Histidine Residues In Proteins Research Articles

Ultrahigh Enzyme Loading Metal-Organic Frameworks for Deep Tissue Pancreatic Cancer Photoimmunotherapy.

Protein drugs hold promise in treating multiple complex diseases, including cancer. The priority of protein drug application is precise delivery of substantial bioactive protein into tumor site. Metal-organic-framework (MOF) is widely considered as a promising carrier to encapsulate protein drug owing to the noncovalent interaction between carrier and protein. However, limited loading efficiency and potential toxicity of metal ion in MOF restrict its application in clinical research. Herein, a tumor targeted collagenase-encapsulating MOF via protein-metal ion-organic ligand coordination (PMOCol ) for refining deep tissue pancreatic cancer photoimmunotherapy is developed. By an expedient method in which the ratio of metal ion, histidine residues of protein and ligand is precisely controlled, PMOCol is constructed with ultrahigh encapsulation efficiency (80.3 wt%) and can release collagenase with high enzymatic activity for tumor extracellular matrix (ECM) regulation after reaching tumor microenvironment (TME). Moreover, PMOcol exhibits intensively poorer toxicity than the zeolitic imidazolate framework-8 biomineralized protein. After treatment, the pancreatic tumor with abundant ECM shows enhanced immunocyte infiltration owing to extracellular matrix degradation that improves suppressive TME. By integrating hyperthermia agent with strong near-infrared absorption (1064 nm), PMOCol can induce acute immunogenicity to host immunity activation and systemic immune memory production to prevent tumor development and recurrence.

A tyrosine, histidine-selective bifunctional cross-linker for protein structure analysis.

Chemical cross-linking mass spectrometry (XL-MS) significantly contributes to the analysis of protein structures and the elucidation of protein-protein interactions. Currently available cross-linkers mainly target N-terminus, lysine, glutamate, aspartate, and cysteine residues in protein. Herein, a bifunctional cross-linker, named [4,4'-(disulfanediylbis(ethane-2,1-diyl)) bis(1-methyl-1,2,4-triazolidine-3,5-dione)] (DBMT) has been designed and characterized aiming to extremely expand the application of XL-MS approach. DBMT is capable of selectively targeting tyrosine residue in protein via an electrochemical click reaction, and/or targeting histidine residue in protein in the presence of 1O2 generated under photocatalytic reaction. A novel cross-linking strategy based on this cross-linker has been developed and demonstrated using model proteins, which provides a complementary XL-MS tool analyzing protein structure, protein complexes, protein-protein interactions, and even protein dynamics.

Histidine phosphorylation in human cells; a needle or phantom in the haystack?

It has been suggested that in mammalian cells histidine residues in proteins may become as frequently phosphorylated as serine, threonine and tyrosine, and may play a key role in mammalian signaling. Here we applied a robust workflow that earlier allowed us to detect histidine phosphorylation in bacteria unambiguously, to probe for histidine phosphorylation in four human cell lines. Initially, seemingly hundreds of protein histidine phosphorylations were picked up in all studied human cell lines. However, careful examination of the data, and several control experiments, led us to the conclusion that >99% of these initially assigned pHis sites were not genuine, and should be site localized to neighboring Ser/Thr residues. Nevertheless, our methods are selective enough to detect just a handful of genuine pHis sites in mammalian cells, representing well-known enzymatic intermediates. Consequently, we do not find any evidence in our data supporting that protein histidine phosphorylation plays a role in mammalian signaling.

Histidine-specific bioconjugation via visible-light-promoted thioacetal activation.

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.

Tetra-nickel substituted polyoxotungsate as an efficient sorbent for the isolation of His6-tagged proteins from cell lysate

By virtue of the flexible structure of polyoxometalates, Ni2+ is encapsulated into trivacant lacunary tungstophosphate ligands by the form of [Ni4] cluster to offer a tetra-nickel substituted polyoxotungsate K6Na4[Ni4(H2O)2(PW9O34)2] (Ni4P2). The Ni4P2 is then immobilized onto the surface of SiO2 nanoparticles by self-assembly under electrostatic interactions to give the product of Ni4P2@SiO2 composites. Due to the specific affinity between substituted Ni2+ in the polyoxotungsate and the histidine residues of protein, Ni4P2@SiO2 composites exhibit highly adsorption selectivity towards histidine protein. This Ni4P2@SiO2 composite is of high stability, and SDS-PAGE assay indicates that it can be used repeatedly as an efficient sorbent for the isolation of His6-tagged proteins from cell lysate with improved performance when compared with commercial NTA-Ni2+ column.

Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics

Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC–MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC–MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research.

A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements

Double quantum coherence (DQC) ESR is a sensitive method to measure magnetic dipolar interactions between spin labels. However, the DQC experiment on Cu2+ centers presents a challenge at X-band. The Cu2+ centers are usually coordinated to histidine residues in proteins. The electron–nuclear interaction between the Cu2+ ion and the remote nitrogen in the imidazole ring can interfere with the electron–electron dipolar interaction. Herein, we report on a modified DQC experiment that has the advantage of reduced contributions from electron–nuclear interactions, which enhances the resolution of the DQC signal to the electron–electron dipolar modulations. The modified pulse-sequence is verified on Cu2+–NO system in a polyalanine-based peptide and on a coupled Cu2+ system in a polyproline-based peptide. The modified DQC data were compared with the DEER data and good agreement was found.

Analysis of conformational changes in rhodopsin by histidine hydrogen-deuterium exchange.

Hydrogen-deuterium exchange (HDX) is a technique that measures the exchange of protein hydrogens for deuteriums in a D2O-containing buffer, providing readout of the structural dynamics. Histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) is a variation of this technique that measures the slow HDX of imidazole C2 hydrogens of histidines. This measurement, when accompanied by pH titration, provides both pK as and half-lives (t 1/2) of the HDX reaction for individual histidine residues in proteins. The pK a and t 1/2 values indicate the electrostatic environment and the degree of side-chain solvent accessibility of the histidine residues, respectively. Herein we describe an experimental protocol to characterize rhodopsin by His-HDX-MS. This technique can be used to monitor different states of rhodopsin and might be useful for monitoring longtime scale events in other GPCRs.

2D IR Spectroscopy of Histidine: Probing Side-Chain Structure and Dynamics via Backbone Amide Vibrations

It is well known that histidine is involved in many biological functions due to the structural versatility of its side chain. However, probing the conformational transitions of histidine in proteins, especially those occurring on an ultrafast time scale, is difficult. Herein we show, using a histidine dipeptide as a model, that it is possible to probe the tautomer and protonation status of a histidine residue by measuring the two-dimensional infrared (2D IR) spectrum of its amide I vibrational transition. Specifically, for the histidine dipeptide studied, the amide unit of the histidine gives rise to three spectrally resolvable amide I features at approximately 1630, 1644, and 1656 cm–1, respectively, which, based on measurements at different pH values and frequency calculations, are assigned to a τ tautomer (1630 cm–1 component) and a π tautomer with a hydrated (1644 cm–1 component) or dehydrated (1656 cm–1 component) amide. Because of the intrinsic ultrafast time resolution of 2D IR spectroscopy, we believe that the current approach, when combined with the isotope editing techniques, will be useful in revealing the structural dynamics of key histidine residues in proteins that are important for function.

Structure of Indole···Imidazole Heterodimer in a Supersonic Jet: A Gas Phase Study on the Interaction between the Aromatic Side Chains of Tryptophan and Histidine Residues in Proteins

In this study, we have investigated the binding motifs between the aromatic side chains of tryptophan and histidine residues in proteins by studying the indole···imidazole heterodimer in a supersonic jet. Different spectroscopic techniques including resonant two-photon ionization (R2PI), UV-UV hole-burning, and resonant ion dip infrared (RIDIR) spectroscopy merged with quantum chemistry calculations have been used for this work. UV-UV hole-burning spectroscopy has been used to confirm the presence of only one structure of the dimer in the experiment. From the comparison of the RIDIR spectrum of the observed dimer with the theoretical IR spectra of different structures of the dimer, it is found that the dimer present in the experiment has a V-shaped structure held by N-H···N hydrogen bond, C-H···π, and weakly present π···π stacking interactions. The most important finding of the present study is that the noncovalent interactions present in the observed dimer have a close resemblance with those present between tryptophan and histidine residues in a nonfluorescent flavoprotein. The present spectroscopic investigation on the indole···imidazole dimer has also immense pharmaceutical significance as this imparts molecular level understanding about the binding motifs of the imidazole drugs with the indole chromophore present in proteins.

Cobalt (III) Complexes as Novel Matrix Metalloproteinase-9 Inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc binding endopeptidases that play crucial roles in various physiological processes and diseases such as embryogenic growth, angiogenesis, arthritis, skin ulceration, liver fibrosis and tumor metastasis. Because of their implications in a wide range of diseases, MMPs are considered as intriguing drug targets. The majority of MMP inhibitors are organic small molecules containing a hydroxamate functionality for the zinc binding group. This hydroxamate group binds to a zinc(II) center in a bidentate fashion and creates a distorted trigonal bipyramidal geometry. Although the hydroxamate group is the most effective zinc binding moiety reported, it has shown two major limitations in clinical trials: low bioavailability and lack of specificity. Because the hydroxamate group is prone to rapid metabolism and has a high binding affinity towards various transition metals, many efforts have been made to develop novel zinc binding groups, including reverse hydroxamate, phosphate, and pyridinone. However, the in vitro activity of these newly developed inhibitors are not as high as the hydroxamate derivatives. It was previously reported that cobalt(III) acacen complexes could interact with histidine residues of proteins and model peptides. The cobalt(III) complexes bind histidine residues in active sites and on enzyme surfaces in a random fashion. Spectroscopic and chromatographic data suggested that the complexes bind to a histidine residue by axial ligand substitution. When an appropriate targeting group is attached, the cobalt(III) complexes can selectively inhibit the histidinecontaining enzymes such as thermolysin, human α-thrombin, and carbonic anhydrase. Since histidine is the most commonly found residue in the active sites of zinc enzymes and zinc-binding proteins, it has been demonstrated that the cobalt(III) complexes can interact with zinc finger proteins, such as HIV-1 nucleocapsid protein NCp7 and human zinc finger transcription factor Sp1. It was also reported that a Co(III) complex with a oligonucleotide targeting Snail family zinc finger transcription factor resulted in a selective inactivation of a transcriptional activity implicated in embryonic development and breast cancer. In this work, a series of the cobalt(III) complexes are prepared as novel MMP inhibitors. Since cobalt(III) complexes have high affinity towards histidine residues and zinc binding proteins, they can be engineered to disrupt the zinc binding active site of MMP. Furthermore, the cobalt(III) complexes may be able to provide better solubility and higher binding affinity than the known organic inhibitors. To test this hypothesis, a series of cobalt(III) complexes were designed and synthesized. For active site directing groups, biphenyl sulfonate and biphenyl amide group were chosen based on the structures of well known MMP inhibitors. In vitro activity of these complexes was evaluated for MMP-9 (gelatinase B), because MMP-9 is one of the most highly expressed MMPs in tumors and has been implicated in tumor aggressiveness. The syntheses of cobalt(III) complexes are shown in Schemes 1-2. The synthesis of 1 and 2 begins with the methylation of commercially available Cbz-Lys(Boc)-OH as

Quantitative Analysis of Acrolein-Specific Adducts Generated during Lipid Peroxidation–Modification of Proteins in Vitro: Identification of Nτ-(3-Propanal)histidine as the Major Adduct

Acrolein, a ubiquitous pollutant in the environment, is endogenously formed through oxidation reactions and is believed to be involved in cytopathological effects observed during oxidative stress. Acrolein exerts these effects because of its facile reactivity with biological materials, particularly proteins. In the present study, we quantitatively analyzed the acrolein-specific adducts generated during lipid peroxidation-modification of proteins and identified the acrolein adduct most abundantly generated in the in vitro oxidized low-density lipoproteins (LDL). Taking advantage of the fact that the acrolein-lysine adducts, N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) and N(ε)-(3-methylpyridinium)lysine (MP-lysine), have stable core structures resistant to the acid hydrolysis condition of proteins, we examined the formation of these adducts in proteins using high performance liquid chromatography with online electrospray ionization tandem mass spectrometry. However, only MP-lysine was detected as a minor product in the iron/ascorbate-mediated oxidation of polyunsaturated fatty acids in the presence of proteins and in the oxidized low-density lipoproteins (LDL). However, using a reductive amination-based pyridylamination method, we analyzed the acrolein-specific adducts with a carbonyl functionality and found that acrolein modification of the protein produced a number of carbonylated amino acids, including an acrolein-histidine adduct. On the basis of the chemical and spectroscopic evidence, this adduct was identified as N(τ)-(3-propanal)histidine. More notably, N(τ)-(3-propanal)histidine appeared to be one of the major adducts generated in the oxidized LDL. These data suggest that acrolein generated during lipid peroxidation may primarily react with histidine residues of proteins to form N(τ)-(3-propanal)histidine.

Acrolein

This special issue of Molecular Nutrition and Food Research is dedicated to acrolein (2-propenal), a constituent of many foods that has poorly understood long-term effects on human health. It is therefore not a surprise that the number of publications on acrolein has steadily increased from 61 publications per year in 2000 to 197 in 2010 (source: http://gopubmed.org/). Acrolein is primarily formed from carbohydrates, lipids and certain amino acids in foods by heat treatment. Besides intake of acrolein via the diet, humans are exposed to acrolein through inhalation of vapors emitted from heated cooking oils, cigarette smoke and inhalation of combustion products of fossil fuels. Finally, acrolein is formed endogenously as a product of polyamine metabolism and oxidative stress. The adverse effects of acrolein on human health originate from its capability to form Michael-type adducts with cysteine, lysine and histidine residues in proteins and with amine groups in DNA bases. These and other aspects are covered in this special issue by nine reviews and three research articles. Abraham et al. (this issue) provide an overview of the metabolism of acrolein, its toxicity and carcinogenicity and its occurrence in various foods and beverages. They conclude that exposure to acrolein via the diet probably does not pose immediate health risks, but, at the same time, point out that a risk assessment is difficult to make due to technical difficulties with measuring acrolein levels in foods. An accurate risk assessment is further complicated by lack of knowledge regarding the immediate and especially the long-term consequences of acrolein's ability to interfere with many metabolic pathways and (redox-sensitive) signaling networks. The contributions by Bein and Leikauf, Conklin et al., Fabisiak et al. and by Myers et al. provide a wealth of new information with respect to the effects of acrolein on gene regulation. Other contributions emphasize the chemical diversity of the cellular damage exerted by acrolein and the molecular consequences of acrolein exposure in cultured cells, laboratory animals and humans. Knowing how and to what extent acrolein modifies DNA and proteins is necessary for elucidating the mechanisms by which acrolein causes cellular dysfunction. The mechanisms of acrolein-related toxicity that affect the nervous system (spinal cord trauma), the brain and the lung are discussed in this issue by experts in the field. Also discussed are strategies aimed at mitigating the deleterious effects of acrolein by using acrolein scavengers such as flavonoids and other (dietary) phytochemicals, vitamins, neutraceuticals and pharmaceuticals. Progress in understanding acrolein's impact on health would not have been possible without the advent of new analytical technologies. Recent analytical breakthroughs include the development of acrolein-specific immunochemical techniques and microarray technologies for assessing acrolein toxicity. Substantial progress has been achieved in assessing acrolein exposure at the molecular level in vivo. Modern mass spectrometry-based techniques enable the analysis of acrolein-mediated DNA damage (reviewed by Chen in this issue) and the analysis of acrolein adducts in mitochondrial proteomes (Wu et al.). This special issue on acrolein offers an overview of the astounding progress that has been made in unraveling the multifaceted effects of acrolein on biological systems and human health. Despite the advances made in understanding the biological consequences of acute acrolein exposure, many questions remain unanswered regarding the health impact of chronic acrolein exposure via the diet and the environment. We agree with the contributors of this issue that further progress in this area requires the development of methods for reliable quantification of acrolein in foods and beverages as well as methods for probing the interaction of acrolein with its biological environment. We are grateful to the Editor-in-Chief for providing us the opportunity to invite distinguished authors to contribute to this issue, to the reviewers for their invaluable comments and to the Editorial Office for tireless support in smoothing the path towards completion of this special issue. Jan Frederik Stevens Oregon State University Claudia S. Maier Oregon State University

Reversible immobilization of rhamnulose-1-phosphate aldolase for biocatalysis: Enzyme loading optimization and aldol addition kinetic modeling

Reusability of the usually expensive immobilization supports by means of reversible immobilization techniques helps reduce the overall cost of the biocatalytic process. One of these techniques, the reversible binding of the histidine residues of proteins with the metal chelated in affinity supports, has been extensively exploited in purification of His-tagged proteins in one-step. The use of these supports for immobilization of His-tagged recombinant enzymes allows performing simultaneously the enzyme purification and immobilization, thus further reducing the number of process steps, enzyme losses and costs. In this work, His-tagged rhamnulose-1-phosphate aldolase (RhuA) was immobilized onto iminodiacetic acid-functionalized agarose charged with cobalt (Co-IDA) for the catalysis of aldol addition reactions of industrial interest. Enzyme loading was optimized to maximize the immobilized activity avoiding diffusion limitations for both the RhuA natural reaction and the synthetic aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal (10 AU/mL of support and 25 AU/mL of support, respectively). RhuA:Co-IDA biocatalysts with high retained activity (92%) and enhanced stability (37 days of half life time) were obtained. Kinetic modeling of the synthetic aldol addition between DHAP and (S)-Cbz-alaninal and the unwanted secondary reaction catalyzed by soluble RhuA was carried out. RhuA:Co-IDA catalysis of the same reactions was found to follow the same kinetic model.

Effects of conformation and hydrogen bonding on the CC and NCN stretching Raman bands of N‐deuterated histidinium

AbstractHistidine is an important and versatile amino acid residue that plays a variety of structural and functional roles in proteins. Although the Raman bands of histidine are generally weak, histidine in the N‐deuterated cationic form with imidazole NπD and NτD bonds (N‐deuterated histidinium) gives two strong Raman bands assignable to the C4C5 stretch (νCC) and the NπC2Nτ symmetric stretch (νNCN) of the imidazole ring. We examined the Raman spectra of N‐deuterated histidinium in 12 crystals with known structures. The observed νCC and νNCN wavenumbers were analyzed to find empirical correlations with the conformation and hydrogen bonding. The effect of conformation on the vibrational wavenumber was expressed as a threefold cosine function of the CαCβC4C5 torsional angle. The effect of hydrogen bonding at Nπ or Nτ was assumed to be proportional to the inverse sixth power of the distance between the hydrogen and acceptor atoms. Multiple linear regression analysis clearly shows that the conformational effect on the vibrational wavenumber is comparable for νCC and νNCN. The hydrogen bond at Nπ weakly lowers the νCC wavenumber and substantially raises the νNCN wavenumber. On the other hand, the hydrogen bond at Nτ strongly raises the νCC wavenumber but does not affect the νNCN wavenumber. These empirical correlations may be useful in Raman spectral analysis of the conformation and hydrogen bonding states of histidine residues in proteins. Copyright © 2010 John Wiley & Sons, Ltd.

Cyclooxygenase-2 generates anti-inflammatory mediators from omega-3 fatty acids

Electrophilic fatty acids are generated during inflammation by non-enzymatic reactions and can modulate inflammatory responses. We used a new mass spectrometry-based electrophile capture strategy to reveal the formation of electrophilic oxo-derivatives (EFOX) from the omega-3 fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA). These EFOX were generated by a cyclooxygenase-2 (COX-2)-catalyzed mechanism in activated macrophages. Modulation of COX-2 activity by aspirin increased the rate of EFOX production and their intracellular levels. Owing to their electrophilic nature, EFOX adducted to cysteine and histidine residues of proteins and activated Nrf2-dependent anti-oxidant gene expression. We confirmed the anti-inflammatory nature of DHA- and DPA-derived EFOX by showing that they can act as peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists and inhibit pro-inflammatory cytokine and nitric oxide production, all within biological concentration ranges. These data support the idea that EFOX are signaling mediators that transduce the beneficial clinical effects of omega-3 fatty acids, COX-2 and aspirin.

Molecular Mechanisms of 4-Hydroxy-2-nonenal and Acrolein Toxicity: Nucleophilic Targets and Adduct Formation

Acrolein and 4-hydroxy-2-nonenal (HNE) are byproducts of lipid peroxidation and are thought to play central roles in various traumatic injuries and disease states that involve cellular oxidative stress, for example, spinal cord trauma, diabetes, and Alzheimer's disease. In this review, we will discuss the chemical attributes of acrolein and HNE that determine their toxicities. Specifically, these aldehydes are classified as type 2 alkenes and are characterized by an alpha,beta-unsaturated carbonyl structure. This structure is a conjugated system that contains mobile pi-electrons. The carbonyl oxygen atom is electronegative and can promote the withdrawal of mobile electron density from the beta-carbon atom causing regional electron deficiency. On the basis of this type of electron polarizability, both acrolein and HNE are considered to be soft electrophiles that preferentially form 1,4-Michael type adducts with soft nucleophiles. Proteomic, quantum mechanical, and kinetic data will be presented, indicating that cysteine sulfhydryl groups are the primary soft nucleophilic targets of acrolein and HNE. This is in contrast to nitrogen groups on harder biological nucleophiles such as lysine or histidine residues. The toxicological outcome of adduct formation is not only dependent upon residue selectivity but also the importance of the targeted amino acid in protein function or structure. In attempting to discern the toxicological significance of a given adduct, we will consider the normal roles of cysteine, lysine, and histidine residues in proteins and the relative merits of corresponding adducts in the manifestations of diseases or toxic states. Understanding the molecular actions of acrolein and HNE could provide insight into many pathogenic conditions that involve initial cellular oxidative stress and could, thereby, offer new efficacious avenues of pharmacological defense.

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of 13C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically 13C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for 13C(epsilon1) nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from 15N backbone relaxation measurements. Compared to measurements of backbone nuclei, 13C(epsilon1) dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the 13C(epsilon1) dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.

Reversible Phosphorylation of Histidine Residues in Proteins from Vertebrates

Signaling by kinases and phosphatases that act on serine, threonine, and tyrosine residues of proteins is among the most extensively studied regulatory mechanisms in mammalian cells, and research focused in this area is ongoing. We are just beginning to appreciate that such signaling mechanisms are extended and enriched by the reversible phosphorylation of histidine residues. The most exciting developments in this field to date come from studies on the beta subunit of heterotrimeric guanosine triphosphate-binding proteins (G proteins), the enzyme adenosine 5'-triphosphate-citrate lyase, and now the Ca(2+)-activated K(+) channel KCa3.1, all of which are targeted by nucleoside diphosphate kinase (which phosphorylates histidines) and protein histidine phosphatase (which dephosphorylates phosphorylated histidines).

Rapid synthesis of Abelson tyrosine kinase inhibitors using click chemistry

Protein kinases catalyze the phosphorylation of serine, threonine, tyrosine and histidine residues in proteins. Aberrant regulation of kinase activity has been implicated in many diseases including cancer. Thus development of new strategies for kinase inhibitor design remains an active area of research with direct relevance to drug development. Abelson (Abl) tyrosine kinase is one of the Src-family of tyrosine kinases and is directly implicated in Chronic Myelogenous Leukemia (CML). In this article, we have, for the first time, developed an efficient method for the construction of small molecule-based bisubstrate inhibitors of Abl kinase using click chemistry. Subsequent biochemical screenings revealed a set of moderately potent inhibitors, a few of which have comparable potency to Imatinib (an FDA-approved drug for treatment of chronic myeloid leukemia) against Abl.