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.     

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II)

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.     

Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy

This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.     

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 α-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 ~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.     

Optically detected magnetic resonance in lysozyme: evidence for three phosphorescent TRP residues

The Optically Detected Magnetic Resonance spectrum of lysozyme has been shown to consist of a multiplet of narrow components, at -1565 MHz, 1585 MHz, and 1620 MHz. The 1585 MHz component is the strongest feature of the spectrum. This is consistent with earlier reports which apparently resolved only this principal component in lysozyme. The linewidths reported here are the narrowest ever reported for tryptophan in proteins. Using Microwave-Induced Phosphorescence techniques, the dominant 1585 MHz line is seen to be coupled to a "narrow" phosphorescence emission component at about 4134A. This component has a bandwidth of about 25A compared to 42A for the normal O-O band for tryptophan in lysozyme.     

Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme.

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.     

The low-temperature luminescence properties of bovine alpha-lactalbumin.

The luminescence of bovine alpha-lactalbumin at 77 K has been studied and compared with that of lysozyme. Alpha-Lactalbumin has several unusual properties, including a fluorescence spectrum showing vibrational fine structure, an abnormal phosphorescence spectrum, a high fluorescence: phosphorescence ratio and an abnormal phosphorescence decay. These properties are largely due to the proximity of tryptophan residues to disulphide bonds. Reduction of all these bonds causes considerable changes in alpha-lactalbumin luminescence, as does denaturation in acid solution. Reduction of a single labile disulphide bond has little effect, and the properties of alpha-lactalbumin III, a variant lacking one disulphide bond and one trypotophan residue, are similar to those of the normal protein. Several differences between alpha-lactalbumin and lysozyme are reported. The results support the suggestion that the two tryptophan residues found in the active site cleft of alpha-lactalbumin may be largely responsible for its luminescence.     

Time-resolved phosphorescence of tyrosine,tyrosine analogs,and tyrosyl residues in oxytocin and small peptides

We present the time-resolved phosphorescence of oxytocin, two oxytocin derivatives, vasopressin and a series of compounds that serve as models for free tyrosine. One of the oxytocin derivatives, desaminodicarbaoxytocin, has the disulfide bridge replaced by an ethylene bridge, and lacks the N-terminus. Similar to the reported fluorescence decays of tyrosine in these peptides, the phosphorescence decays generally are not single exponentials, but can be fit as biexponentials. The decay times for the oxytocin peptides are shorter than for desaminodicarbaoxytocin or the model compounds, and this we attribute to enhanced spin-orbit coupling due to the presence of sulfur. We measured the phosphorescence decay of the model cyclic pentapeptide that contains tyrosine and compared it to that observed for the same cyclic pentapeptide in which tyrosine is replaced by tryptophan. We also report the phosphorescence of 2-tryptophan-oxytocin, and deamino-2-tryptophan-oxytocin in which biexponential phosphorescence decay is also observed.     

Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme.

The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.     

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.     

Phase-resolved spectral measurements with several two tryptophan containing proteins

We have used frequency domain fluorescence techniques to resolve the component emission spectra for several two tryptophan containing proteins (e.g., horse liver alcohol dehydrogenase, sperm whale apomyoglobin, yeast 3-phosphoglycerate kinase, apoazurin from Alcaligenes denitricans). We have first performed multifrequency phase/modulation measurements and have found the fluorescence of each of these proteins to be described by a double exponential. Then, using phase-sensitive detection and the algorithm of Gratton and Jameson [Gratton, E., & Jameson, D. M. (1985) Anal. Chem. 57, 1694-1697], we have determined the emission spectrum associated with each decay time for these proteins. We have compared these phase-resolved spectra with the fractional contributions of the component fluorophores determined by selective solute quenching experiments. Reasonably good agreement is seen in most cases, which argues that the individual Trp residues emit independently. In the case of apoazurin, however, a negative amplitude is seen for the phase-resolved spectrum of the short-lifetime component. This pattern is consistent with the occurrence of energy transfer from the internal Trp residue to the surface Trp of this protein. We also present multifrequency lifetime measurements, phase-resolved spectra, and solute quenching data for a few protein-ligand complexes, to illustrate the utility of this approach for the study of changes in the fluorescence of proteins.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Triplet state energy transfer in several proteins.

Energy transfer between excited triplet states of aromatic amino acid residues was observed at 1.4 degrees K. The distance necessary for energy transfer between monomeric tyrosine and tryptophan residues was determined to be roughly 63 A. Total phosphorescence decay rate constants for several proteins were determined while emission corresponding to tyrosine and tryptophan residues was monitored. The observed decay rate constants are interpreted in terms of intramolecular interactions of the polypeptide residues.     

Protein structural changes induced by glutathione-coated CdS quantum dots as revealed by Trp phosphorescence

We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins involved in interaction with nanoparticles. Characterization of protein–nanoparticle interaction is crucial in assessing biological hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular formula of GSH₁₈S₅₆Cd₆₀. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe, and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS. Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated affinities were comparable to those observed for specific protein–ligand interactions and suggest that protein–nanoparticle interaction may have a biological impact.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.     

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.     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Time-dependent phosphorescence anisotropy measurements of the slow rotational motions of proteins in viscous solution

A method of monitoring slow rotational motions of proteins from the decay of the intrinsic phosphorescence is described. The phosphorescence is excited with a 10-μsec pulse of vertically polarized light from an air gap lamp, and the anisotropy was computed as a function of time from the simultaneously detected vertically and horizontally polarized components of the emission. The approach is illustrated with time-dependent measurements of the anisotropy of the tryptophan phosphorescence of Staphylococcus aureus nuclease, bovine carbonic anhydrase, and liver alcohol dehydrogenase in glycerol-phosphate buffer between ?90 and ?70°C. The temperature- and molecular-weight dependence of the exponential decays in the anisotropy indicate that overall rotation of the proteins is at the origin of the depolarization. The potential of the approach as a probe of the slow rotational motions of proteins in membranes and other macromolecular complexes is stressed.