Two-Site Ionic Labeling with Pyranine:  Implications for Structural Dynamics Studies of Polymers and Polypeptides by Time-Resolved Fluorescence Anisotropy

Abstract

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine). TRFA analysis shows that such labeling provides a more rigid attachment of the fluorophore to the macromolecule than the covalent or single-point ionic labeling of amino groups, leading to time-resolved anisotropy decays that better reflect the backbone motion of the labeled polymer segment. Optimal coupling of pyranine to biomolecule dynamics is shown to be obtained for appropriately spaced Arg groups, and in such cases the ionic binding is stable up to 150 mM ionic strength. TRFA was used to monitor the behavior of pyranine-labeled poly(allylamine) (PAM) and poly-d-lysine (PL) in sodium silicate derived sol-gel materials and revealed significant restriction of backbone motion upon entrapment for both polymers, an observation that was not readily apparent in a previous study with entrapped fluorescein-labeled PAM and PL. The implications of these findings for fluorescence studies of polymer and biomolecule dynamics are discussed.

NMR Spectroscopy Can Characterize Proteins Encapsulated in a Sol-Gel Matrix

“…Indeed, with only a few peaks and such large linewidths in the SG spectrum, we were unable to measure T 1 for the encapsulated protein. These observations parallel those upon encapsulation of small cationic molecules, where the electrostatic attraction between probe and the negatively charged SG walls causes a large population of the probes to become effectively immobilized and/or to undergo transient immobilization. ,− …”

“…[24][25][26] These findings parallel earlier ones that show negative and neutral (or nearly neutral) solutes are free to tumble when encapsulated, while cationic solutes interact strongly with the anionic silicate walls. 21,[27][28][29][30][31][32] The resolved peaks of SG-encapsulated cyt b 5 and Mb have nearly 2-fold greater linewidths than those of the solution spectra (∼1.6× for cyt b 5 ; ∼1.9× for Mb). There are two likely sources of this increase: magnetic susceptibility dispersion introduced by inhomogeneities in the SG; increases in rotational correlation times (τ c ) caused by encapsulation.…”

“…These observations parallel those upon encapsulation of small cationic molecules, where the electrostatic attraction between probe and the negatively charged SG walls causes a large population of the probes to become effectively immobilized and/or to undergo transient immobilization. 21,[27][28][29][30][31] In addition to broadened lines, the contact-shifted peaks of the encapsulated proteins show small, but significant, shifts of as much as ∼1.9 ppm (Figure 2). 34 As shown in Figure 2, shifted 1 H-peaks are not associated with the protein surface and hence likely reflect small structural changes upon encapsulation.…”

Switching on the Phosphorescence of Pyrene by Cycloplatination

“…Pyrene has been commonly used as a fluorescent probe because of its intense fluorescence, excimeric formation, , and fluorescence anisotropy . Most of the applications of pyrene focus on its lowest energy singlet state (S 1 ), but the corresponding triplet excited state (T 1 ) and its phosphorescence have rarely been harnessed in chemical sensing and photochemical reactions .…”

Aqueous Anion Receptors through Reduction of Subcomponent Self‐Assembled Structures

“…This property allows it to be used for measuring intracellular pH values in vivo. [17] By 1 H NMR spectroscopy, two different sets of peaks were observed as G6 was titrated into host 4 ( Figure S24). The first set of peaks that appears was assigned to the 1:1 hostguest complex, as its complete formation was reached upon addition of one equivalent of G6.…”