Proteomic analysis of fatty-acylated proteins in mammalian cells with chemical reporters reveals S-acylation of histone H3 variants.

Bioorthogonal chemical reporters are useful tools for visualizing and identifying post-translational modifications on proteins. Here we report the proteomic analysis of mammalian proteins targeted by a series of fatty acid chemical reporters ranging from myristic to stearic acid. The large-scale analysis of total cell lysates from fully solubilized Jurkat T cells identified known fatty-acylated proteins and many new candidates, including nuclear proteins and in particular histone H3 variants. We demonstrate that histones H3.1, H3.2, and H3.3 are modified with fatty acid chemical reporters and identify the conserved cysteine 110 as a new site of S-acylation on histone H3.2. This newly discovered modification of histone H3 could have implications for nuclear organization and chromatin regulation. The unbiased proteomic analysis of fatty-acylated proteins using chemical reporters has revealed a greater diversity of lipid-modified proteins in mammalian cells and identified a novel post-translational modification of histones.

Tandem fluorescence imaging of dynamic S-acylation and protein turnover.

The functional significance and regulation of reversible S-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other S-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.

Robust fluorescent detection of protein fatty-acylation with chemical reporters.

Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

Comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics.

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na2S2O4-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.

Seeing small molecules in action with bioorthogonal chemistry.

Chemical probes that target specific protein families offer powerful tools to accelerate drug discovery. Small molecules modified with uniquely reactive functional groups and detection tags provide novel tools to characterize complex proteomes functionally and also to help determine the specificity of small molecule inhibitors toward various enzyme/protein classes. This review highlights the application of bioorthogonal chemistries in combination with chemical probes, which together are offering unprecedented opportunities to dissect the functions of enzyme/protein families in vivo and enabling more precise target identification of small molecules. Advances in chemical probes and bioorthogonal reactions are poised to reveal new therapeutic targets and to facilitate the discovery and characterization of small molecules aimed at disease.

Thermal [1,5] hydrogen sigmatropic shifts in cis,cis-1,3-cyclononadienes probed by gas-phase kinetic studies and density functional theory calculations.

The kinetics of gas-phase thermal [1,5] hydrogen shifts interconverting the five isomeric mono-deuterium-labeled cis,cis-1,3-cyclononadienes have been followed at four temperatures from 240 to 287 degrees C. The activation parameters found were Ea = 37.1 +/- 0.8 kcal/mol, log A = 11.6 +/- 0.3, DeltaH++ = 36.0 +/- 0.8 kcal/mol, and DeltaS++ = -9.0 +/- 0.3 eu. Density functional theory based calculations have provided geometries and energies for the ground-state cyclononadiene conformational isomers, for the transition states linking one to another, and for the transition states for [1,5] hydrogen shifts responsible for isomerizations among the five labeled dienes. A generalized formulation of the Winstein-Holness equation is presented and applied to the complex system, one that involves 11 ground-state conformers, 10 transition states separating them, and five transition states for [1,5] hydrogen shifts. The value for the empirical Ea derived from calculated mole fractions of ground-state conformers and calculated energies for specific ground-state conformers and [1,5] hydrogen shift transition structures was 37.5 kcal/mol, in excellent agreement with the experimentally obtained activation energy. The significance of conformational options in various ground states and transition structures for the [1,5] hydrogen shifts is considerable, an inference that may well have general applicability.

Gas-phase kinetics and activation parameters for thermal [1,5] hydrogen shifts interconverting monodeuterium- labeled 1,3-cycloheptadienes.

The kinetics of thermal equilibrations among monodeuterium-labeled 1,3-cycloheptadienes in the gas phase followed from 154 to 190 degrees C provide activation parameters for the [1,5] shift of a single hydrogen: E(a) = (27.5 +/- 0.9) kcal/mol and log A = 9.7 [corrected] +/- 0.4. These activation parameters imply a comparatively low E(a) barrier balanced by demandingly specific geometric constraints, for DeltaS (170 degrees C) = -17 [corrected] e.u.

Deuterium kinetic isotope effects and mechanism of the thermal isomerization of bicyclo[4.2.0]oct-7-ene to 1,3-cyclooctadiene.

The thermal conversion of cis-bicyclo[4.2.0]oct-7-ene to cis,cis-1,3-cyclooctadiene might involve a direct disrotatory ring opening, or it might possibly take place by way of cis,trans-1,3-cyclooctadiene. This cis,trans-diene might possibly form the more stable cis,cis isomer through a [1,5] hydrogen shift or a trans-to-cis isomerization about the trans double bond. Deuterium kinetic isotope effect determinations for the isomerizations of 2,2,5,5-d(4)-bicyclo[4.2.0]oct-7-ene and 7,8-d(2)-bicyclo[4.2.0]oct-7-ene rule out these two alternatives because the observed effects are much smaller than would be anticipated for these mechanisms: k(H)/k(D)(d(4)) at 250 degrees C is 1.17 (1.04 per D), and k(H)/k(D)(d(2)) at 238 degrees C is 1.20 (1.10 per D). The direct disrotatory ring opening route remains the preferred mechanism.