Atmospheric Pressure Photoionization with Halogen Anion Attachment for Mass Spectrometric Analysis of Hydrocarbons and Hydrocarbon-Based Polymers.

Aliphatic hydrocarbons and hydrocarbon-based synthetic polymers are of interest in many fields, but their characterization by mass spectrometric methods is generally limited due to their poor ionizability. Recently, atmospheric pressure photoionization (APPI), combined with halogen anion attachment in negative-ion mode, has drawn attention as a potential method for ionizing various polymers without extensive fragmentation or other unwanted side reactions. In this work, the applicability of halogen anion attachment with APPI was studied using several synthetic polymers, including polyethylene, polypropylene, polyisoprene, and polystyrene, as well as simple -alkanes of various chain lengths.

Structural insights into ternary immunocomplex formation and cross-reactivity: binding of an anti-immunocomplex FabB12 to Fab220-testosterone complex.

Anti-immunocomplex (Anti-IC) antibodies have been used in developing noncompetitive immunoassays for detecting small molecule analytics (haptens). These antibodies bind specifically to the primary antibody in complex with hapten. Although several anti-IC antibody-based immunoassays have been developed, structural studies of these systems are very limited.

Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily.

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid.

Robust Approach for Quantifying Glucocorticoid Binding to the Anti-Cortisol Fab Fragment via Native Mass Spectrometry.

In the development of proteins, aptamers, and molecular imprints for diagnostic purposes, a major goal is to obtain a molecule with both a high binding affinity and specificity for the target ligand. Cushing syndrome or Addison's disease can be diagnosed by cortisol level tests. We have previously characterized and solved the crystal structure of an anti-cortisol (17) Fab fragment having a high affinity to cortisol but also significant cross-reactivity to other glucocorticoids, especially the glucocorticoid drug prednisolone.

Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids.

Cortisol is a steroid hormone that is produced by the adrenal gland. It is a primary stress hormone that increases glucose levels in the blood stream. High concentrations of cortisol in the body can be used as a biomarker for acute and chronic stress and related mental and physiological disorders.

Calculation and Visualization of Binding Equilibria in Protein Studies.

A set of simulation applets has been developed for visualizing the behavior of the association and dissociation reactions in protein studies. These reactions are simple equilibrium reactions, and the equilibrium constants, most often dissociation constant , are useful measures of affinity. Equilibria, even in simple systems, may not behave intuitively, which can cause misconceptions and mistakes.

Three-dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6-bladed β-propeller hydrolase family.

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d-xylonolactone. We have determined crystal structures of Cc XylC in complex with d-xylonolactone isomer analogues d-xylopyranose and (r)-(+)-4-hydroxy-2-pyrrolidinone at high resolution. Cc XylC has a 6-bladed β-propeller architecture, which contains a central open channel having the active site at one end.

Xylonolactonase from Is a Mononuclear Nonheme Iron Hydrolase.

xylonolactonase ( XylC, EC 3.1.1.

Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact.

Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.

Allergens and their associated small molecule ligands-their dual role in sensitization.

Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small-molecule ligands. Ligand-binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds.

Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase.

Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the JCM12802 endo-polygalacturonase PGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C.

Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods.

In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.

Development of hypoallergenic variants of the major horse allergen Equ c 1 for immunotherapy by rational structure based engineering.

The use of recombinant allergens is a promising approach in allergen-specific immunotherapy (AIT). Considerable limitation, however, has been the ability of recombinant allergens to activate effector cells leading to allergic reactions. Recombinant hypoallergens with preserved protein folding and capacity to induce protective IgG antibodies binding effectively to the native allergen upon sensitization would be beneficial for safer AIT.

Thermokinetic Analysis of Protein Subunit Exchange by Variable-Temperature Native Mass Spectrometry.

Many protein complexes are assembled from a varying number of subunits, which are continuously exchanging with diverse time scales. This structural dynamics is considered to be important for many regulatory and sensory adaptation processes that occur . We have developed an accurate method for monitoring protein subunit exchange by using native electrospray ionization mass spectrometry (ESI-MS), exemplified here for an extremely stable Rad50 zinc hook (Hk) dimer assembly, Zn(Hk).

Quantitation of Thyroid Hormone Binding to Anti-Thyroxine Antibody Fab Fragment by Native Mass Spectrometry.

Thyroid hormones are important regulatory hormones, acting on nearly every cell in the body. The two main thyroid hormones are l-thyroxine (tetraiodo-l-thyronine, T) and 3,3',5-triiodo-l-thyronine (T), which are produced in the thyroid gland and secreted into the blood stream. Other important thyroid hormone metabolites are 3,3'-diiodo-l-thyronine (T) and l-thyronine (T), which may show increased levels in circulation due to dietary iodine deficiency or other medical disorders.

Bioconjugation with Aminoalkylhydrazine for Efficient Mass Spectrometry-Based Detection of Small Carbonyl Compounds.

Bioconjugation through oxime or hydrazone formation is a versatile strategy for covalent labeling of biomolecules in vitro and in vivo. In this work, a mass spectrometry-based method was developed for the bioconjugation of small carbonyl compounds (CCs) with an aminoalkylhydrazine to form stable hydrazone conjugates that are readily detectable with electrospray ionization mass spectrometry (ESI-MS). Out of all hydrazine reagents tested, 2-(dimethylamino)ethylhydrazine (DMAEH) was selected for further analysis due to the fastest reaction rates observed.

The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry.

The SCP2 (sterol carrier protein 2)-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the β-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized.

Unraveling Substrate Specificity and Catalytic Promiscuity of Aspergillus oryzae Catechol Oxidase.

Catechol oxidases and tyrosinases are coupled binuclear copper enzymes that oxidize various o-diphenolic compounds to corresponding o-quinones. Tyrosinases have an additional monooxygenation ability to hydroxylate monophenol to o-diphenol. It is still not clear what causes the difference in the catalytic activities.

A new crystal form of Aspergillus oryzae catechol oxidase and evaluation of copper site structures in coupled binuclear copper enzymes.

Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.

The crystal structure of D-xylonate dehydratase reveals functional features of enzymes from the Ilv/ED dehydratase family.

The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.

Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose.

Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme Xyn10C/Cel48B from is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated.

The Crystal Structure of a Bacterial l-Arabinonate Dehydratase Contains a [2Fe-2S] Cluster.

We present a novel crystal structure of the IlvD/EDD family enzyme, l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.

Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria.

Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is caused by genetic deficiency of the enzyme aspartylglucosaminidase (AGA) which is involved in glycoprotein degradation. AGU is a progressive disorder that results in severe mental retardation in early adulthood. No curative therapy is currently available for AGU.

Crystallization and X-ray diffraction analysis of an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.

L-Arabinonate dehydratase (EC 4.2.1.

Structural and functional features of the NAD(P) dependent Gfo/Idh/MocA protein family oxidoreductases.

The Gfo/Idh/MocA protein family contains a number of different proteins, which almost exclusively consist of NAD(P)-dependent oxidoreductases that have a diverse set of substrates, typically pyranoses. In this study, to clarify common structural features that would contribute to their function, the available crystal structures of the members of this family have been analyzed. Despite a very low sequence identity, the central features of the three-dimensional structures of the proteins are surprisingly similar.