Nanosecond time-resolved circular polarization of fluorescence: study of NADH bound to horse liver alcohol dehydrogenase.

Abstract

Circularly polarized luminescence (CPL) spectroscopy provides information on the excited-state chirality of a lumiphore analogous but complementary to information regarding the ground-state chirality derived from circular dichroism. While CPL spectra are sensitive to the structure surrounding the lumiphore, their applicability to studies of complex biomolecules is limited by the fact that as a rule these molecules contain several identical lumiphores in different environments (e.g., tryptophan residues in a protein). Moreover, the ensemble of biomolecules in solution frequently exists in an equilibrium among different conformational states that interconvert on a time scale longer than that involved in the emission of luminescence. Hence, a typical CPL spectrum is a superposition of several independent contributions with a high degree of spectral overlap. One effective approach for the resolution of such composite spectra is by monitoring the time evolution of the CPL signal and the luminescence decay after electronic excitation of the lumiphore and correlating the CPL components to those obtained from analysis of the luminescence decay. Individual terms in the CPL may then be assigned distinct decay components. Time-dependent CPL may also arise from excited-state interactions of the lumiphore that affect the local chiral properties during the excited state lifetime. In such cases each lumiphore may possess a timedependent CPL, and the time-resolved CPL (TR-CPL) then can be used to obtain information on the dynamics of excitedstate interactions.We have recently utilized time-resolved optical activity and demonstrated that we could assign each of the four components obtained in the analysis of the decay of the roomtemperature phosphorescence of bacterial glucose-6-phosphate dehydrogenase (G6PDH) with a time-independent gem derived from the analysis of the time-resolved circularly polarized phosphorescence from the enzyme's tryptophan residues (4). This assignment allowed us to conclude that the room-temperature phosphorescence of G6PDH originates in three or four tryptophan residues, each with a unique, timeindependent CPL contribution.In

Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level

“…The multiexponential 2P-fluorescence decay of NADH (PBS, pH 7.4), as a function of mMDH and LDH concentration (Supplementary Figure S2A and S2B), suggest the presence of multiple structural conformations, as was proposed previously [68, 77]. In addition, the fluorescence intensity (data not shown) and average lifetime of NADH-mMDH is twice (Supplementary Figure S2C) that of the unbound cofactor [78, 79].…”

“…At fully occupied LDH sites [68, 80], the average fluorescence lifetime is increased by 3 fold as compared with free NADH (Supplementary Figure S1C). The observed enhancement of NADH fluorescence in the protein environment is due to the unfolded conformation of adenine and nicotinamide ring [9, 60, 77]. It is worth mentioning that the slow component (1.57 ns, 6%) is significantly faster than that of cellular autofluorescence decays (~3.3 ns, ~15%).…”

Rapid time-resolved Circular Polarization Luminescence (CPL) emission spectroscopy

“…This is particularly advantageous for applications such as security and biosensing 37 . Time-gating of SM-CPL spectrometer acquisition has previously been achieved and used by others to study chiral molecular interactions [45][46][47][48][49] , but the required SM-CPL spectrometer modifications were technically challenging and resulted in severe restrictions with regards to waveband scan range, poor acquisition rate, and poor signal to noise ratios 50,51 . Here, we achieve full-spectra time-gated detection with the SS-CPL spectrometer.…”

Circularly polarized lanthanide luminescence for advanced security inks

“…For example, our custom-built PEM-SM-CPL spectrometer requires ~45 minutes to acquire a scan of a typical Eu III complex with default scan settings (150 nm spectral window, 1 ms dwell time / 0.5 nm increments, 5 spectral accumulations). 72 Time-gated CPL measurement has been achieved with some PEM-SM-CPL spectrometers, [81][82][83][84][85] but only over a limited wavelength window, with a poor signal-to-noise ratio (SNR) and substantially increased scan times. 86,87 Given that time-gated measurements are already challenging over a small wavelength window we can safely say that time-gated full-spectrum CPL measurements are impractical with PEM-SM-CPL spectrometers.…”