Strategy for selective chemical cross-linking of tyrosine and lysine residues (original) (raw)
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Ab Initio Studies on the Mechanism of Tyrosine Coupling
The Journal of Physical Chemistry A, 2001
Oxidative stress is considered to be a major contributor to dysfunction in a host of disease states. Reactive oxygen species (ROS) mediate distinct oxidative alterations in biopolymers, including DNA, proteins, lipids, and lipoproteins. Currently, the mechanisms of biochemical reactions underlying oxidative stress are poorly understood because of the instability of ROS. One of the consequences of oxidative stress is one-electron oxidation of tyrosine (Tyr) residues in proteins, which represents a hallmark of this insult and is implicated in the pathogenesis of a number of pathological processes leading to atherosclerosis, inflammatory conditions, multiple system atrophy and several neurodegenerative diseases. Major products of oxidation of Tyr include protein-bound dityrosine and isodityrosine. In this report, the mechanism of tyrosine coupling (including structure and stability of a number of proposed reaction intermediates) is studied by high-level density functional and conventional ab initio methods including B3LYP, MP2, CASSCF, and CASPT2. It is demonstrated that dityrosine and isodityrosine are the most stable structures at all theoretical levels applied. In addition to classical structures of the reaction intermediates, evidence is found for a novel transient structure of Tyr dimer, stacked dityrosyl. This dimer is predicted to exist because of strong electron correlation between two tyrosyl moieties. The counterpoise corrected energy of stacked dityrosyl is below the energy of two tyrosyl radicals by about 95 kJ/mol at the PUMP2/6-31G** level. High proton affinity of tyrosyl radical (about 9.4 eV) suggests that positively charged amino acids in the vicinity of a solvent-exposed Tyr residue may increase the probability of tyrosine coupling.
Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains
The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrop...
Scientific Reports, 2014
Recent studies have indicated that different post-translational modifications (PTMs) synergistically orchestrate specific biological processes by crosstalks. However, the preference of the crosstalk among different PTMs and the evolutionary constraint on the PTM crosstalk need further dissections. In this study, the in situ crosstalk at the same positions among three tyrosine PTMs including sulfation, nitration and phosphorylation were systematically analyzed. The experimentally identified sulfation, nitration and phosphorylation sites were collected and integrated with reliable predictions to perform large-scale analyses of in situ crosstalks. From the results, we observed that the in situ crosstalk between sulfation and nitration is significantly under-represented, whereas both sulfation and nitration prefer to co-occupy with phosphorylation at same tyrosines. Further analyses suggested that sulfation and nitration preferentially co-occur with phosphorylation at specific positions in proteins, and participate in distinct biological processes and functions. More interestingly, the long-term evolutionary analysis indicated that multi-PTM targeting tyrosines didn't show any higher conservation than singly modified ones. Also, the analysis of human genetic variations demonstrated that there is no additional functional constraint on inherited disease, cancer or rare mutations of multiply modified tyrosines. Taken together, our systematic analyses provided a better understanding of the in situ crosstalk among PTMs. T hrough modification of proteins by covalent attachment of other functional groups or by proteolytic cleavage, post-translational modifications (PTMs) temporally and spatially affect protein activity, stability and trafficking, regulate most of biological and physiological functions, and determine the cellular dynamics and plasticity 1-3. In particular, one PTM can crosstalk with other PTMs to synergistically orchestrate specific processes through three distinct mechanisms 4-13. First, different PTMs can co-occur in the same proteins and crosstalk in a cis-regulatory manner 4,5. For example, the phosphorylation of the motif YKXEXXpSP (Y is a hydrophobic residue, X is any amino acid, pS is a phosphorylatable serine) at S303 of HSF1, a heat shock transcription factor, enhances the adjacent lysine sumoylation at K298 4 (Fig. 1a). Also, a recently computational analysis suggested that a considerable proportion of acetylated lysines might influence the PTMs such as phosphorylation, methylation and ubiquitination of adjacent sites 6. Second, one PTM can regulate another PTM by modifying its cognate enzymes and vice versa, in a trans-regulatory mode 7-9. For example, the E3 ubiquitin ligase complex of Rictor/Cullin-1/Rbx1 ubiquitinates an AGC kinase of SGK1 and promote its degradation, whereas the T1135 of Rictor can be phosphorylated by multiple AGC kinases including SGK1, and such a phosphorylation disrupts the interaction of Rictor and Cullin-1 to inhibit the ubiquitination of SGK1 8 (Fig. 1b). Third, multiple PTMs can ''in situ'' interplay with each other by competitively modifying same residues 10-13. For example, a circadian clock protein PER2 is competitively O-GlcNAcylated and phosphorylated at S662, while the in situ crosstalk precisely regulates the PER2 repressor activity 13 (Fig. 1c). In addition, different types of PTM crosstalks can simultaneously occur and regulate biological functions in a complicated manner. For example, PKCd phosphorylates Caspase-3 14 , which reciprocally cleaves PKC-d as a trans-crosstalk 15 (Fig. 1d). Also, p53 can be activated through the PKCd-mediated phosphorylation of S46 16 , and such a phosphorylation promotes its
Mass spectral characterization of organophosphate-labeled lysine in peptides
Analytical …, 2009
Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237. 1, 220.0, 192.0, 163.9, 128.9, and 83.9 amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8 amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.
Oxidative Dimerization of Proteins: Role of Tyrosine Accessibility
Archives of Biochemistry and Biophysics, 2000
To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. Materials and methods. Aqueous solutions of proteins were oxidized by OH • or N 3 • free radicals produced by ␥ or pulse irradiation in an atmosphere of N 2 O. Protein dimers were quantified by SDS-PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. Results. Using N 3 • free radicals, the initial yields of dimerization are equal to (8.6 ؎ 0.7) ؋ 10 ؊9 mol J ؊1 for both proteins. Using OH • free radicals, they become equal to (1.23 ؎ 0.1) ؋ 10 ؊8 and (4.42 ؎ 0.1) ؋ 10 ؊8 mol J ؊1 for hen and turkey egg-white lysozymes, respectively (␥ radiolysis). Discussion. N 3 • radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH • may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH • could easily be followed by intermolecular dimerization.
Journal of Biochemical and Biophysical Methods, 2008
The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.
Tyrosine Analogues for Probing Proton-Coupled Electron Transfer Processes in Peptides and Proteins
Journal of the American Chemical Society, 2009
A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK a of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK a 's that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning one type of aryloxyl in the presence of other possible aryloxyl radicals in peptides and proteins with multiple tyrosine residues between which electrons and protons can be transferred.
Chemo- and Regioselective Lysine Modification on Native Proteins
Journal of the American Chemical Society, 2018
Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Kwas the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those case...