Reactivity of Functional Groups on the Protein Surface:  Development of Epoxide Probes for Protein Labeling

Chen, G.; Heim, Alexander; Riether, Doris; Yee, Dominic J.; Milgrom, Yelena; Gawinowicz, Mary Ann; Sameš, Dalibor

doi:10.1021/ja034287m

Cited by 129 publications

(98 citation statements)

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“…Here, we show that distinct classes of reactive carbon electrophiles demonstrate widely divergent amino acid preferences in proteomes. The promiscuity of the SE probe designates it as a highly versatile electrophile for ABPP, as well as potentially related chemical biology endeavors, such as ligand-guided protein surface labeling 14 , which aims to convert reversible ligands into covalent probes by proximity-induced reactivity with nucleophilic amino acids neighboring protein active sites. One could envision improving the target selectivity of SE probes by combining this electrophile with high-affinity binding groups for individual proteins of interest.…”

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confidence: 99%

Disparate proteome reactivity profiles of carbon electrophiles

2008

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Insights into the proteome reactivity of electrophiles are crucial for designing activity-based probes for enzymes lacking cognate affinity labels. Here, we show that different classes of carbon electrophiles exhibit markedly distinct amino acid labeling profiles in proteomes, ranging from selective reactivity with cysteine to adducts with several amino acids. These data thus specify electrophilic chemotypes with restricted and permissive reactivity profiles to guide the tailored design of next-generation functional proteomics probes.The field of activity-based protein profiling (ABPP) applies reactive chemical probes to profile the functional state of enzymes in native proteomes1. Original ABPP probes incorporated well-defined affinity labels as reactive groups to target enzyme classes such as the serine2 and cysteine3 hydrolases. Many enzymes, however, do not possess cognate affinity labels, and the design of ABPP probes for these proteins remains challenging. Structural insights into the substrate-binding pocket of enzyme classes can reveal nucleophilic residues for targeting with appropriate electrophiles. Recent work in the design of protein kinase probes positioned α-fluoromethyl ketone and acyl-phosphate electrophiles within an adenosine triphosphate (ATP) scaffold to exploit the nucleophilicity of proximal cysteine4 and lysine5 residues respectively. Differentiating among electrophilic chemotypes that show restricted and permissive amino acid reactivity profiles should streamline such endeavors to design ABPP probes for a wide range of enzyme classes.A variety of electrophiles are available for incorporation into ABPP probes. The proteome reactivity profiles of iodoacetamide and maleimide reactive groups have been extensively investigated 6 . Here, we expand on these studies by investigating the reactivity of a panel of carbon electrophiles (Fig. 1a), comprising a phenylsulfonate ester (SE, 1), linear-(EP, 2) and spiro-epoxides (SP, 5), an α-chloroacetamide (CA, 3) and an α,β-unsaturated ketone (UK, 4) in complex proteomes. An alkyne was incorporated into these electrophilic frameworks to provide a click chemistry handle for gel and mass spectrometric analysis 7 . Application of these electrophiles to a soluble mouse proteome, followed by click chemistry with a rhodamine azide (Rh-N 3 ) reporter tag and visualization of labeled proteins by SDS-PAGE and in-gel fluorescence scanning, demonstrated that the panel of electrophiles exhibit a range of protein reactivities (see Supplementary Information Fig. 1). Highest reactivity was observed for the UK probe, which demonstrated substantial protein labeling at concentrations as low as 1 μM. The CA and SE electrophiles demonstrated moderate levels of reactivity, whereas, the EP and SP probes displayed little to no protein labeling even at concentrations up to 20 μM. We then examined in greater depth the protein and amino-acid labeling profiles for the three probes that displayed the highest levels of proteome reactivity (SE, CA and UK). To address this ...

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confidence: 99%

Disparate proteome reactivity profiles of carbon electrophiles

2008

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“…A next generation of ligands that demonstrates high affinities (higher than ligands with hydrophobic SREs) and validation of metal ion coordination to surface His residues of CA by X-ray crystallography is necessary to prove the validity and utility of this approach. 604,605 They demonstrated that their ligands selectively labeled one (or two closely spaced) His residues of HCA II, and that the covalent reaction was directed by binding of the ligand to the active site of the enzyme: no labeling of HCA II was observed when a large excess of a high-affinity arylsulfonamide competitor was included in the reaction with the epoxide probe. Chen et al demonstrated that their probe (220) labeled His64, a catalytically essential residue located in the conical cleft of HCA II (see section 4.6), and that it labeled HCA II selectively in the yeast proteome (when an extra, controlled amount of HCA II was added to the proteome).…”

Section: Hydrophilic or Charged Secondary Recognition Elementsmentioning

confidence: 99%

“…Chen et al demonstrated that their probe (220) labeled His64, a catalytically essential residue located in the conical cleft of HCA II (see section 4.6), and that it labeled HCA II selectively in the yeast proteome (when an extra, controlled amount of HCA II was added to the proteome). 604 Using a probe (221) that lacked the fluorescein moiety (Fluor) of, and that was slightly longer than, 220, Takaoka et al were able to label a His residue at the amino terminus of HCA II (His3 or His4), outside of the conical cleft. 605 They were able to regenerate the catalytic activity of the enzyme by removal of the sulfonamide moiety of the tethered molecule by reaction with an alkoxyamine.…”

Section: Hydrophilic or Charged Secondary Recognition Elementsmentioning

confidence: 99%

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

et al. 2008

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I. Introduction to Carbonic Anhydrase (CA) and to the Review 948 1. Introduction: Overview of CA as a Model 948 1.1. Value of Models 950 1.2. Objectives and Scope of the Review 950 2. Overview of Enzymatic Activity 950 3. Medical Relevance 951 II. Structure and Structure−Function Relationships of CA 953 4. Global and Active-Site Structure 953 4.1. Structure of Isoforms 953 4.2. Isolation and Purification 954 4.3. Crystallization 954 4.4. Structures Determined by X-ray Crystallography and NMR 955 4.4.1. Structures Determined by X-ray Crystallography 955 4.4.2. Structure Determined by NMR 955 4.5. Global Structural Features 963 4.6. Structure of the Binding Cavity 964 4.7. Zn II -Bound Water 965 5. Metalloenzyme Variants 966 6. Structure−Function Relationships in the Catalytic Active Site of CA 968 6.1. Effects of Ligands Directly Bound to Zn II 968 6.2. Effects of Indirect Ligands 969 7. Physical-Organic Models of the Active Site of CA 969 III. Using CA as a Model to Study Protein−Ligand Binding 970 8. Assays for Measuring Thermodynamic and Kinetic Parameters for Binding of Substrates and Inhibitors 970 8.1. Overview 970 8.

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“…Erlanson and S.K Hansen in 2008, [80] after Sames and colleagues demonstrated an epoxide as a suitable electrophile during work to develop a selective affinity probe. [81] Benzenesulfonamide, a potent nanomolar inhibitor of carbonic anhydrase II (CAII), was separately modified with twelve electrophiles including an epoxide, in addition to a fluorescent tag, to yield 12 chemical probes. These were individually incubated with a selection of proteins and subsequently analysed by in-gel fluoresence.…”

Section: Activity Based Protein Profiling and Affinity Labellingmentioning

confidence: 99%

Kinetic Template‐Guided Tethering of Fragments

2012

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This thesis is composed of two separate projects: Kinetic Template-Guided Tethering of Fragments and Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1 Degradation after DNA Damage. Kinetic Template-Guided Tethering of FragmentsThe development of a novel methodology for the site-directed discovery of small molecule, protein-binding ligands is described. The protein of interest, with a cysteine thiol (either native or engineered) adjacent to the desired binding pocket, is incubated with mixtures of low molecular weight compounds (fragments) modified with either an acrylamide or a vinyl sulfonamide capture group. Any ligand within the mixture that binds within the pocket brings the capture group into close proximity with the cysteine thiol, promoting a conjugate addition reaction at an increased rate over the background reaction. The capture reaction is designed to be slow, such that during the time course of an experiment, no adduct formation is observed unless the reaction is templated by the protein. By this method, binding ligands are rapidly identified by mass spectrometry analysis of the crude reaction mixtures. The methodology has been termed 'kinetic template-guided tethering'. Design and Synthesis of a Chemical Probe to Dissect the Cellular Signalling Cascade leading to Cyclin D1Degradation after DNA Damage An inhibitor described within the literature was found to attenuate the reduction of cyclin D1 after DNA damage to cells. In order to implement a two-step chemical proteomics strategy to find the molecular target of this inhibitor, a synthesis of the compound with an alkyne appendage was required. The alkyne acts as a functional handle for attachment of a reporter molecule in cells via the Huisgen cycloaddition ('Click') reaction. The design and synthesis of this inhibitor with the alkyne appendage is described.

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Reactivity of Functional Groups on the Protein Surface: Development of Epoxide Probes for Protein Labeling

Cited by 129 publications

References 13 publications

Disparate proteome reactivity profiles of carbon electrophiles

Disparate proteome reactivity profiles of carbon electrophiles

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

Kinetic Template‐Guided Tethering of Fragments

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