ and z •‐type ions by cleavage of the peptide backbone at the N─Cα bond, although neutral losses from amino acid side chains have also been observed. Residue‐specific neutral side chain losses provide useful information when conducting database searching and de novo sequencing. Here, we use a combination of infrared ion spectroscopy and quantum‐chemical calculations to assign the structures of two ETD‐generated w‐type fragment ions. These ions are spontaneously formed from ETD‐generated z •‐type fragments by neutral loss of 33 Da in peptides containing a cysteine residue. Analysis of the infrared ion spectra confirms that these z •‐ions expel a thiol radical (SH•) and that a vinyl C═C group is formed at the cleavage site. z •‐type fragments containing a Cys residue but not at the cleavage site do not spontaneously expel a thiol radical, but only upon additional collisional activation after ETD.

Cα–Cβ bond cleavage occurs at the side chain of the N-terminal phenylalanine residue during CID of tyrosine-containing peptide radical cations

Molecular radical cationic peptides (M+) display rich and diverse gas-phase ion chemistry that can differ substantially from that of their protonated counterparts. Dissociation of M+ radical cations can provide relevant structural information for protein sequencing, often complementary to that obtained using traditional approaches. In this study, low-energy collision-induced dissociation (CID) of a series of tyrosine-containing peptide molecular radical cations having an N-terminal phenylalanine residue led to new products resulting from radical-mediated Cα–Cβ bond cleavage at the side chain of the phenylalanine residue. The chemical identity of the species formed through side chain loss (91 Da) occurring via N-terminal Cα–Cβ bond cleavage was examined using a combination of low-energy CID experiments, isotopic labeling mass spectrometric experiments, and density functional theory calculations. This unusual dissociation process occurs with fascinating unimolecular gas phase ion chemistry and requires a π-radical cationic tyrosine residue with significant spin density delocalized into the N-terminal amino group. The radical character at the resulting –NH2+ moiety then induces β-scission at the side chain Cα–Cβ bond of the phenylalanine residue. This discovery also demonstrates the role of the NH2 group in facilitating Cα–Cβ bond cleavage of the phenylalanine residue by the proximal tyrosine π-radical cation, probably involving a through-space electron transfer process.

Side-chain loss reactions of collisionally activated protonated peptoids: A mechanistic insight

Peptoids constitute an emergent class of synthetic biocompatible macromolecules that are best described as polymers of N-substituted glycines. Peptides and peptoids are regioisomers with the side chains appended in peptoids to the nitrogen atoms rather than to the α-carbon atoms as in peptides. Peptide sequencing now takes great advantage of collision-induced dissociation experiments, based on the elucidation of the decomposition pathways of protonated peptides. In contrast, data on peptoid ion decompositions are to date scarcely present in the literature. Upon CID, protonated peptoids were recently shown to mostly dissociate by cleavages at the backbone amide bonds yielding B- and Y-fragment ions. In addition, the loss of the side-chain group and/or the formation of the side-chain fragment ion are common reactions for peptoids containing protonated N-α-aromatic side chain.In the present paper, we submitted protonated tailor-made peptoids to (energy-resolved) collision-induced dissociation experiments to investigate the side-chain loss reaction mechanisms. We also used orthogonal methods, such as quantum chemistry, ion mobility spectrometry and infrared photodissociation spectroscopy to establish the structures of the fragment ions. We ended up with different mechanistic scenarios consistent with the nature of the fragment ions and the kinetic energy dependence of decomposition reactions. Our mechanistic proposals associate the proton mobile model, proton bound dimer intermediacy and concerted rearrangement reactions.

Synthetic Polyampholytes as Macromolecular Cryoprotective Agents.

A series of polyampholytes based on different molar ratios on N, N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA), and optionally, N- tert-butylacrylamide ( t-BuAAm), were prepared by free radical copolymerization, and tested as DMSO-free cryoprotective agents for 3T3 fibroblast cells by using a standard freeze-rethaw protocol. Polybetaines prepared by reaction of DMAPMA homo and copolymers with 1,3-propane sultone were used as additional controls. Results showed strong effects of copolymer composition, molecular weight, polymer and NaCl concentrations, on post-thaw cell viability. Binary (DMAPMA/AA) copolymers showed best post-thaw cell viability of 70% at a 30/70 mol % ratio of DMAPMA/AA, which increased to 90% upon introduction of 9 mol % t-BuAAm while maintaining the 30/70 mol % cation/anion ratio. The use of acrylamide linkages in DMAPMA ensures absence of hydrolytic loss of cationic side chains. These polyampholytes were found to decrease ice crystal size and to form a polymer-rich, ice-free layer around cells, reducing damage from intercellular ice crystals during both freezing and thawing steps. These polyampholytes also dehydrate cells during freezing, which helps protect cells from intracellular ice damage. While cell viability immediately after thawing was high, subsequent culturing revealed poor attachment and long-term viability, which is attributed to residual cell damage from intracellular ice formation. Addition of 2 wt % DMSO or 1% BSA to the polymer-based freeze medium was found to mitigate this damage and result in post-thaw viabilities matching those achieved with 10 wt % DMSO.

An Outward-Facing Aromatic Amino Acid Is Crucial for Signaling between the Membrane-Spanning and Nucleotide-Binding Domains of Multidrug Resistance Protein 1 (MRP1; ABCC1).

The 190-kDa human MRP1 is an ATP-binding cassette multidrug and multiorganic anion efflux transporter. The 17 transmembrane helices of its three membrane-spanning domains, together with its two nucleotide binding domains (NBDs), form a stabilizing network of domain-domain interactions that ensure substrate binding in the cytoplasm is efficiently coupled to ATP binding and hydrolysis to effect solute efflux into the extracellular milieu. Here we show that Ala substitution of Phe583 in an outward-facing loop between the two halves of the transporter essentially eliminates the binding of multiple organic anions by MRP1. Conservative substitutions with Trp and Tyr had little or no effect. The F583A mutation also caused a substantial increase in orthovanadate-induced trapping of azidoADP by the cytoplasmic NBDs of MRP1, although the binding of ATP was unaffected. These observations indicate that the loss of the aromatic side chain at position 583 impairs the release of ADP and thus effectively locks the transporter in a low-affinity solute binding state. Phe583 is the first outward-facing amino acid in MRP1 found to be critical for its transport function. Our data provide evidence for long-range coupling, presumably via allosteric interaction, between this outward-facing region of MRP1 and both the solute binding and nucleotide binding regions of the transporter. Cryoelectron microscopy structural and homology models of MRP1 indicate that the orientation of the Phe583 side chain is altered by ATP binding but are currently unable to provide insights into the molecular mechanism by which this long-range signaling is propagated.

Synthetic cannabinoid BB-22 (QUCHIC): Human hepatocytes metabolism with liquid chromatography-high resolution mass spectrometry detection

Clandestine laboratories continue producing new synthetic cannabinoids that mimic and magnify natural cannabinoids effects to circumvent drug scheduling legislation. New synthetic cannabinoids are highly potent and responsible for many acute intoxications and deaths. Characterization of metabolic pathways is critical to identify metabolite markers whose detection can prove intake. BB-22 is a new potent synthetic cannabinoid whose toxicological and metabolic properties are currently unavailable. Analytical methods require constant updating and are challenging due to extensive synthetic cannabinoid metabolism and low marker concentrations. A single non-specific BB-22 metabolite was previously identified in incubations with human liver microsomes (BB-22 3-carboxyindole). Clear characterization of BB-22’s metabolism is required to help toxicologists document BB-22 consumption in clinical and forensic cases. We incubated 10 μmol/L BB-22 with cryopreserved human hepatocytes for 3 h. Samples were analyzed by liquid chromatography on a biphenyl column and high resolution mass spectrometry. Results were processed with data mining software, identifying ten metabolites. Loss of the quinolinyl side-chain via ester hydrolysis was the main biotransformation. All other metabolites were produced by further indole or cyclohexylmethyl hydroxylation or glucuronidation. We recommend BB-22 3-carboxyindole and two BB-22 3-carboxyindole-hydroxycyclohexylmethyl isomers as metabolite targets for documenting BB-22 intake. Hydrolysis of biological samples before analysis is strongly suggested to improve detection of phase I metabolites. BB-22 3-carboxyindole is not specific for BB-22 intake, as it was previously detected as a minor MDMB-CHMICA and ADB-CHMICA metabolite. Consumption of these two synthetic cannabinoids should be ruled out to confirm BB-22 intake.

Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan.

RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

Abstract LB-066: Cysteine mutant location affects chemotype lability in site-specific antibody drug conjugates

Abstract Antibody Drug Conjugates (ADCs) combine the specificity of antibody therapies with the potency of highly toxic payloads and have become an established therapeutic tool for the treatment of multiple types of cancer. The design process for novel ADCs requires careful attention to the biological target and chemotype selection. Increasingly, the importance of linker attachment and location is also recognized as a critical variable for optimal therapeutic efficacy. For ADCs employing cysteine conjugation, the payload to be delivered has commonly been attached to the antibody backbone via reaction with the native cysteines of the inter-chain disulfides. These cysteines are found in the constant domain of the light chains, the CH1 domain and hinge region of the heavy chains. Alternatively, the chemotype can be conjugated to discrete locations on the antibody backbone by engineering cysteine mutations into the protein sequence, allowing for conjugation in a site-specific manner. For certain chemotypes, site-specific conjugation has demonstrated several distinct advantages over native disulfide conjugation, including homogenous ADC loading, malemide attachment stability and hydrophobic masking of the payload. These effects are particularly applicable to conjugates made at mutation site S239C, which is found on the interior face of the CH2 of the Fc region. Moreover, we have recently discovered that ADCs using the antimitotic tubulysin M show greatly improved chemotype stability when conjugated specifically to the S239C site. In plasma, the tubulysin M molecule is susceptible to the loss of an acetate side chain on the tubuvaline subunit, a change which greatly diminishes tubulin binding and cytotoxicity. Using ex vivo methods and mass spectroscopy we show that, compared to tubulysin M conjugates made to the native interchain disulfide bonds, conjugation to S239C protects the lability of this acetate group and demonstrates improved in vitro cytotoxicity following ADC plasma incubation. With the aim of identifying further protective conjugation locations, we generated and analyzed 20 additional cysteine mutation sites predicted to share similar biophysical properties to S239C. Top performing single mutations were further combined with S239C and evaluated for a combined effect on tubulysin M acetate stability. Surprisingly, several of the new cysteine mutation sites demonstrate a greater effect on tubulysin M stability when used in conjunction with S239C than when used alone. The results on acetate stability following plasma incubation are further reflected by enhanced cytotoxic potency in vitro using pre incubated tubulysin M ADCs. This data demonstrates that appropriate site-specific conjugation can protect against chemotype degradation, and in addition to the advantages of hydrophobic masking and malemide stability, these findings underline the importance of location of chemotype conjugation for optimal ADC design and engineering. Citation Format: Andrew Waight, Chris Leiske, Travis Biechele, Patrick Burke, Rory Rohm, Peter Senter, Dennis Benjamin, Django Sussman. Cysteine mutant location affects chemotype lability in site-specific antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-066. doi:10.1158/1538-7445.AM2017-LB-066

Generation and Characterization of Gas-Phase Doubly Charged Biradical Peptide Ions (M2+••).

The gas-phase chemistry of peptide radical ions is attracting considerable interest in the fields of biology and mass spectrometry owing to its capability to provide sequence information on peptides and proteins. In this study, we observed that doubly charged peptide ions (M2+) can be produced from the collision-induced dissociation (CID) of Hg(II)-adducted peptide ions. The chemical nature and, thus, the dissociation pathways of this hydrogen-deficient biradical M2+ species is intriguing. We investigated the generation and dissociation behavior of this M2+ species under electron-capture dissociation (ECD) and CID conditions. The side-chain loss in the CID of the charge-reduced M+• ions formed by single-electron capture suggested that M2+ existed as a biradical ion. This ion underwent the combination of the two radical sites and conversion to hydrogen surplus species through structural rearrangement with increased energies. This study demonstrated a promising method to generate reactive doubly charged biradical precursor ions and, thus, help characterize novel biomolecules.

Nanostructured Alpha-NiCe Mixed Hydroxide for Highly Sensitive Amperometric Prednisone Sensors

Nickel hydroxide nanoparticles with enhanced electrocatalytic activity and stability in the alpha phase were prepared by incorporation of 20 atom% of Ce(III) ions, that were shown to be oxidized to the catalytic active Ce(IV) species in the mixed hydroxide/oxide nanomaterial. Alpha-NiCe is constituted by smaller nanoparticles as compared with pure nickel hydroxide and can interact with prednisone, a widely prescribed medicine that is controlled by the International Anti-Doping Agency because of its use as anabolic agent. This compound is more or less easily hydroxilated by oxygen and then oxidized to the respective keto species, that was shown to be oxidized further by the modified electrodes upon loss of the alkyl side chain. Thus, a sensitive prednisone sensor was demonstrated exploring the electrocatalytic oxidation reaction instead of the reduction of prednisone to prednisolone, as previously reported in the literature. Sensitivity as high as 3.9×10−2Amol−1L/cm2 and limit of detection as low as 8.4×10−9molL−1 were achieved using flow injection analyses.