A Pyrene Maleimide with a Flexible Linker for Sampling of Longer Inter-Thiol Distances by Excimer Formation

Pyrene-containing compounds are commonly used in a number of fluorescence-based applications because they can form excited-state dimers (excimers) by stacking interaction between excited-state and ground-state monomers. Their usefulness arises from the facts that excimer formation requires close proximity between the pyrenes and that the excimer emission spectrum is very different from that of the monomers. One of many applications is to assess proximity between specific sites of macromolecules labeled with pyrenes. This has been done using pyrene maleimide, a reagent that reacts with reduced thiols of cysteines, but its use for structural studies of proteins has been rather limited. This is because the introduction of two cysteines at sufficiently close distance from each other to obtain excimer fluorescence upon labeling with pyrene maleimide requires detailed knowledge of the protein structure or extensive site-directed mutagenesis trials. We synthesized and tested a new compound with a 4-carbon methylene linker placed between the maleimide and the pyrene (pyrene-4-maleimide), with the aim of increasing the sampling distance for excimer formation and making the use of excimer fluorescence simpler and more widespread. We tested the new compound on thiol-modified oligonucleotides and showed that it can detect proximity between thiols beyond the reach of pyrene maleimide. Based on its spectroscopic and chemical properties, we suggest that pyrene-4-maleimide is an excellent probe to assess proximities between cysteines in proteins and thiols in other macromolecules, as well as to follow conformational changes

Schematic representation of the pyrene-4-maleimide synthesis and structure.

<p>A. Pyrene maleimide structure. B. Pyrene-4-maleimide synthesis route and structure. Ph₃P: triphenylphosphine; DIAD: diisopropyl azodicarboxylate.</p

Long lifetime of pyrene-4-maleimide excimer emission.

<p>DNA₁₄: reduced single-stranded DNA₁₄ adduct. DNA_14-14c: double-stranded DNA_14-14c adduct. IRF: instrument response function. The red lines are fits of the data to multi-exponential functions, with the two weighted residuals (R_i) <i>vs.</i> time plots corresponding to the double-stranded (top) and single-stranded (bottom) data fits.</p

Double-stranded DNA models with pyrene maleimides attached <i>via</i> thiol linkers.

<p>A. Stick representation view along the DNA long axis. Pyrene-4-maleimide attached to DNA_14-14c and DNA_14-12c is shown in green and red, respectively. Pyrene maleimide attached to DNA_14-14c is shown in blue. The 5′ thiol was present in the 14-bp long strand in all cases. The 3′ thiol was present in the 14-bp or 12-bp long complementary strands. B. Stick representation view perpendicular to that in panel A. Only the pyrenes are shown for clarity. Color coding as in panel A. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026691#s3" target="_blank">Materials and Methods</a> for details.</p

Emission of pyrene compounds reacted with thiol-modified DNA.

<p>A. Emission spectra of single-stranded DNA containing a 5′ end thiol group (DNA₁₄) reacted with pyrene-4-maleimide. Data were normalized to peak 1 intensity from reduced DNA₁₄. [DNA₁₄] was 0.5 µM, and [pyrene-4-maleimide] was 2 µM. B. Emission spectra of double-stranded DNA reacted with pyrene maleimide. DNA_14-14c: DNA₁₄ annealed to fully complementary DNA with a 3′ end thiol group (DNA_14c). DNA_14-12c: DNA₁₄ annealed to a 3′ end two-base shorter complementary oligonucleotide with a 3′ end thiol group. DNA_14-10c: DNA₁₄ annealed to a 3′ end four-base shorter complementary oligonucleotide with a 3′ end thiol group. C. Emission spectra of double-stranded DNA reacted with pyrene-4-maleimide. Insert: schematic representation of the experimental system showing the double-stranded DNAs labeled with pyrene-4-maleimide. The pyrenes are represented by green rhomboids. Data in panels B and C were normalized to peak 1 intensity. The labels in panel B also apply to panel C. D. Excimer/monomer emission ratio. The values were calculated as: excimer/monomer = I_{peak 4}/I_{peak 1}, where I is the highest intensity of the peak, and peak 1 and peak 4 correspond to the excimer and monomer emission peaks. Averages ± SEM from experiments such as those shown in panels B and C (n = 3 for pyrene maleimide, and n = 4 for pyrene-4-maleimide). The asterisk denotes P<0.05 for the pyrene-4-maleimide DNA_14-14c adduct <i>vs.</i> each of the other adducts presented in panel D. The concentrations of the fluorescent probes and double-stranded DNA were 4 µM and 0.5 µM, respectively.</p