Patrik R. Callis

Mechanisms of Tryptophan Fluorescence Shifts in Proteins

Tryptophan fluorescence wavelength is widely used as a tool to monitor changes in proteins and to make inferences regarding local structure and dynamics. We have predicted the fluorescence wavelengths of 19 tryptophans in 16 proteins, starting with crystal structures and using a hybrid quantum mechanical-classical molecular dynamics method with the assumption that only electrostatic interactions of the tryptophan ring electron density with the surrounding protein and solvent affect the transition energy. With only one adjustable parameter, the scaling of the quantum mechanical atomic charges as seen by the protein/solvent environment, the mean absolute deviation between predicted and observed fluorescence maximum wavelength is 6 nm. The modeling of electrostatic interactions, including hydration, in proteins is vital to understanding function and structure, and this study helps to assess the effectiveness of current electrostatic models.

Fluorometric determination of the neutral lipid content of microalgal cells using Nile Red

The fluorophore Nile Red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) has been used to determine neutral lipid in microalgal cells. Cellular fluorescence of stained cells and gravimetrically or chromatographically determined lipid were linearly correlated when Nile Red was excited at 488 – 525 nm and the fluorescent emision measured at 570 – 600 nm. Nile Red is a vital stain which allowed flow cytometric sorting of live microalgal populations based on their lipid content.

Perturbation selection rules for multiphoton electronic spectroscopy of neutral alternant hydrocarbonsa)

A one‐electron perturbation which is to significantly enhance a ‘‘forbidden’’ single‐photon or multiphoton optical transition between any two states of an alternant hydrocarbon must satisfy pseudoparity selection rules in addition to the usual group theoretical selection rules. Pseudoparity predicts whether vibronic or inductive perturbations, e.g., will be successful, while group theory specifies the proper perturbation symmetries. A principal result is that for a given electronic transition, the roles of vibronic, and inductive perturbations are complementary. Their roles reverse depending on whether the transition requires an even or odd number of photons.

Hybrid simulations of solvation effects on electronic spectra: Indoles in water

Solute–solvent interactions and dynamics are simulated with a fully molecular hybrid method consisting of a semiempirical quantum mechanical method with singly excited configurations for the solute and classical molecular dynamics (MD) for the solvent (H2O). The interactions are purely electrostatic, with the solute being polarizable and sharing its charge information with the MD at 5 fs intervals. The solvent charges are fixed and the results are not sensitive to the point charges used. For the solute, the results depend on the dipole moment much more than on the point charge magnitudes leading to a given dipole. This method is applied to the spectral shifts, dynamics, linewidths, and free energies of indole and 3‐methylindole (3MI) in water at 300 K, including the effect of geometry changes and clarifications concerning vertical vs 0–0 transition predictions. Large fluorescence Stokes shifts are predicted, in fair agreement with observed values. The 1La excited state dipole is calculated to be about 12 ...

Molecular orbital theory of the 1Lb and 1La states of indole

A comprehensive study of the 1Lb and 1La excited states of indole, using a spectroscopically calibrated semiempirical molecular orbital method [the spectroscopic version of the intermediate neglect of differential overlap‐configuration interaction (INDO/S‐CI)], is reported. Results from two standard parametrizations and procedures are identified as giving satisfactory agreement with several experimental properties. Diagrams of MOs, π transition densities, and π density changes are provided from these two calculations. In addition, transition energies, oscillator strengths, transition moment directions, dipoles, and two‐photon properties are presented for 24 additional calculations representing variations in the key parameters.

Resolution of La and Lb bands in methyl indoles by two-photon spectroscopy

Polarized two-photon fluorescence excitation spectra were obtained for indole, 5-methylindole, 3-methylindole and 2,3-dimeth-ylindole in cyclohexane and butanol solutions. The polarization ratios clearly indicate the onset of La absorption and directly verify established models regarding the relative response of La and Lb state energies to substitution and solvent perturbations (prior to excited-state relaxations). The two-photon properties are successfully modeled by INDO/S CI computation.

Understanding the variable fluorescence quantum yield of tryptophan in proteins using QM-MM simulations. Quenching by charge transfer to the peptide backbone

Abstract A reasonable basis for the puzzling variation of tryptophan (Trp) fluorescence quantum yields in proteins arises naturally through quantum mechanics-molecular dynamics simulations in which the energy of the lowest Trp ring-to-amide backbone charge transfer (CT) state is monitored during dynamics trajectories for 16 Trps in 13 proteins. The energy, fluctuations, and relaxation of the high lying CT state are highly sensitive to protein environment (local electric field) and rotamer conformation, leading to large variations in 1 L a fluorescence yield and lifetime.

Two-photon fluorescence excitation spectra of aromatic amino acids

Abstract Two-photon excited fluorescence excitation spectra using circularly and linearly polarized light are presented for the aromatic amino acids tryptophan, tyrosine (amide form) and phenylalanine in neutral aqueous solution at room temperature. The wave-length range was 440–620 nm, corresponding to 220–310 nm in the UV. In contrast to one-photon (UV) excitation, phenylalanine absorption is about the same as that of tyrosine and both are shifted about 1500 cm −1 to higher energy relative to their UV bands. Thus, observing phenylalanine photophysics in proteins may be more feasible with two-photon excitation. Semiempirical theory gives a good account of the results.

Acridine orange staining reaction as an index of physiological activity in Escherichia coli

The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

Mechanism of the Very Efficient Quenching of Tryptophan Fluorescence in Human γD- and γS-Crystallins: The γ-Crystallin Fold May Have Evolved To Protect Tryptophan Residues from Ultraviolet Photodamage†

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated β-sheet Greek key domains of β- and γ-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human γD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552−11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human γS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n − 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.