Room temperature phosphorescence study of phosphate binding in Escherichia coli alkaline phosphatase.

The phosphorescence spectrum and decay of Trp109 in Escherichia coli alkaline phosphatase was measured for the enzyme in 10 mM Tris/HCl, pH 7.4, at 21 degrees C. Changes in the spectrum and decay from the steady-state in response to non-covalent phosphate binding suggested a phosphate-induced alteration in the local environment surrounding Trp109 which lies buried below the active site. The seemingly inflexible structure in the region of Trp109, as judged by its very long phosphorescence lifetime, appeared unaltered when the enzyme was symmetrically bound with phosphate. However, the protein with phosphate bound to only one site displayed a marked increase in flexibility that extended over both subunits. For ratios of phosphate/enzyme (mol/mol) between 1.0 and 2.0, the observation of exponential phosphorescence decays with lifetimes that are a function of dilution provided evidence for the rapid exchange between phosphate half-saturated and fully-saturated enzymes consistent with observed enzyme turnover rates. The lifetimes under these conditions result in the calculation of a Kd for the dissociation of phosphate from the doubly occupied enzyme of 1.1 +/- 0.1 microM. The non-exponential decays at P/Ed (phosphate/dimeric enzyme) ratios less than 1.0 revealed that the exchange of phosphate between phosphate-free and half-saturated enzymes was not occurring on the timescale of the phosphorescence decay times, which implied that the half-saturated molecule cannot be contributing significantly to catalysis under steady-state conditions. The observation that the phosphorescence decay at a P/Ed ratio of 1.0 is exponential with a lifetime characteristic of the half-saturated species indicates that the binding of the first phosphate is significantly greater than the second, or that the binding exhibits negative cooperativity.

Temperature dependence of the disulfide perturbation to the triplet state of tryptophan.

Variability in the temperature dependence of disulfide quenching of the tryptophan phosphorescence of globular proteins in rigid glasses is illustrated with lysozyme and alpha-bungarotoxin. A laser-pulsed phosphorescence study of this short-range interaction with a model indole-disulfide system is described. The perturbation of secondary dibutyl disulfide on the triplet state of the indole moiety in 2-(3-indolyl)ethyl phenyl ketone in rigid media is found to display a bimodal temperature dependence. The quenching rate constant at contact between chromophore and perturber is observed to be temperature independent below 30 K, but to increase with temperature between 30 and 100 K with an activation energy of approximately 200 cm(-1). The results suggest that the underlying quenching interaction involves a photo-induced one-electron transfer from the excited state of indole to the disulfide.

Room temperature phosphorescence of Trp-314 as a monitor of subunit communications in alcohol dehydrogenase from horse liver.

The phosphorescence properties of liver alcohol dehydrogenase from horse were characterized at limiting concentrations of coenzyme and coenzyme analogues. The emission decay kinetics of Trp-314 in strong, slowly exchanging, ternary complexes with NADH/isobutyramide, NAD/pyrazole, and NADH/dimethyl sulfoxide displays a markedly nonexponential character. The analysis of decay components over the saturation curve reveals that the phosphorescence from singly bound protein molecules has a lifetime from 1 to 1.3 s, which is 2-3 times larger than observed with fully bound and unliganded enzyme. The remarkably tighter configuration reported by the triplet probe for the coenzyme-binding domain in half-saturated macromolecules is not exclusive of strongly inhibited ternary complexes. Measurements on binary complexes with NADH, ADPR, and the inactive coenzyme analogue 1,4,5,6-tetrahydronicotinamide adenine dinucleotide confirm that binding of the ligand to one subunit has qualitatively the same influence on protein structure. If the lifetime of Trp-314 provides clear evidence for an appreciable change in conformation at half-binding that is apparently triggered by the ADPR fragment of the coenzyme, such communication between subunits does not lead to allosteric phenomena in coenzyme binding.

Evidence for ligand-induced conformational changes in proteins from phosphorescence spectroscopy.

Phosphorescence spectroscopy on mouse myeloma IgA J539 in rigid solution at 77K revealed the type of anomalous short-lived component in the tryptophan decay originally observed with lysozyme (Churchich, J.E., 1966. Biochim. Biophys. Acta. 120:406-412) and seen in a large number of Bence Jones proteins (Longworth, J.W., C.L. McLaughlin, and A. Solomon. 1976. Biochemistry. 15:2953-2958). The decay time of the anomalous component that results from the interaction between tryptophan side chains and disulfide linkages in proteins was observed to significantly lengthen in J539 in response to binding of a galactan antigen. With hen egg-white lysozyme in which the type of fluorescence enhancement on ligand binding seen with J539 has also been observed, phosphorescence measurements revealed a similar lengthening of the decay time of the disulfide-induced anomalous component in the tryptophan decay. These perturbations are interpreted as ligand-induced changes to the tryptophan-disulfide proximities that have been shown to exist in these structures. Given the short-range nature of the disulfide perturbation (see following article) the observations suggest, in particular when combined with x-ray crystallographic data, that phosphorescence decay-time measurements of disulfide perturbations can serve as a sensitive spectroscopic indicator of subtle conformational changes in immunoglobulins and other tryptophan-disulfide containing proteins.

Distance dependence of the tryptophan-disulfide interaction at the triplet level from pulsed phosphorescence studies on a model system.

In the present study the distance dependence of tryptophan-disulfide interaction is examined with a view to both utilizing the interaction as a more quantitative indicator of subtle conformational changes in proteins as well as elucidating the interaction mechanism. To examine perturbations specifically at the indole triplet level 2-(3-indolyl)-ethyl phenyl ketone (IEPK) in which excitation is transferred with high efficiency to the triplet state of the indole moiety was employed. Phosphorescence decays of IEPK excited by a laser pulse in 70/30 (vol/vol) ethanolether at 77 K were measured in the presence of various concentrations of simple disulfides. The nonexponential phosphorescence decays arising from a distribution of fixed chromophoreperturber separations and the steady-state quenching of IEPK were accounted for with an exponential dependence of the quenching rate constant with distance. The small effective Bohr radius (0.8 A) that appears in the exponent emphasizes the localized nature of the interaction. Comparison of the triplet quenching rate constant obtained at quencher contact with IEPK to that estimated in proteins suggests a dependence on the triplet energy of the indole moiety and an endothermic nature for the quenching process. The study predicts that in proteins tryptophan-disulfide interactions are very localized in nature and should give rise to detectable anomalous decays only out to 2 A beyond van der Waals contact between the interacting partners.

Perturbations to the intersystem crossing of proflavin upon binding to DNA and poly d(A-IU) from triplet-delayed emission spectroscopy.

The steady-state prompt fluorescence, phosphorescence and delayed fluorescence spectra and triplet lifetimes of free proflavin and proflavin bound to native DNA and alternating poly d(A-IU) were obtained as a function of temperature in a buffer-glycerol solvent. The intensity of the proflavin E-type delayed fluorescence (DF) relative to both the phosphorescence (Ph) and the prompt fluorescence (F) was observed to increase with temperature, and plots of both ln (DF/Ph) and ln (DF/(F.tau T] as a function of 1/T were linear over a wide range of temperatures. Although the activation energies for the thermal repopulation of the proflavin excited singlet state from the triplet obtained from the slopes of these plots were essentially unchanged on binding, perturbations to the S1----T1 intersystem crossing rate constants extracted from the intercepts at infinite temperature were observed. The marked enhancement of the intersystem crossing that occurs with binding to the iodinated polynucleotide reflects an external heavy atom perturbation upon the intercalated dye which also induces a shortening in the triplet lifetime. With proflavin bound to DNA an enhancement to the S1----T1 intersystem crossing, though lesser in magnitude than for poly d(A-IU), is observed but with no change to the triplet lifetime. The well-studied fluorescence quenching of DNA-bound proflavin is a result of this increase in the intersystem crossing. It is proposed that these non-heavy atom enhancements in the intersystem crossing are due to distortions of the molecular plane of the bound proflavin molecule. In total these analyses provide a complete description of the excited state processes of the proflavin molecule and their variations with temperature.

Rotational mobility associated with the protein moiety of human serum lipoproteins from tryptophan phosphorescence anisotropy measurements.

The steady-state phosphorescence anisotropy of human low density (LDL) and very low density (VLDL) serum lipoproteins in a glycerol--buffer solvent has been investigated. By virtue of the long lifetime of the triplet state, depolarization of the emission resulting from slow rotational motions is observed between -90 and -50 degrees C. Despite the number of emitting residues within these complexes, the rotational behavior can be represented within experimental error by a single correlation time. The magnitude and temperature dependence of the rotational correlation times observed for LDL are consistent with overall rotation of essentially spherical rigid particles as the source of the phosphorescence depolarization. Preliminary data for VLDL indicate the presence of internal peptide motions which are more rapid than those of the overall complex.

Unusual emission properties of the tryptophans at the surface of short ragweed allergen Ra5.

The fluorescence and low-temperature phosphorescence of short ragweed pollen allergen Ra5 have been examined. Both the fluorescence of Ra5 in aqueous solution at room temperature and the phosphorescence of Ra5 in rigid glycol-water solvents at low temperature result entirely from the tryptophan residues in the molecule. The heterogeneity in the low-temperature phosphorescence spectra indicates that the two tryptophans in Ra5 are located in distinct environments and can be studied independently. The fluorescence wave-length maximum and large solvent-relaxation shift in the fluorescence of the allergen in 1:1 ethylene glycol-water reveal that at least one of the residues is exposed to the polar solvent. Studies of the iodide and cesium ion quenching of the fluorescence of Ra5 are consistent with this assignment but suggest that one of the tryptophans may be shielded by a negative charge in the protein. Phosphorescence data obtained in the presence of iodide and with increasing temperature confirm that both aromatic residues are readily subject to perturbation but identify the more susceptible one with one of the two spectral components that manifest themselves in the phosphorescence of Ra5. The phosphorescence wavelength maxima and the triplet-state lifetimes of the distinct tryptophans reveal that both residues lie in unusual local environment at the protein surface. While one aromatic side chain appears to be stabilized by an intramolecular interaction with a polar function, the other is perturbed quite possibly through an interaction with a disulfide linkage in the molecule.

Phosphorescence studies of the interaction of myelin basic protein with phosphatidylserine vesicles.

Phosphorescence from the lone tryptophan residue has been studied to monitor the interaction of myelin basic protein with phosphatidylserine vesicles. Spectral shifts in the phosphorescence of the protein in a glycerol-buffer (70:30 w/w) solvent at low temperature are consistent with fluorescence data obtained under ambient conditions, indicating that the tryptophan side chain is exposed to the solvent in the free protein but is buried on interaction with a lipid bilayer. Measurements of the phosphorescence intensity and lifetime as a function of temperature reveal a marked protection of the tryptophan to thermally induced quenching in the presence of phosphatidylserine vesicles. Steady-state anisotropy measurements on the tryptophan phosphorescence were used to follow the slow motions of the protein associated with the synthetic bilayer. The observations that the rotational correlation time for the membrane-associated protein is 4 X 10(3) times that anticipated for a molecule the size of basic protein reflects its partial intrinsic character in the membrane.

Phosphorescence evidence for the role of solvent--protein interactions in the energetics of conformational flexibility of liver alcohol dehydrogenase.

When tryptophyl side chains are hidden within relatively inflexible domains of globular proteins, the lifetime of the phosphorescence from these residues provides a measure of the local conformational flexibility. The phosphorescence decay from the tryptophan buried at the base of the nucleotide-binding domain in liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was monitored between 1 and 40 degrees C to determine the energetics associated with the rate of local unfolding. The slow rate at which this process takes place is found to result from a high entropic barrier rather than from the disruption of strong intramolecular interactions. This observation along with the response of the system to solvent perturbations points to the significance of solvent-protein interactions in determining conformational flexibility.

Room temperature phosphorescence and the dynamic aspects of protein structure.

While the phosphorescence of aromatic chromophores in solution is normally quenched through diffusion of dissolved oxygen and other solvent-mediated processes, the phosphorescence of some proteins in solution is observed at room temperature. The tryptophan phosphorescence arises from residues which are hindered from interaction with oxygen by the folding of the polypeptide chains. Measurements of the phosphorescence lifetime of horse liver alcohol dehydrogenase (alcohol: NAD(+) oxidoreductase, EC 1.1.1.1) as a function of oxygen concentration indicate that internal tryptophan residues are periodically exposed to oxygen. This permits the calculation of rate constants for conformational oscillations in the enzyme. The present article illustrates the feasibility of employing phosphorescence in the study of proteins in solution in general and the utility of such experiments in probing the dynamic aspects of protein structure.

Spin-orbital probes of biomolecular structure. A model DNA-acridine system.

Heavy atoms, such as bromine or iodine, perturb the excited-state properties of aromatic chromophores through a spin-orbital coupling mechanism. In the present work the use of specifically directed spin-orbital probes to study subtle structural relationships in biopolymers is described. Heavy atoms are introduced into defined sites in biochemical systems and the emission spectrum of a ligand or intrinsic chromophore is monitored for perturbation by the bound heavy atom. This technique is illustrated by a study of acridine dye binding to the copolymer poly(dA-BrdU). The results are interpreted in terms of an "externally" bound dye fraction whose emission is perturbed by the heavy atom in the polymer and an intercalated dye component unperturbed by bromine.

Role of heterogeneity of the solvation site in electronic spectra in solution.

The emission spectra of polar aromatic molecules in rigid, polar solution are shown to depend on the exciting wavelength. Occurrence of the phenomenon depends on both the excited-state lifetime of the chromophore and the degree of rigidity of the medium. The results are interpreted in terms of a model which stresses the contribution of micro-environmental heterogeneity to electronic absorption and emission spectra.