scholarly journals Halomethyl-Triazoles for Rapid, Site-Selective Protein Modification

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5461
Author(s):  
Richard C. Brewster ◽  
Alison N. Hulme
Keyword(s):  
Protein Modification ◽  
Protein Translation ◽  
Cysteine Residue ◽  
Amino Acid Residues ◽  
Post Translational Modifications ◽  
Select Amino Acid ◽  
Methionine Residues ◽  
And Function ◽  
Alkylating Reagents ◽  
Site Selective

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.

scholarly journals Partial amino acid sequence of rat pre-prolactin

1977 ◽  
Vol 161 (1) ◽  
pp. 189-192 ◽  
Author(s):  
R A Maurer ◽  
J Gorski ◽  
D J McKean
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Cysteine Residue ◽  
Amino Acid Residues ◽  
Partial Amino Acid Sequence ◽  
Considerable Similarity ◽  
Rat Pituitary ◽  
N Terminus ◽  
Methionine Residues

Rat pituitary mRNA was used to direct the cell-free synthesis of pre-prolactin labelled with [4,5-3H]leucine and either [35S] methioninc or [35S] cystine. Sequence analysis of the labelled protein indicates that pre-prolactin has 29 amino acid residues joined to the N-terminus of the prolactin sequence. Leucine residues were found at positions 13, 14, 15, 16, 21 and 22, methionine residues at positions 1, 17 and 18, and a cysteine residue at position 24 of the precursor sequence, and this partial sequence shows considerable similarity with other precursors that have been sequenced.

scholarly journals Mutually exclusive locales for N-linked glycans and disorder in glycoproteins

2018 ◽  
Author(s):  
Alvina Singh ◽  
Indu Kumari ◽  
Dharma Pally ◽  
Shyamili Goutham ◽  
Sujasha Ghosh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Stability ◽  
Amino Acid Residue ◽  
Tertiary Structure ◽  
Amino Acid Residues ◽  
Mutual Exclusivity ◽  
Post Translational Modifications ◽  
Phylogenetic Conservation ◽  
And Function ◽  
Disordered Regions

AbstractSeveral post-translational modifications of proteins lie within regions of disorder, stretches of amino acid residues that exhibit a dynamic tertiary structure and resist crystallization. Such localization has been proposed to expand the binding versatility of the disordered regions, and hence, the repertoire of interacting partners for the proteins. However, investigating a dataset of 500 human N-linked glycoproteins, we observed that the sites of N-linked glycosylations, or N-glycosites, lay predominantly within the regions of predicted order rather than their unstructured counterparts. This mutual exclusivity between disordered stretches and N-glycosites could not be reconciled merely through asymmetry in distribution of asparagines, serines or threonines residues, which comprise the minimum-required signature for conjugation by N-linked glycans, but rather by a contextual enrichment of these residues next to each other within the ordered portions. In fact, N-glycosite neighborhoods and disordered stretches showed distinct sets of enriched residues suggesting their individualized roles in protein phenotype. N-glycosite neighborhood residues also showed higher phylogenetic conservation than disordered stretches within amniote orthologs of glycoproteins. However, a universal search for residue-combinations that are putatively domain-constitutive ranked the disordered regions higher than the N-glycosite neighborhoods. We propose that amino acid residue-combinations bias the permissivity for N-glycoconjugation within ordered regions, so as to balance the tradeoff between the evolution of protein stability, and function, contributed by the N-linked glycans and disordered regions respectively.

scholarly journals Post-translational Modifications in Oral Bacteria and Their Functional Impact

2021 ◽  
Vol 12 ◽  
Author(s):  
Qizhao Ma ◽  
Qiong Zhang ◽  
Yang Chen ◽  
Shuxing Yu ◽  
Jun Huang ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Regulatory Networks ◽  
Oral Bacteria ◽  
Amino Acid Residues ◽  
Post Translational Modifications ◽  
Physiological Processes ◽  
The Face ◽  
Domains Of Life ◽  
And Function ◽  
External Stimuli

Oral bacteria colonize the oral cavity, surrounding complex and variable environments. Post-translational modifications (PTMs) are an efficient biochemical mechanism across all domains of life. Oral bacteria could depend on PTMs to quickly regulate their metabolic processes in the face of external stimuli. In recent years, thanks to advances in enrichment strategies, the number and variety of PTMs that have been identified and characterized in oral bacteria have increased. PTMs, covalently modified by diverse enzymes, occur in amino acid residues of the target substrate, altering the functions of proteins involved in different biological processes. For example, Ptk1 reciprocally phosphorylates Php1 on tyrosine residues 159 and 161, required for Porphyromonas gingivalis EPS production and community development with the antecedent oral biofilm constituent Streptococcus gordonii, and in turn Php1 dephosphorylates Ptk1 and rapidly causes the conversion of Ptk1 to a state of low tyrosine phosphorylation. Protein acetylation is also widespread in oral bacteria. In the acetylome of Streptococcus mutans, 973 acetylation sites were identified in 445 proteins, accounting for 22.7% of overall proteins involving virulence factors and pathogenic processes. Other PTMs in oral bacteria include serine or threonine glycosylation in Cnm involving intracerebral hemorrhage, arginine citrullination in peptidylarginine deiminases (PADs), leading to inflammation, lysine succinylation in P. gingivalis virulence factors (gingipains, fimbriae, RagB, and PorR), and cysteine glutathionylation in thioredoxin-like protein (Tlp) in response to oxidative stress in S. mutans. Here we review oral bacterial PTMs, focusing on acetylation, phosphorylation, glycosylation, citrullination, succinylation, and glutathionylation, and corresponding modifying enzymes. We describe different PTMs in association with some examples, discussing their potential role and function in oral bacteria physiological processes and regulatory networks. Identification and characterization of PTMs not only contribute to understanding their role in oral bacterial virulence, adaption, and resistance but will open new avenues to treat oral infectious diseases.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

Unrestrictive protein modification localization and quality control for open search of mass spectra

2018 ◽  
Author(s):  
Zhiwu An ◽  
Fuzhou Gong ◽  
Yan Fu
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Tandem Mass Spectrometry ◽  
Mass Spectra ◽  
Protein Modification ◽  
Software Tool ◽  
Tandem Mass ◽  
Simulation Data ◽  
Post Translational Modifications

We have developed PTMiner, a first software tool for automated, confident filtering, localization and annotation of protein post-translational modifications identified by open (mass-tolerant) search of large tandem mass spectrometry datasets. The performance of the software was validated on carefully designed simulation data. <br>

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

2018 ◽  
Author(s):  
Daniel D. Brauer ◽  
Emily C. Hartman ◽  
Daniel L.V. Bader ◽  
Zoe N. Merz ◽  
Danielle Tullman-Ercek ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Modification ◽  
Positive Charge ◽  
Specific Protein ◽  
High Yield ◽  
Site Specific ◽  
Protein Cage ◽  
Protein Carriers ◽  
Site Selective ◽  
Targeted Library

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

The Involvement of Post-Translational Modifications in Alzheimer's Disease

2018 ◽  
Vol 15 (4) ◽  
pp. 313-335 ◽  
Author(s):  
Serena Marcelli ◽  
Massimo Corbo ◽  
Filomena Iannuzzi ◽  
Lucia Negri ◽  
Fabio Blandini ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Protein Modification ◽  
Neurodegenerative Disorder ◽  
Amyloid Β ◽  
Normal Function ◽  
Ageing Population ◽  
Covalent Bonds ◽  
Post Translational Modifications ◽  
Related Proteins

Background: Alzheimer's disease (AD) is a neurodegenerative disorder recognized as the most common cause of chronic dementia among the ageing population. AD is histopathologically characterized by progressive loss of neurons and deposits of insoluble proteins, primarily composed of amyloid-β pelaques and neurofibrillary tangles (NFTs). Methods: Several molecular processes contribute to the formation of AD cellular hallmarks. Among them, post-translational modifications (PTMs) represent an attractive mechanism underlying the formation of covalent bonds between chemical groups/peptides to target proteins, which ultimately result modified in their function. Most of the proteins related to AD undergo PTMs. Several recent studies show that AD-related proteins like APP, Aβ, tau, BACE1 undergo post-translational modifications. The effect of PTMs contributes to the normal function of cells, although aberrant protein modification, which may depend on many factors, can drive the onset or support the development of AD. Results: Here we will discuss the effect of several PTMs on the functionality of AD-related proteins potentially contributing to the development of AD pathology. Conclusion: We will consider the role of Ubiquitination, Phosphorylation, SUMOylation, Acetylation and Nitrosylation on specific AD-related proteins and, more interestingly, the possible interactions that may occur between such different PTMs.

scholarly journals Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B

2021 ◽  
Vol 22 (5) ◽  
pp. 2501
Author(s):  
Sonja Hinz ◽  
Dominik Jung ◽  
Dorota Hauert ◽  
Hagen S. Bachmann
Keyword(s):  
Protein Function ◽  
Tumor Development ◽  
Cysteine Residue ◽  
Type I ◽  
Pharmacological Characterization ◽  
Anti Cancer ◽  
And Function ◽  
Caax Motif ◽  
Important Drug

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

scholarly journals The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

2021 ◽  
Vol 22 (5) ◽  
pp. 2591
Author(s):  
Pengfei Ma ◽  
Jie Li ◽  
Lei Qi ◽  
Xiuzhu Dong
Keyword(s):  
Heat Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Small Heat Shock Protein ◽  
Phase Cell ◽  
Molecular Weights ◽  
Oxidative Inactivation ◽  
Methionine Residues ◽  
Shock Proteins ◽  
And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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