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Genetically encoded probes with red-shifted absorption and fluorescence are highly desirable for imaging applications because they can report from deeper tissue layers with lower background and because they provide additional colors for multicolor imaging. Unfortunately, red and especially far-red fluorescent proteins have very low quantum yields, which undermines their other advantages. Elucidating the mechanism of nonradiative relaxation in red fluorescent proteins (RFPs) could help developing ones with higher quantum yields. Here we consider two possible mechanisms of fast nonradiative relaxation of electronic excitation in RFPs. The first, known as the energy gap law, predicts a steep exponential drop of fluorescence quantum yield with a systematic red shift of fluorescence frequency. In this case the relaxation of excitation occurs in the chromophore without any significant changes of its geometry. The second mechanism is related to a twisted intramolecular charge transfer in the excited state, followed by an ultrafast internal conversion. The chromophore twisting can strongly depend on the local electric field because the field can affect the activation energy. We present a spectroscopic method of evaluating local electric fields experienced by the chromophore in the protein environment. The method is based on linear and two-photon absorption spectroscopy, as well as on quantum-mechanically calculated parameters of the isolated chromophore. Using this method, which is substantiated by our molecular dynamics simulations, we obtain the components of electric field in the chromophore plane for seven different RFPs with the same chromophore structure. We find that in five of these RFPs, the nonradiative relaxation rate increases with the strength of the field along the chromophore axis directed from the center of imidazolinone ring to the center of phenolate ring. Furthermore, this rate depends on the corresponding electrostatic energy change (calculated from the known fields and charge displacements), in quantitative agreement with the Marcus theory of charge transfer. This result supports the dominant role of the twisted intramolecular charge transfer mechanism over the energy gap law for most of the studied RFPs. It provides important guidelines of how to shift the absorption wavelength of an RFP to the red, while keeping its brightness reasonably high., Competing Interests: JNS is the owner of the company JNScott Consulting, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Drobizhev, Molina, Callis, Scott, Lambert, Salih, Shaner and Hughes.)

Time-resolved fluorescence emission and resonance-enhanced second harmonic generation (SHG) spectra were collected from 4-dimethylaminobenzonitrile (DMABN) adsorbed to the aqueous-silica interface in order to identify how strongly associating solvent-substrate interactions change DMABN's photoisomerization properties. In bulk polar solution, DMABN forms an excited twisted intramolecular charge-transfer (TICT) state that emits with a distinctive, solvatochromic fluorescent signature. At the silica-aqueous interface, the TICT fluorescence disappears, similar to DMABN's behavior in nonpolar environments. SHG spectra confirm that the interface is, in fact, polar, and DMABN's unusual fluorescence emission acquired from the interface is attributed to strong hydrogen bonding associations between the water molecules and the silica surface that prevent adsorbate isomerization. Additionally, SHG spectra show a strong resonance at long wavelengths that is unexpected based on bulk absorbance spectra and selection rules for nonlinear hyperpolarizabilities. Using both Zerner's INDO semiempirical and TD-DFT calculations, this spectroscopic behavior is attributed to a combination of strong electric fields present at the aqueous-silica interface and surface-induced changes to DMABN's ground-state molecular structure.

Fluorescent DNA base analogs and intrinsic fluorophores are gaining importance for multiphoton microscopy and imaging, however, their quantitative nonlinear excitation properties have been poorly documented. Here we present the two-photon absorption (2PA) spectra of 2-aminopurine (2AP), 7-methyl guanosine (7MG), isoxanthopterin (IXP), 6-methyl isoxanthopterin (6MI), as well as L-tryptophan (L-trp) and 3-methylindole (3MI) in aqueous solution and some organic solvents measured in the wavelength range 550 - 810 nm using femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods. The peak 2PA cross section values range from 0.1 GM (1 GM = 10 -50 cm 4 s photon -1 ) for 2AP to 2.0 GM for IXP and 7MG. Assuming typical excitation conditions for a scanning 2PEF microscope, we estimate a maximum image frame rate of ~175 frames per second (FPS)., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

We use TD-DFT to calculate the one-photon absorption (1PA) and two-photon absorption (2PA) properties of C153 and Prodan in toluene and DMSO, and benchmark different methods relative to accurate experimental data available from the literature on these particular systems. As the first step, we modify the range-separated TD-DFT to provide the best prediction for the peak 1PA wavelength, and then apply the optimized functionals to achieve quantitative predictions of the corresponding two-photon absorption cross section, σ 2PA , with an accuracy ∼10-20% in C153 and ∼20-30% in Prodan. To elucidate the origin of residual discrepancies between the theory and experimental observations, we invoked the two essential states model for σ 2PA , which allows us to verify not only the transition wavelength and the σ 2PA value, but also to quantitatively benchmark the calculation of key molecular parameters such as the transition dipole moment and the change of the permanent dipole moment. Such comprehensive cross-checking indicates that a larger discrepancy in Prodan is most likely caused by a noted failure of DFT to predict the relative intensity and relative ordering of closely lying excited states with different degrees of intramolecular charge transfer, which we further support by analyzing the predictions obtained by high-level coupled-cluster calculations in the gas phase. Our results highlight the utility of benchmarking the calculations not only relative to other theoretical methods, but also in comparison to the experimental measurements, wherever such data are available.

The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement.

The protein-water interface is a critical determinant of protein structure and function, yet the precise nature of dynamics in this complex system remains elusive. Tryptophan fluorescence has become the probe of choice for such dynamics on the picosecond time scale (especially via fluorescence "upconversion"). In the absence of ultrafast ("quasi-static") quenching from nearby groups, the TDFSS (time-dependent fluorescence Stokes shift) for exposed Trp directly reports on dipolar relaxation near the interface (both water and polypeptide). The small protein GB1 contains a single Trp (W43) of this type, and its structure is refractory to pH above 3. Thus, it can be used to examine the dependence of dipolar relaxation upon charge reconfiguration with titration. Somewhat surprisingly, the dipolar dynamics in the 100 fs to 100 ps range were unchanged with pH, although nanosecond yield, rates, and access all changed. These results were rationalized with the help of molecular dynamics (including QM-MM) simulations that reveal a balancing, sometimes even countervailing influence of protein and water dipoles. Interestingly, these simulations also showed the dominant influence of water molecules which are associated with the protein interface for up to 30 ps yet free to rotate at approximately "bulk" water rates.

Principles behind quenching of tryptophan (Trp) fluorescence are updated and extended in light of recent 100-ns and 1-μs molecular dynamics (MD) trajectories augmented with quantum mechanical (QM) calculations that consider electrostatic contributions to wavelength shifts and quenching. Four studies are summarized, including (1) new insight into the single exponential decay of NATA, (2) a study revealing how unsuspected rotamer transitions affect quenching of Trp when used as a probe of protein folding, (3) advances in understanding the origin of nonexponential decay from 100-ns simulations on 19 Trps in 16 proteins, and (4) the correlation of wavelength with lifetime for decay-associated spectra (DAS). Each study strongly reinforces the concept that-for Trp-electron transfer-based quenching is controlled much more by environment electrostatic factors affecting the charge transfer (CT) state energy than by distance dependence of electronic coupling. In each case, water plays a large role in unexpected ways.

The steady state fluorescence spectral maximum (λmax) for tryptophan 140 of Staphylococcal nuclease remains virtually unchanged when nearby charged groups are removed by mutation, even though large electrostatic effects on λmax might be expected. To help understand the underlying mechanism of this curious result, we have modeled λmax with three sets of 50-ns molecular dynamics simulations in explicit water, equilibrated with excited state and with ground state charges. Semiempirical quantum mechanics and independent electrostatic analysis for the wild-type protein and four charge-altering mutants were performed on the chromophore using the coordinates from the simulations. Electrostatic contributions from the nearby charged lysines by themselves contribute 30-90 nm red shifts relative to the gas phase, but in each case, contributions from water create compensating blue shifts that bring the predicted λmax within 2 nm of the experimental value, 332 ± 0.5 nm for all five proteins. Although long-range collective interactions from ordered water make large blue shifts, crucial for determining the steady state λmax for absorption and fluorescence, such blue shifts do not contribute to the amplitude of the time dependent Stokes shift following excitation, which comes from nearby charges and only ∼6 waters tightly networked with those charges. We therefore conclude that for STNase, water and protein effects on the Stokes shift are not separable.

Here, we present a new all-optical method of interrogation of the internal electric field vector inside proteins. The method is based on experimental evaluation of the permanent dipole moment change upon excitation and the pure electronic transition frequency of a fluorophore embedded in a protein matrix. The permanent dipole moment change can be obtained from two-photon absorption measurements. In addition, permanent dipole moment change, tensor of polarizability change, and transition frequency for the free chromophore should be calculated quantum-mechanically. This allows obtaining the components of the electric field by considering the second-order Stark shift. We use the fluorescent protein mCherry as an example to demonstrate the applicability of the method.

Protein folding kinetics is commonly monitored by changes in tryptophan (Trp) fluorescence intensity. Considerable recent discussion has centered on whether the fluorescence of the single Trp in the much-studied, fast-folding villin headpiece C-terminal domain (HP35) accurately reflects folding kinetics, given the general view that quenching is by a histidine cation (His(+)) one turn away in an α-helix (helix III) that forms early in the folding process, according to published MD simulations. To help answer this question, we ran 1.0 μs MD simulations on HP35 (N27H) and a faster-folding variant in its folded form at 300 K and used the coordinates and force field charges with quantum calculations to simulate fluorescence quenching caused by electron transfer to the local amide and to the His(+). The simulations demonstrate that quenching by His(+) in the fully formed helix III is possible only during certain Trp and His(+) rotamer and solvent conformations, the propensity of which is a variable that can allow Trp fluorescence to report the global folding rate, as recent experiments imply.

Rapid photobleaching of fluorescent proteins can limit their use in imaging applications. The underlying kinetics is multi-exponential and strongly depends on the local chromophore environment. The first, reversible, step may be attributed to a rotation around one of the two exocyclic C-C bonds bridging phenol and imidazolinone groups in the chromophore. However it is not clear how the protein environment controls this motion - either by steric hindrances or by modulating the electronic structure of the chromophore through electrostatic interactions. Here we study the first step of the photobleaching kinetics in 13 red fluorescent proteins (RFPs) with different chromophore environment and show that the associated rate strongly correlates with the bond length alternation (BLA) of the two bridge bonds. The sign of the BLA appears to determine which rotation is activated. Our results present experimental evidence for the dominance of electronic effects in the conformational dynamics of the RFP chromophore.

Tryptophan (Trp) fluorescence is potentially a powerful probe for studying the conformational ensembles of proteins in solution, as it is highly sensitive to the local electrostatic environment of the indole side chain. However, interpretation of the wavelength-dependent complex fluorescence decays of proteins has been stymied by controversy about two plausible origins of the typical multiple fluorescence lifetimes: multiple ground-state populations or excited-state relaxation. The latter naturally predicts the commonly observed wavelength-lifetime correlation between decay components, which associates short lifetimes with blue-shifted emission spectra and long lifetimes with red-shifted spectra. Here we show how multiple conformational populations also lead to the same strong wavelength-lifetime correlation in cyclic hexapeptides containing a single Trp residue. Fluorescence quenching in these peptides is due to electron transfer. Quantum mechanics-molecular mechanics simulations with 150-ps trajectories were used to calculate fluorescence wavelengths and lifetimes for the six canonical rotamers of seven hexapeptides in aqueous solution at room temperature. The simulations capture most of the unexpected diversity of the fluorescence properties of the seven peptides and reveal that rotamers having blue-shifted emission spectra, i.e., higher average energy, have an increased probability for quenching, i.e., shorter average lifetime, during large fluctuations in environment that bring the nonfluorescent charge transfer state and the fluorescing state into resonance. This general mechanism should also be operative in proteins that exhibit multiexponential fluorescence decays, where myriad other sources of conformational heterogeneity besides rotamers are possible.

Use of fluorescence in biology and biochemistry for imaging and characterizing equilibrium and dynamic processes is growing exponentially. Much progress has been made in the last few years on the microscopic understanding of the underlying principles of what controls the wavelength and quenching of fluorescence in biopolymers, both of which are central to the utility of fluorescent probes. This chapter is concerned with the quantitative microscopic understanding and prediction of the fluorescence wavelength and/or intensity of a fluorescent probe molecule attached to a biopolymer as revealed by hybrid quantum and classical mechanical computation procedures. The aim is not only to provide a recipe, but also even more importantly, to communicate the qualitative basic concepts of interpretation of fluorescence. These are surprisingly simple, although not broadly appreciated at this time. In addition, an effort has been made to show how these techniques have led to an emerging understanding of the relation between time-dependent wavelengths shifts due to solvent relaxation and population decay of conformational sub-ensembles., (© 2011 Elsevier Inc. All rights reserved.)

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 beta-sheet Greek key domains of beta- and gamma-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 gammaD-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 gammaS-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.

Hybrid quantum mechanical/molecular mechanics (QM-MM) calculations [Callis and Liu, J. Phys. Chem. B 2004, 108, 4248-4259] make a strong case that the large variation in tryptophan (Trp) fluorescence yields in proteins is explained by ring-to-backbone amide electron transfer, as predicted decades ago. Quenching occurs in systems when the charge transfer (CT) state is brought below the fluorescing state (1L(a)) as a result of strong local electric fields. To further test this hypothesis, we have measured the fluorescence quantum yield in solvents of different polarity for the following systems: N-acetyl-L-tryptophanamide (NATA), an analogue for Trp in a protein; N-acetyl-L-tryptophan ethyl ester (NATE), wherein the Trp amide is replaced by an ester group, lowering the CT state energy; and 3-methylindole (3MI), a control wherein this quenching mechanism cannot take place. Experimental yields in water are 0.31, 0.13, and 0.057 for 3MI, NATA, and NATE, respectively, whereas, in the nonpolar aprotic solvent dioxane, all three have quantum yields near 0.35, indicating the absence of electron transfer. In alkyl alcohols the quantum yield for NATA and NATE is between that found for water and that found for dioxane, and it is surprisingly independent of chain length (varying from methanol to decanol), revealing that microscopic H-bonding, and not the bulk dielectric constant, dictates the electron transfer rate. QM-MM calculations indicate that, when averaged over the six rotamers, the greatly increased quenching found in water relative to dioxane can be attributed mainly to the larger fluctuations of the energy gap in water. These experiments and calculations are in complete accord with quenching by a solvent stabilized charge transfer from ring to amide state in proteins.

We report quantum mechanical-molecular mechanical (QM-MM) predictions of fluorescence quantum yields for 20 tryptophans in 17 proteins, whose yields span the range from 0.01 to 0.3, using ab initio computed coupling matrix elements for photoinduced electron transfer from the 1La excited indole ring to a local backbone amide. The average coupling elements span the range 140-1000 cm-1, depending on tryptophan rotamer conformation. The matrix elements were from the singles configuration interaction matrix, and were largely insensitive to which of the three basis sets was used. Large fluctuations were seen on the time scale of tens of femtoseconds, caused primarily by side chain and backbone torsional variations for 150 ps of dynamics at 300 K. The largest coupling occurs for the chi1 = -60 degrees rotamer and is purely through-bond. There is no apparent correlation between the coupling magnitude and quantum yield, which is still dominated by energy gap and reorganization energy. The source of error bars for predicted quenching rates using the weak coupling golden rule may be due to inaccurate averaged Franck-Condon weighted densities because of inadequate simulation times and parameters and/or to failure of the weak coupled golden rule used in these predictions because of the broad distribution of Landau-Zener probabilities arising from the large variable coupling.

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.

The wavelength of maximum emission of tryptophan depends on the local electrostatic environment of the indole chromophore. The time-resolved emission spectra of seven rigid cyclic hexapeptides containing a single tryptophan residue were measured. The emission maxima of the three decay-associated spectra for the seven peptides ranged from 341 to 359 nm, suggesting that different tryptophan rotamers have different emission maxima even in the case of solvent-exposed tryptophans. This conclusion is supported by quantum mechanical/molecular dynamics simulations of the six canonical side chain rotamers of tryptophan in solvated hexapeptides. The calculated range of emission maxima for the tryptophan rotamers of the seven peptides is 344-365 nm. The precision of the wavelength calculations and the peptide, water, and charged side chain contributions to the spectral shifts are examined. The results indicate that the emission maxima of decay-associated spectra can aid in the assignment of fluorescence lifetimes to tryptophan rotamers.

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.

This work reports an explanation for the unusual monoexponential fluorescence decay of 5-fluorotryptophan (5FTrp) in single-Trp mutant proteins [Broos, J.; Maddalena, F.; Hesp, B. H. J. Am. Chem. Soc. 2004, 126, 22-23] and substantially clarifies the origin of the ubiquitous nonexponential fluorescence decay of tryptophan in proteins. Our results strongly suggest that the extent of nonexponential fluorescence decay is governed primarily by the efficiency of electron transfer (ET) quenching by a nearby amide group in the peptide bond. Fluoro substitution increases the ionization potential (IP) of indole, thereby suppressing the ET rate, leading to a longer average lifetime and therefore a more homogeneous decay. We report experimental IPs for a number of substituted indoles including 5-fluoroindole, 5-fluoro-3-methylindole, and 6-fluoroindole, along with accurate ab initio calculations of the IPs for these and 20 related molecules. The results predict the IP of 5-fluorotryptophan to be 0.19 eV higher than that of tryptophan. 5-Fluoro substitution does not measurably alter the excitation-induced change in permanent dipole moment nor does it change the fluorescent state from 1La to 1Lb. In combination with electronic structure information this argues that the increased IP and the decreased excitation energy of the 1La state, together 0.3 eV, are solely responsible for the strong reduction of electron transfer quenching. 6-Fluoro substitution is predicted to increase the IP by a mere 0.09 eV. In agreement with our conclusions, the fluorescence decay curves of 6-fluorotryptophan-containing proteins are well fit using only two decay times compared to three required for Trp.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

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.

Nonresonant two-photon electronic spectroscopy of polyatomic molecules is reviewed for the period since 1979. Emphasis is placed on studies that expose patterns in the two-photon fluorescence (also ionization, optoacoustic) excitation spectra of aromatic hydrocarbons and the effect of vibrations and substitution, particularly within the framework of pseudoparity rules. A section is devoted to biological molecules and the emerging use of two-photon-induced fluorescence anisotropy. Relevant theoretical results are discussed, with emphasis on quantum chemical predictions of vibronic coupling and substituent effects on two-photon absorptivity and tensor properties of individual molecules. This chapter includes higher-order spectroscopy, and a limited number of three- and four-photon studies are discussed.

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.