2-Ureido-4[1H]-pyrimidinones are known to dimerize via a strong quadruple hydrogen bond array. A detailed study of the dimerization constant and lifetime of the dimer is presented here. Excimer fluorescence of pyrene-labeled 2-ureido-4[1H]-pyrimidinone 1b was used to determine a dimerization constant Kdim of 6 × 107 M-1 in CHCl3, 1 × 107 M-1 in chloroform saturated with water, and 6 × 108 M-1 in toluene (all at 298 K). Under these conditions, the preexchange lifetime of the similar dimers of both 1d and 1e is 170 ms in CDCl3, 80 ms in wet CDCl3, and 1.7 s in toluene-d8, as determined by dynamic NMR spectroscopy. Association rate constants were calculated from the Kdim values and the preexchange lifetimes. The resulting values are significantly lower than the diffusion-controlled association rate constants calculated using the Stokes−Einstein and the Debeije equations. This difference is ascribed to a tautomeric equilibrium of the monomer between the dimerizing 4[1H]-pyrimidinone and nondimerizing 6[1H]-py...

Stability and Lifetime of Quadruply Hydrogen Bonded 2-Ureido-4[1H]-pyrimidinone Dimers

Analysis of time-resolved fluorescence anisotropy decays.

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.

/pdf/detection-of-actin-assembly-by-fluorescence-energy-transfer-3p0ts5bzg4.pdf

Detection of actin assembly by fluorescence energy transfer.

Analysis of fluorescence anisotropy decays by a least square method

Photoinduced electron transfer processes in fullerene–organic chromophore systems

G actin, labelled presumably on cysteine-373 with the fluorescent chromophore N-iodoacetyl-N'-(5 sulfo-1-napthyl)-ethylenediamine and purified by Sephacryl S-200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations. It was found that these anisotropy decays could be fitted by a sum of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5 degrees C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5 degrees C) were introduced in order to characterize the F actin motions. The lower of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions. The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb12836.x

Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2880%2981090-8

Pulse fluorimetric study of labelled actin—DNase I complex

Excimer fluorescence to monitor transitions of the association dissociation equilibria induced by dilution of proteins composed of subunits.

Fluorescence-detected circular dichroism (FDCD) was introduced into the study of protein conformation changes. Actin was used as a model protein which undergoes dynamic conformation changes as it polymerizes. Actin labeled with N-(1-pyrene)iodoacetamide (PIA) showed monomer fluorescence peak at 386 and 410 nm, and excimer fluorescence peak at around 480 nm. Excimer was formed by PIA-dimers labeled to different sites of amino acid residues. New information concerned with actin structural changes were monitored by fluorescence emission spectra excited with left- and right-circulary polarized light at 355 nm. FDCD intensities were shown as the difference in the fluorescence emission ΔF, where ΔF=(F
 L−F
 R)/(F
 L+F
 R) denoting F
 L and F
 R as emissions obtained by excitation with left- and right-circulary polarized light. When solvent conditions of PIA-actin were changed by addition of NaCl, TFE, or ATP, ΔF showed sensitive responses to these compounds. From the analysis of ΔF
 M and ΔF
 E which represent the peaks of ΔF at the monomer- and excimer-emission band, the information concerned with the actin intrastructural changes were obtained. This method based on monitoring the excimer fluorescence with FDCD could be used for other proteins to extract finer structural changes that cannot be detected by the normal fluorescence spectroscopy.

Excimer Fluorescence as a Tool for Monitoring Protein Domain Dynamics Applied to Actin Conformation Changes Based on Circulary Polarized Fluorescence Spectroscopy

A system to use fluorometry for the search of protein crystallization buffers was developed. The screening of candidates was done with a use of short gel-filtration column (Sephacryl S-100 HR) linked to the fluorometer. Protein elution was monitored by intrinsic fluorescence or emission from its labels. This method was applied to actin and actin complexes. When nuclei were formed in actin solution, preceding the peak of actin, a new peak appeared on the elution curve. It was found that the fluorescence allows detection of even small amount of nuclei formed in the buffer. Using the screened buffers, crystal growths were attempted. The images of crystals were captured by fluorescence microscope. The monitoring of nuclei with this method will accelerate the screening of crystallization buffers. This system is applicable to the crystallization of other proteins and their complexes.

Takamitsu Ikkai

Papers

Anisotropy decay of labelled actin. Evidence of the flexibility of the peptide chain in F-actin molecules.

Pulse fluorimetric study of labelled actin—DNase I complex

Excimer fluorescence to monitor transitions of the association dissociation equilibria induced by dilution of proteins composed of subunits.

Excimer Fluorescence as a Tool for Monitoring Protein Domain Dynamics Applied to Actin Conformation Changes Based on Circulary Polarized Fluorescence Spectroscopy

Introduction of Fluorometry to the Screening of Protein Crystallization Buffers