Chemical Modification Reagents Research Articles

Fabrication of superhydrophobic self-cleaning Ni-Co-MoS2/Ni composite coating

This work investigated the parallel magnetically assisted jet electrodeposition of Ni-Co-MoS2/Ni superhydrophobic composite coatings. As a comparison, the effect of the magnetic field on Ni-Co alloy coatings was analyzed. Then, micro/nano MoS2/Ni particles were further added to study the performance of Ni-Co-MoS2/Ni composite coatings under different concentrations of micro/nano particles. Research indicated that the Ni-Co-MoS2/Ni composite coating surface exhibits coral like micro/nano structures, and the micro/nano structures became denser as the concentration of micro/nano particles increased. The superhydrophobic composite coatings were obtained without any chemical modification reagents. The contact angle of the superhydrophobic composite coating was as high as 153.2° and the sliding angle was 8.7° when the amount of micro/nano particles was added at 5 g/L. The coral like micro/nano structures of the composite coating provided additional air cushion to support surface water droplets and exhibited excellent self-cleaning performance. In addition, the composite coating also showed excellent corrosion resistance and wear resistance, which was still relatively intact after friction, only exhibiting few wears on coral like micro/nano structures, it still maintained a good silver mirror effect after friction. This work suggested that parallel magnetically assisted jet electrodeposition could fabricate superhydrophobic composite coatings with excellent performance.

Thermodynamic Overview of Bioconjugation Reactions Pertinent to Lysine and Cysteine Peptide and Protein Residues

Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities.

Proteome-Derived Peptide Libraries for Deep Specificity Profiling of N-terminal Modification Reagents.

Protein and peptide N termini are important targets for selective modification with chemoproteomics reagents and bioconjugation tools. The N-terminal ⍺-amine occurs only once in each polypeptide chain, making it an attractive target for protein bioconjugation. In cells, new N termini can be generated by proteolytic cleavage and captured by N-terminal modification reagents that enable proteome-wide identification of protease substrates through tandem mass spectrometry (LC-MS/MS). An understanding of the N-terminal sequence specificity of the modification reagents is critical for each of these applications. Proteome-derived peptide libraries in combination with LC-MS/MS are powerful tools for profiling the sequence specificity of N-terminal modification reagents. These libraries are highly diverse, and LC-MS/MS enables analysis of the modification efficiencies of tens of thousands of sequences in a single experiment. Proteome-derived peptide libraries are a powerful tool for profiling the sequence specificities of enzymatic and chemical peptide labeling reagents. Subtiligase, an enzymatic modification reagent, and 2-pyridinecarboxaldehyde (2PCA), a chemical modification reagent, are two reagents that have been developed for selective N-terminal peptide modification and can be studied using proteome-derived peptide libraries. This protocol outlines the steps for generating N-terminally diverse proteome-derived peptide libraries and for applying these libraries to profile the specificity of N-terminal modification reagents. Although we detail the steps for profiling the specificity of 2PCA and subtiligase in Escherichia coli and human cells, these protocols can easily be adapted to alternative proteome sources and other N-terminal peptide labeling reagents. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of N-terminally diverse proteome-derived peptide libraries from E. coli Alternate Protocol: Generation of N-terminally diverse proteome-derived peptide libraries from human cells Basic Protocol 2: Characterizing the specificity of 2-pyridinecarboxaldehyde using proteome-derived peptide libraries Basic Protocol 3: Characterizing the specificity of subtiligase using proteome-derived peptide libraries.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: β-mercaptoethanol (βME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20–95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146–1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Synthesis and structure regulation of armor-wearing biomass-based porous carbon: Suppression the leakage current and self-discharge of supercapacitors

Biomass-based porous carbon is a very promising electrode material for supercapacitors, but adverse leakage current and self-discharge has hindered its practical applications. Herein, high-performance armor-wearing biomass-based porous carbon (NP-RHPC) was prepared with finely adjustable surface properties and micro-structure using rice husk as carbon source and coal pitch as chemical modification reagent. The synergetic effect of the stable biochar skeleton and the pitch carbon armor ensures NP-RHPC has low leakage current and self-discharge, long cyclic stability, and good rate performance. The leakage current and open circuit voltage attenuation rate of NP-RHPC are lower than those of N-RHPC without pitch carbon armor and the commercial capacitor carbon of YP-50F. The synthesized NP-RHPC has ideal specific capacitance of 209.2 F g−1 and the capacitance retention rate is up to 95.0% over 20,000 cycles at a high current density of 1.0 A g−1, which is higher than that of N-RHPC. This impressively long-term cyclic performance of NP-RHPC can be ascribed to the excellent electrical conductivity and especially the reduction of the unstable oxygen-containing groups. This study provides insights for the rational design of high-performance armor-wearing biomass-based porous carbon electrode material towards the advanced application of biomass and coal pitch in energy storage.

Intranuclear Delivery of DNA Nanostructures via Cellular Mechanotransduction.

DNA nanostructures are attractive gene carriers for nanomedicine applications, yet their delivery to the nucleus remains inefficient. We present the application of extracellular mechanical stimuli to activate cellular mechanotransduction for boosting the intranuclear delivery of DNA nanostructures. Treating mammalian cells with polythymidine-rich spherical nucleic acids (poly(T) SNAs) under gentle compression by a single coverslip leads to up to ∼50% nuclear accumulation without severe endosomal entrapment, cytotoxicity, or long-term membrane damage; no chemical modification or transfection reagent is needed. Gentle compression activates Rho-ROCK mechanotransduction and causes nuclear translocation of YAP. Joint compression and treatment with poly(T) oligonucleotides upregulate genes linked to myosin, actin filament, and nuclear import. In turn, Rho-ROCK, myosin, and importin mediate the nuclear entry of poly(T) SNAs. Treatment of endothelioma cells with poly(T) SNAs bearing antisense oligonucleotides under compression inhibits an intranuclear oncogene. Our data should inspire the marriage of DNA nanotechnology and cellular biomechanics for intranuclear applications.

Structured sludge derived multifunctional layer for simultaneous separation of oil/water emulsions and anions contaminants

The effective treatment of complex oily wastewater is of great significance but still a considerable challenge, since single-function, expensive reagents, and complicated process have emerged as shackles for practical applications. Herein, with the objective to waste-control-waste, we proposed a facile and sustainable strategy to fabricate a low-cost multifunctional layer from hazardous waste aluminum sludge (WAS) for complex oily wastewater management. The as-designed layered double oxides/WAS (LDOs/WAS) layer with three-dimensional (3D) hierarchical rough surface exhibited excellent underwater superoleophobicity even under corrosive conditions and low adhesion to oil without any chemical modification reagent treatment. Significantly, the layer can be applied to gravity-directed simultaneous efficient oil-in-water emulsions and anions (taking phosphate as an example) separation with a separation efficiency for emulsion and phosphate up to 99.4% and 99.1%, respectively, and a high separation flux of above 2585 L m−2 h−1. Notably, the flux can be controlled simply and flexibly by adjusting the thickness of the layer. Furthermore, the layer also displayed excellent thermal stability, chemical stability, durability and recyclability. Therefore, this work not only presents a promising approach to design sludge-based multifunctional materials for complex oily wastewater remediation, but also shows great potential and value in environmental pollutions reduction and industrial applications.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis.

In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) inEscherichia coli cells. During purification using Ni-affinity chromatography, the 35kDa protein was degraded via several intermediates to form a 24kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35kDa protein to the 24kDa protein was investigated. Reagents reacting with sulphhydryl groups exerted significant effects strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibitedGraphical Abstract the degradation of the 24kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the MEROPS peptidase database.

Preparation and Characterization of Halloysite-Based Carriers for Quercetin Loading and Release

AbstractHalloysite nanotubes (HNTs) have attracted much attention as delivery carriers for various drugs, but the loading of one such drug, quercetin, on HNTs has been investigated only rarely and usually involved cyclic vacuum pumping. The main objective of the present study was to develop a novel carrier system based on HNTs for quercetin delivery without a vacuum process and to investigate the effect of chemical modification of HNTs on the loading and release of quercetin. For this purpose, comparative studies of five chemical modification reagents (sodium lauroamphoacetate, cocoamidopropyl betaine, 1-hydroxyethyl 2-nonyl imidazoline betaine, triethanolamine, and dipicolinic acid) functionalized on HNTs were investigated for quercetin loading and in vitro release. Characterization of raw halloysite, modified halloysite, and quercetin-loaded halloysite were done by X-ray diffraction (XRD), Fourier-transform infrared spectrometry (FTIR), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM). The results indicated that chemical modification could improve the interactions between HNTs and quercetin. After chemical modification, quercetin was anchored to both the inner and outer surfaces of HNTs by electrostatic attraction, hydrogen bonding, and van der Waals forces. Sodium lauroamphoacetate-modified HNTs were given the highest loading of 1.96 wt.% among the five reagents. Cocamidopropyl betaine-modified HNTs exhibited the best sustained-release profile with only 29.07% for initial burst release and 480 h of consecutive release. Carboxyl groups of the modification reagent improved the loading capacity of quercetin. Amide groups prolonged drug release due to the strong affinity between amine and phenolic hydroxyl groups of quercetin. The release of quercetin from the cocamidopropyl betaine-modified HNTs fitted a first-order kinetics model well. The present study suggested that cocamidopropyl betaine-modified HNTs offer promise as vehicles for delivery of quercetin and for extending the application of quercetin.

Preparation of novel photosensitive indium niobate gel films and their characteristics

In this study, preparation of photosensitive indium niobate (InNbO4) gel films by using a chemical modification reagent and their characteristics were investigated. In the electronic absorption spectra of the InNbO4 gel films, the absorption peak assigned a chelate ring was observed at 340 nm, this peak intensity decreased by UV irradiation. From this result, the obtained gel films exhibit photosensitivity. About 10 μm patterns could be fabricated. The InNbO4 film heat-treated at 600–900 °C exhibited diffraction peaks of monoclinic crystals and the photoinduced hydrophillic properties.

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs).

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 μM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

Chemical labelling for visualizing native AMPA receptors in live neurons

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

A protease-resistant α-galactosidase from Pleurotus djamor with broad pH stability and good hydrolytic activity toward raffinose family oligosaccharides

An acidic α-galactosidase designated as PDGI (Pleurotus djamor α-galactosidase) was purified to homogeneity with 290-fold purification and a specific activity of 52.18 units/mg by means of ion exchange chromatography and gel filtration chromatography. PDGI is a monomeric protein exhibiting a molecular mass of 60kDa in SDS-PAGE and gel filtration. The optimum pH and temperature of the enzyme with pNPGal as substrate were 5.0 and 53.5°C, respectively. It displayed great pH stability within the pH range 3.0–10.0. Besides, the enzyme harbored remarkable resistance to acid protease and varying degrees of tolerance to other proteases: trypsin>collagenase Type-I>α-chymotrypsin neutral protease>proteinaseK. It was strongly inhibited by K+, Cd2+, Cu2+, Hg2+, Al3+, Fe3+ and Ag + ions. The chemical modification reagents diethypyrocarbonate (DEPC), 2,3-butanedione (DIC) and trinitrophenol (TNBS) increased the activity of PDGI 1.5-fold whereas N-bromosuccinimide (NBS) and parachloro-mercuri-benzoate (PCMB) drastically suppressed its activity. PDGI displayed activity toward stachyose and raffinose. The Km values for hydrolysis of pNPGal, stachyose and raffinose were 0.76, 7.63 and 6.29mM, respectively. Furthermore, PDGI degraded raffinose and sthachyose. These results suggest that PDGI has great potential for elimination of the non-digestible and flatulence-causing oligosaccharides stachyose and raffinose from legumes.

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk

An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate–phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50°C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a Km value of 2.8mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H2O2 in milk, the lower limit of determination was 0.1μM, and the recoveries of added H2O2 were 96–109%.

Purification and characterisation of two acidic peroxidase isoforms from the sheaths of bamboo shoots

SummaryThree isoforms of peroxidase (POD) were isolated from the sheaths of bamboo shoots by chromatofocusing on Polybuffer exchanger PBE 94. POD‐A was partially purified, and POD‐B and POD‐C were purified and characterised. POD‐A was a basic peroxidase, whereas POD‐B and POD‐C were acidic peroxidases with different isoelectric points. Using o‐phenylenediamine (OPD) as a hydrogen‐donor, the optimum temperatures for function of POD‐B and POD‐C were 55 and 45–55 °C, respectively, while both had the same optimum pH of 4.5. Both isoforms were stable between 30 and 60 °C and between pH 4.5 and 10. The activities of the POD isoforms towards hydrogen‐donors were both pH and concentration dependent. Under optimal conditions, POD‐B and POD‐C catalysed the oxidation of catechol, pyrogallol, and OPD at higher rates than guaiacol, o‐dianisidine and 2, 2’‐azino‐bis‐ (3‐ethylbenzothiazoline‐6‐sulphonic acid). Both isoforms were almost completely inhibited by chemical modification reagent diethyl pyrocarbonate (4.5 mM).

Catalytic characteristics of plant-esterase from wheat flour

A plant-esterase extracted from wheat flour and purified with a PEG1000/NaH(2)PO(4) aqueous two-phase system was characterized for its catalytic characteristics. The optimal condition for plant-esterase to catalyze 1-naphthyl acetate was at 30°C, pH 6.5. It kept stability at 20°C during 120min and at pH 5.5 during 60h. The effects of metal ions, chemical modification reagents and pesticides on plant-esterase activity were investigated. It was found that Ba(2+) and Pb(2+) at concentrations of 20mM significantly inhibited the activity of plant-esterase while Mg(2+), Ca(2+) and Fe(2+) at the same concentration enhanced the enzyme activity. Chemical modification reagents significantly influenced the activity of plant-esterase. Particularly, PMSF (4.5mM) and N-bromosuccinimide (11mM) inhibited by 5.40-19.87% of the enzyme activity. It is implied that serine and tryptophan are related to the enzyme activity. Plant-esterase were displayed concentration-dependent inhibition by dichlorvos, carbofuran and carbendazim (IC50=0.31-63.12ppm). All these results indicated that catalytic efficiency of plant-esterase strongly depends on reaction conditions, activity effectors and amino acid residues at the active site. It makes meaningful guidance on further design of sensing material in monitoring pesticides.

Tethering Chemistry and K+ Channels

Voltage-gated K+ channels are dynamic macromolecular machines that open and close in response to changes in membrane potential. These multisubunit membrane-embedded proteins are responsible for governing neuronal excitability, maintaining cardiac rhythmicity, and regulating epithelial electrolyte homeostasis. High resolution crystal structures have provided snapshots of K+ channels caught in different states with incriminating molecular detail. Nonetheless, the connection between these static images and the specific trajectories of K+ channel movements is still being resolved by biochemical experimentation. Electrophysiological recordings in the presence of chemical modifying reagents have been a staple in ion channel structure/function studies during both the pre- and post-crystal structure eras. Small molecule tethering agents (chemoselective electrophiles linked to ligands) have proven to be particularly useful tools for defining the architecture and motions of K+ channels. This Minireview examines the synthesis and utilization of chemical tethering agents to probe and manipulate the assembly, structure, function, and molecular movements of voltage-gated K+ channel protein complexes.

Purification and biochemical characterization of a superoxide dismutase from the soluble fraction of the cyanobacterium, Spirulina platensis

A superoxide dismutase (SOD) was purified from Spirulina platensis sonicate. The SOD was purified to homogeneity (48-fold and 0.24% yield) through ammonium sulphate precipitation and DEAE-52 anion exchange chromatography. The SOD from S. platensis appeared to be a homodimer with a molecular weight of 30kDa and a subunit MW of 15kDa as determined by both native polyacrylamide gel electrophoresis and mass spectrometry. The enzyme activity was stable at pH 6.5-10.0 and 50°C. Using group-specific chemical modifying reagents, the amino acids arginine, histidine, tryptophan, tyrosine and aspartic acid were identified to be essential for S. platensis SOD activity. The amino acid composition was found to lack methionine and cysteine. The inhibition of activity by H2O2 suggests that the enzyme may be an iron containing SOD.

Reactivity-Based Analysis of Domain Structures in Native Replication Protein A

Replication protein A (RPA) is an essential heterotrimeric ssDNA binding protein that participates in DNA repair, replication, and recombination. Though X-ray and NMR experiments have been used to determine three-dimensional structure models of the protein's domain fragments, a complete RPA structural model has not been reported. To test whether the fragment structures faithfully represent the same portions in the native solution-state protein, we have examined the structure of RPA under biologically relevant conditions. We have probed the location of multiple amino acids within the native RPA three-dimensional structure using reactivity of these amino acids toward proteolytic and chemical modification reagents. In turn, we evaluated different structural models by comparing the observed native RPA reactivities with anticipated reactivities based on candidate structural models. Our results show that our reactivity analysis approach is capable of critically assessing structure models and can be a basis for selecting the most relevant from among alternate models of a protein structure. Using this analytical approach, we verified the relevance of RPA fragment models to the native protein structure. Our results further indicate several important features of native RPA's structure in solution, such as flexibility at specific locations in RPA, particularly in the C-terminal region of RPA70. Our findings are consistent with reported DNA-free structural models and support the role of conformational change in the ssDNA binding mechanism of RPA.

LYS70 of E. coli Quinolinate Phosphoribosyltransferase Is Protected from Chemical Modification by Formation of an Inhibitor Complex.

Quinolinate phosphoribosyltransferase was examined for susceptibility to different chemical modification reagents. Loss of enzyme activity with trinitrobenzenesulfonate (TNBS) occurred when 1.1 lysines per subunit were modified. Tryptic digestion of the modified enzyme followed by HPLC-MS analysis of the peptides showed Lys70 reacts with TNBS. Based on x-ray studies, this amino acid participates in a conformational change distant from the active site.