Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

Repair of diverse diabetic defects of ß-cells in man and mouse by pharmacological glucokinase activation.

Glucokinase activators (GKAs) are being developed and clinically tested for potential antidiabetic therapy. The potential benefits and limitations of this approach continue to be intensively debated. To contribute to the understanding of experimental pharmacology and therapeutics of GKAs, we have tested the efficacy of one of these agents (Piragliatin) in isolated islets from humans with type 2 diabetes mellitus (T2DM), from mice with glucokinase (GK) mutations induced by ethyl-nitroso-urea (ENU) as models of Maturity Onset Diabetes of the Young linked to GK and Permanent Neonatal Diabetes Mellitus linked to GK (PNDM-GK) and finally of islets rendered glucose insensitive by treatment with the sulphonyl urea compound glyburide in organ culture. We found that the GKA repaired the defect in all three instances as manifest in increased glucose-induced insulin release and elevated intracellular calcium responses. The results show the remarkable fact that acute pharmacological activation of GK reverses secretion defects of ß-cells caused by molecular mechanism that differ vastly in nature, including the little understood multifactorial lesion of ß-cells in T2DM of man, the complex GK mutations in mice resembling GK disease and acute sulphonylurea failure of mouse ß-cells in tissue culture. The implications of these results are to be discussed on the theoretical basis underpinning the strategy of developing these drugs and in light of recent results of clinical trials with GKAs that failed for little understood reasons.

Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators.

We performed genome-wide mutagenesis in C57BL/6J mice using N-ethyl-N-nitrosourea to identify mutations causing high blood glucose early in life and to produce new animal models of diabetes. Of a total of 13 new lines confirmed by heritability testing, we identified two semi-dominant pedigrees with novel missense mutations (Gck(K140E) and Gck(P417R)) in the gene encoding glucokinase (Gck), the mammalian glucose sensor that is mutated in human maturity onset diabetes of the young type 2 and the target of emerging anti-hyperglycemic agents that function as glucokinase activators (GKAs). Diabetes phenotype corresponded with genotype (mild-to-severe: Gck(+/+) < Gck(P417R/+), Gck(K140E)(/+) < Gck(P417R/P417R), Gck(P417R/K140E), and Gck(K140E/K140E)) and with the level of expression of GCK in liver. Each mutant was produced as the recombinant enzyme in Escherichia coli, and analysis of k(cat) and tryptophan fluorescence (I(320/360)) during thermal shift unfolding revealed a correlation between thermostability and the severity of hyperglycemia in the whole animal. Disruption of the glucokinase regulatory protein-binding site (GCK(K140E)), but not the ATP binding cassette (GCK(P417R)), prevented inhibition of enzyme activity by glucokinase regulatory protein and corresponded with reduced responsiveness to the GKA drug. Surprisingly, extracts from liver of diabetic GCK mutants inhibited activity of the recombinant enzyme, a property that was also observed in liver extracts from mice with streptozotocin-induced diabetes. These results indicate a relationship between genotype, phenotype, and GKA efficacy. The integration of forward genetic screening and biochemical profiling opens a pathway for preclinical development of mechanism-based diabetes therapies.

Mutational analysis of allosteric activation and inhibition of glucokinase.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

Research and development of glucokinase activators for diabetes therapy: theoretical and practical aspects.

Glucokinase Glucokinase (GK GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis glucose homeostasis . Most importantly, it serves as glucose sensor glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs antidiabetic drugs . Small molecules called glucokinase activators (GKAs) glucokinase activators (GKAs) which bind to an allosteric activator allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme's affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM T2DM ) type 2 diabetes mellitus (T2DM) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.

Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy.

The influence of proteins and solutes on hysteresis of freezing and melting of water was measured by infrared (IR) spectroscopy. Of the solutes examined, poly-L-arginine and flounder antifreeze protein produced the largest freezing point depression of water, with little effect on the melting temperature. Poly-L-lysine, poly-L-glutamate, cytochrome c and bovine serum albumin had less effect on the freezing of water. Small compounds used to mimic non-polar (trimethylamine N-oxide, methanol), positively charged (guanidinium chloride, NH(4)Cl, urea) and negatively charged (Na acetate) groups on protein surfaces were also examined. These molecules and ions depress water's freezing point and the melting profiles became broad. Since infrared absorption measures both bulk solvent and solvent bound to the solutes, this result is consistent with solutes interacting with liquid water. The amide I absorption bands of antifreeze protein and poly-L-arginine do not detectably change with the phase transition of water. An interpretation is that the antifreeze protein and poly-L-arginine order liquid water such that the water around the group is ice-like.

Phosphate assisted proton transfer in water and sugar glasses: a study using fluorescence of pyrene-1-carboxylate and IR spectroscopy.

The role of water's H-bond percolation network in acid-assisted proton transfer was studied in water and glycerol solutions and in sugar glasses. Proton transfer rates were determined by the fluorescence of pyrene-1-carboxylate, a compound with a higher pK in its excited state relative to the ground state. Excitation of pyrene-1-COO- produces fluorescence from pyrene-1-COOH when a proton is accepted during the excited singlet state lifetime of pyrene-1-COO-. The presence of glycerol as an aqueous cosolvent decreases proton transfer rates from phosphoric and acetic acid in a manner that does not follow the Stokes relationship on viscosity. In sugar glass composed of trehalose and sucrose, proton transfer occurs when phosphate is incorporated in the glass. Sugar glass containing phosphate retains water and it is suggested that proton transfer requires this water. The infrared (IR) frequency of water bending mode in sugar glass and in aqueous solution is affected by the presence of phosphate and the IR spectral bands of all phosphate species in water are temperature dependent; both results are consistent with H-bonding between water and phosphate. The fluorescence results, which studied the effect of cosolvent, highlight the role of water in assisting proton transfer in reactions involving biological acids, and the IR results, which give spectroscopic evidence for H-bonding between water and phosphate, are consistent with a mechanism of proton transfer involving H-bonding. The possibility that the phosphate-rich surface of membranes assists in proton equilibration in cells is discussed.

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence.

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.

Pyrene-1-carboxylate in water and glycerol solutions: origin of the change of pK upon excitation.

Pyrene-1-carboxy acid has a pK of 4 in the ground state, and a pK of 8 in the excited state. Fluorescence spectra of the acid and base forms are presented as a function of solvent and temperature. Ab initio quantum calculations indicate that the bond between the ring system and the carboxyl group has aromatic character that becomes stronger upon excitation. This stabilization helps to account for the increase in pK upon excitation.

Temperature dependence for fluorescence of beta-NADH in glycerol/water solution and in trehalose/sucrose glass.

Fluorescence imaging of cells and tissue can be used to evaluate beta-NADH redox and location. At low temperature, beta-NADH fluorescence intensity increases and therefore sensitivity of imaging increases. In this paper, the temperature dependence of fluorescence was evaluated for beta-NADH in glycerol/water solution and in trehalose/sucrose glass. The average fluorescence lifetime for NADH in glycerol/water is 0.66 ns, compared with 5.3 ns in trehalose/ sucrose at 20 degrees C. Emission spectra were recorded from 290 to 12 K. The fluorescence of beta-NADH in glycerol/water increases approximately 16 fold and the emission shifts about 35 nm to the blue as temperature decreases. Much smaller change is seen for fluorescence of beta-NADH in sugar glass. Below 77 K, the beta-NADH spectral features did not change significantly with temperature change, and so no increase in sensitivity is obtained by going to very low temperatures. It is suggested that the sensitivity of beta-NADH fluorescence is related to water relaxation around the excited state molecule. Differences in water in various tissues may contribute to beta-NADH fluorescence changes when cells are altered.

Protonation of excited state pyrene-1-carboxylate by phosphate and organic acids in aqueous solution studied by fluorescence spectroscopy.

Pyrene-1-carboxylic acid has a pK of 4.0 in the ground state and 8.1 in the singlet electronic excited state. In the pH range of physiological interest (pH approximately 5-8), the ground state compound is largely ionized as pyrene-1-carboxylate, but protonation of the excited state molecule occurs when a proton donor reacts with the carboxylate during the excited state lifetime of the fluorophore. Both forms of the pyrene derivatives are fluorescent, and in this work the protonation reaction was measured by monitoring steady-state and time-resolved fluorescence. The rate of protonation of pyrene-COO(-) by acetic, chloroacetic, lactic, and cacodylic acids is a function of DeltapK, as predicted by Marcus theory. The rate of proton transfer from these acids saturates at high concentration, as expected for the existence of an encounter complex. Trihydrogen-phosphate is a much better proton donor than dihydrogen- and monohydrogen-phosphate, as can be seen by the pH dependence. The proton-donating ability of phosphate does not saturate at high concentrations, but increases with increasing phosphate concentration. We suggest that enhanced rate of proton transfer at high phosphate concentrations may be due to the dual proton donating and accepting nature of phosphate, in analogy to the Grotthuss mechanism for proton transfer in water. It is suggested that in molecular structures containing multiple phosphates, such as membrane surfaces and DNA, proton transfer rates will be enhanced by this mechanism.

Temperature excursion infrared (TEIR) spectroscopy used to study hydrogen bonding between water and biomolecules.

Water is a highly polar molecule that is capable of making four H-bonding linkages. Stability and specificity of folding of water-soluble protein macromolecules are determined by the interplay between water and functional groups of the protein. Yet, under some conditions, water can be replaced with sugar or other polar protic molecules with retention of protein structure. Infrared (IR) spectroscopy allows one to probe groups on the protein that interact with solvent, whether the solvent is water, sugar or glycerol. The basis of the measurement is that IR spectral lines of functional groups involved in H-bonding show characteristic spectral shifts with temperature excursion, reflecting the dipolar nature of the group and its ability to H-bond. For groups involved in H-bonding to water, the stretching mode absorption bands shift to lower frequency, whereas bending mode absorption bands shift to higher frequency as temperature decreases. The results indicate increasing H-bonding and decreasing entropy occurring as a function of temperature, even at cryogenic temperatures. The frequencies of the amide group modes are temperature dependent, showing that as temperature decreases, the amide group H-bonds to water strengthen. These results are relevant to protein stability as a function of temperature. The influence of solvent relaxation is demonstrated for tryptophan fluorescence over the same temperature range where the solvent was examined by infrared spectroscopy.

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations.

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.

[Aladan3]TIPP: a fluorescent delta-opioid antagonist with high delta-receptor binding affinity and delta selectivity.

Fluorescent analogues of the potent and highly selective delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid beta-(6'-dimethylamino-2'-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald3 analogues of TIPP and TIP all retained delta-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K(e) value of 2.03 nM in the mouse vas deferens assay and five times higher delta vs. mu selectivity (K(i)mu/K(i)delta = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) delta-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.

Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase.

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656-666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 28.7 mm Hg(-1) s(-1) in the presence of BHA. The quenching rate of BHA is 4000 s(-1). The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O(2) diffusion and flexibility in heme proteins.

Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters.

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.

Fluorescence polarization studies of B-phycoerythrin oriented in polymer film.

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 --> State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 --> State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.