PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5-Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions.

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable ß-hairpin "zipper" peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid ß-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 ß-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance.

The Color of Cation-π Interactions: Subtleties of Amine-Tryptophan Interaction Energetics Allow for Radical-like Visible Absorbance and Fluorescence.

Several peptides and a protein with an inter- or intramolecular cation-π interaction between tryptophan (Trp) and an amine cation are shown to absorb and fluoresce in the visible region of the spectrum. Titration of indole with sodium hydroxide or ammonium hydroxide yields an increasing visible fluorescence as well. Visible absorption and multipeaked fluorescence excitation spectra correlate with experimental absorption spectra and the vibrational modes of calculated absorption spectra for the neutral Trp radical. The radical character of the cation-indole interaction is predicted to stem from the electrostatic dislocation of indole highest occupied molecular orbital (HOMO) charge density toward the cation with a subsequent electronic transition from the HOMO-2 to the HOMO. Because this is a vertical transition, fluorescence is possible. Hydrogen bonding at the indole amine most likely stabilizes the radical-like state. These results provide new spectroscopic tools for the investigation of cation-π interactions in numerous biological systems, among them, proteins and their myriad ligands, and show that one, or at most, two, point mutations with natural amino acids are all that is required to impart visible fluorescence to proteins.

Excited State Electron Distribution and Role of the Terminal Amine in Acidic and Basic Tryptophan Dipeptide Fluorescence.

The results of quantum yield (QY) study of tryptophanyl glutamate (Trp-Glu), tryptophanyl lysine (Trp-Lys) and lysinyl tryptophan (Lys-Trp) dipeptides over the pH range, 1.5 - 13, show that the charge state of the N-terminal amine, and not the nominal molecular charge determines the QY. When the terminal amine is protonated, QY is low (10-2) for all three dipeptides. As the terminal amine cation is found proximal to the indole ring in Trp-Glu and Trp-Lys conformers but not in those for Lys-Trp, its effect may lie only in the partitioning of energy between nonradiative processes, not on QY reduction. QY is also low when both the N-terminal amine and indole amine are deprotonated. These two low QY states can be distinguished by fluorescence lifetime measurement. Molecular dynamics simulation shows that the Chi 1 conformers persist for tens of nanoseconds such that 100 - 101 nanosecond lifetimes may be associated with individual Chi 1 conformers. The ground state electron density or isosurface of high QY (0.30) 3-methyindole has a uniform electron density over the indole ring as do the higher QY Trp dipeptide conformers. This validates the association of ground state isosurfaces with QY. Excited state orbitals from calculated high intensity, low energy absorption transitions are typically centered over the indole ring for higher QY dipeptide species and off the ring in lower QY species. Thus excited state orbitals substantiate the earlier finding that the ground state isosurface charge density pattern on the indole ring can be predictive of QY.

The broken ring: reduced aromaticity in Lys-Trp cations and high pH tautomer correlates with lower quantum yield and shorter lifetimes.

Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK(1), pK(2), and pK(R)) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the (1)L(a) transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes.

Relating Trp-Glu dipeptide fluorescence to molecular conformation: the role of the discrete Chi 1 and Chi 2 angles.

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp-Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time-resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp-Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles--60°, 180°, and 300°--play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH3 cation-π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp-Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence.

A spectroscopic survey of substituted indoles reveals consequences of a stabilized 1Lb transition.

Although tryptophan is a natural probe of protein structure, interpretation of its fluorescence emission spectrum is complicated by the presence of two electronic transitions, (1)L(a) and (1)L(b). Theoretical calculations show that a point charge adjacent to either ring of the indole can shift the emission maximum. This study explores the effect of pyrrole and benzyl ring substitutions on the transitions' energy via absorption and fluorescence spectroscopy, and anisotropy and lifetime measurements. The survey of indole derivatives shows that methyl substitutions on the pyrrole ring effect (1)L(a) and (1)L(b) energies in tandem, whereas benzyl ring substitutions with electrophilic groups lift the (1)L(a)/(1)L(b) degeneracy. For 5- and 6-hydroxyindole in cyclohexane, (1)L(a) and (1)L(b) transitions are resolved. This finding provides for (1)L(a) origin assignment in the absorption and excitation spectra for indole vapor. The 5- and 6-hydroxyindole excitation spectra show that despite a blue-shifted emission spectrum, both the (1)L(a) and (1)L(b) transitions contribute to emission. Fluorescence lifetimes of 1(0) ns for 5-hydroxyindole are consistent with a charge acceptor-induced increase in the nonradiative rate (1).

Correlation of TrpGly and GlyTrp Rotamer Structure with W7 and W10 UV Resonance Raman Modes and Fluorescence Emission Shifts.

Tryptophyl glycine (TrpGly) and glycyl tryptophan (GlyTrp) dipeptides at pH 5.5 and pH 9.3 show a pattern of fluorescence emission shifts with the TrpGly zwitterion emission solely blue shifted. This pattern is matched by shifts in the UV resonance Raman (UVRR) W10 band position and the W7 Fermi doublet band ratio. Ab initio calculations show that the 1340 cm(-1) band of the W7 doublet is composed of three modes, two of which determine the W7 band ratios for the dipeptides. Molecular dynamics simulations show that the dipeptides take on two conformations: one with the peptide backbone extended; one with the backbone curled over the indole. The dihedral angle critical to these conformations is χ(1) and takes on three discrete values. Only the TrpGly zwitterion spends an appreciable amount of time in the extended backbone conformation as this is stabilized by two hydrogen bonds with the terminal amine cation. According to a Stark effect model, a positive charge near the pyrrole keeps the (1)L(a) transition at high energy, limiting fluorescence emission red shift, as observed for the TrpGly zwitterion. The hydrogen bond stabilized backbone provides a rationale for the C(methylene)-C(α)-C(carbonyl) W10 symmetric stretch that is unique to the TrpGly zwitterion.

Extension of the tryptophan chi2,1 dihedral angle-W3 band frequency relationship to a full rotation: correlations and caveats.

The correlation of the UVRR nuW3 mode with the tryptophan chi(2,1) dihedral angle [Maruyama and Takeuchi (1995) J. Raman Spectrosc. 26, 319; Miura et al. (1989) J. Raman Spectrosc. 20, 667; Takeuchi (2003) Biopolymers 72, 305] has been extended to a full, 360 degrees rotation. The 3-fold periodicity of the relationship (cos 3chi(2,1)) over 360 degrees results in up to six dihedral angles for a given nuW3. Consideration of a Newman plot of dihedral angles for proteinaceous tryptophans taken from the Protein Data Bank shows that sterically hindered ranges of dihedral angle reduce the possible chi(2,1) to one or two. However, not all proteinaceous tryptophans follow the nuW3-chi(2,1) relationship. Hydrogen bonding at the indole amine, weaker, electrostatic cation-pi and anion-quadrapole interactions, and environmental hydrophobicity are examined as possible contributing factors to noncompliance with the relationship. This evaluation suggests that cumulative weak electrostatic and nonpolar interactions, contributing to steric hindrance, characterize the environment of tryptophans that obey the nuW3-chi(2,1) relationship, matching that of the crystalline tryptophan derivatives used to formulate the relationship. In the absence of methods to quantify these weak interactions, measurement of the full width half-maximum bandwidth (fwhm) of the W3 band is suggested as a primary screen for evaluating the applicability of the nuW3-chi(2,1) relationship.

Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation.

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.

A pathway of structural changes produced by monastrol binding to Eg5.

Monastrol is a small molecule inhibitor that is specific for Eg5, a member of the kinesin 5 family of mitotic motors. Crystallographic models of Eg5 in the presence and absence of monastrol revealed that drug binding produces a variety of structural changes in the motor, including in loop L5 and the neck linker. What is not clear from static crystallographic models, however, is the sequence of structural changes produced by drug binding. Furthermore, because crystallographic structures can be influenced by the packing forces in the crystal, it also remains unclear whether these drug-induced changes occur in solution, at physiologically active concentrations of monastrol or of other drugs that target this site. We have addressed these issues by using a series of spectroscopic probes to monitor the structural consequences of drug binding. Our results demonstrated that the crystallographic model of an Eg5-ADP-monastrol ternary complex is consistent with several solution-based spectroscopic probes. Furthermore, the kinetics of these spectroscopic signal changes allowed us to determine the temporal sequence of drug-induced structural transitions. These results suggested that L5 may be an element in the pathway that links the state of the nucleotide-binding site to the neck linker in kinesin motors.

Intersubunit interactions associated with Tyr42 alpha stabilize the quaternary-T tetramer but are not major quaternary constraints in deoxyhemoglobin.

Previous mutational studies on Tyr42alpha variants as well as the current studies on the mutant hemoglobin alphaY42A show that the intersubunit interactions associated with Tyr42alpha significantly stabilize the alpha1beta2 interface of the quaternary-T deoxyhemoglobin tetramer. However, crystallographic studies, UV and visible resonance Raman spectroscopy, CO combination kinetic measurements, and oxygen binding measurements on alphaY42A show that the intersubunit interactions formed by Tyr42alpha have only a modest influence on the structural properties and ligand affinity of the deoxyhemoglobin tetramer. Therefore, the alpha1beta2 interface interactions associated with Tyr42alpha do not contribute significantly to the quaternary constraints that are responsible for the low oxygen affinity of deoxyhemoglobin. The slight increase in the ligand affinity of deoxy alphaY42A correlates with small, mutation-induced structural changes that perturb the environment of Trp37beta, a critical region of the quaternary-T alpha1beta2 interface that has been shown to be the major source of quaternary constraint in deoxyhemoglobin.

Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels.

Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm(-1). The low and high values correspond to the initial deoxy T state and liganded R (R(2)) state species, respectively. The 222 and 227 cm(-1) species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the alpha(1)beta(2) interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.

Comparative vibrational spectroscopy of intracellular tau and extracellular collagen I reveals parallels of gelation and fibrillar structure.

The N-terminal tau 2-19 peptide undergoes gelation, syneresis, and aggregation over a period of years. These changes may be approximated on a shorter time scale by agitation and partial dehydration. The anomalously enhanced (229 nm) ultraviolet resonance Raman (UVRR) imide II band reveals a common structural feature for gels of nondehydrated tau 2-19 and collagen I and insoluble paired helical filaments (PHFs) and collagen I of weak hydrogen bonding at proline carbonyls. Anomalous UVRR enhancement of amide bands at 229 nm results from gel structure, as demonstrated by increased amide absorption at the red edge for tau 2-19 gel and implies the involvement of water in gel structure. In aged, dehydrated tau 2-19 gel, proline carbonyls lose their bonds to water and tyrosine becomes deprotonated, as demonstrated by UVRR spectroscopy. The Fourier transform infrared (FTIR) amide I band shows that antiparallel beta-sheet structure increases with syneresis in the tau 2-19 hydrogel. The comparison of FTIR results for PHFs with collagen I gel and polyproline demonstrates that the secondary structure of PHFs is polyproline II. One implication of this assignment is that the fibrillation of hydrophilic tau is thermodynamically driven by the entropy gained as hydrogen-bonded water is freed, as for collagen I. The FTIR results also show that peptide domains culled from a longer protein do not necessarily fold into identical secondary structures. A pathological, sequential mechanism of gelation, syneresis, and fibrillation for tau in AD is suggested and is supported by the observation of amorphous neurofibrillary tangle development and fibrillation in vivo.

Conformational changes in hemoglobin S (betaE6V) imposed by mutation of the beta Glu7-beta Lys132 salt bridge and detected by UV resonance Raman spectroscopy.

The impact upon molecular structure of an additional point mutation adjacent to the existing E6V mutation in sickle cell hemoglobin was probed spectroscopically. The UV resonance Raman results show that the conformational consequences of mutating the salt bridge pair, betaGlu(7)-betaLys(132), are dependent on which residue of the pair is modified. The betaK132A mutants exhibit the spectroscopic signatures of the R --> T state transition in both the "hinge" and "switch" regions of the alpha(1)beta(2) interface. Both singly and doubly mutated hemoglobin (Hb) betaepsilon7Alpha exhibit the switch region signature for the R --> T quaternary state transition but not the hinge signature. The absence of this hinge region-associated quaternary change is the likely origin of the observed increased oxygen binding affinity for the Hb betaepsilon7Alpha mutants. The observed large decrease in the W3 alpha14beta15 band intensity for doubly mutated Hb betaepsilon7Alpha is attributed to an enhanced separation in the A helix-E helix tertiary contact of the beta subunits. The results for the Hb A betaGlu(7)-betaLys(132) salt bridge mutants demonstrate that attaining the T state conformation at the hinge region of the alpha(1)beta(2) dimer interface can be achieved through different intraglobin pathways; these pathways are subject to subtle mutagenic manipulation at sites well removed from the dimer interface.

UV resonance Raman study of beta93-modified hemoglobin A: chemical modifier-specific effects and added influences of attached poly(ethylene glycol) chains.

The reactive sulfhydryl group on Cys beta93 in human adult hemoglobin (HbA) has been the focus of many studies because of its importance both as a site for synthetic manipulation and as a possible binding site for nitric oxide (NO) in vivo. Despite the interest in this site and the known functional alterations associated with manipulation of this site, there is still considerable uncertainty as to the conformational basis for these effects. UV resonance Raman (UVRR) spectroscopy is used in this study to evaluate the conformational consequences of chemically modifying the Cys beta93 sulfhydryl group of both the deoxy and CO-saturated derivatives of HbA using different maleimide and mixed disulfide reagents. Included among the maleimide reagents are NEM (n-ethylmaleimide) and several poly(ethylene glycol) (PEG)-linked maleimides. The PEG-based reagents include both different sizes of PEG chains (PEG2000, -5000, and -20000) and different linkers between the PEG and the maleimide. Thus, the effect on the conformation of both linker chemistry and PEG size is evaluated. The spectroscopic results reveal minimal perturbation of the global structure of deoxyHbA for the mixed disulfide modification. In contrast, maleimide-based modifications of HbA perturb the deoxy T state of HbA by "loosening" the contacts associated with the switch region of the T state alpha(1)beta(2) interface but do not modify the hinge region of this interface. When the NEM-modified HbA is also subjected to enzymatic treatment to remove the C-terminal Arg alpha141 (yielding NESdes-ArgHb), the resulting deoxy derivative exhibits the spectroscopic features associated with a deoxy R state species. All of the CO-saturated derivatives exhibit spectra that are characteristic of the fully liganded R structure. The deoxy and CO derivatives of HbA that have been decorated on the surface with large PEG chains linked to the maleimide-modified sulfhydryl through a short linker group all show a general intensity enhancement of the tyrosine and tryptophan bands in the UVRR spectrum. It is proposed that this effect arises from the osmotic impact of a large, close PEG molecule enveloping the surface of the protein.