Fluorescence lifetime distributions in proteins.

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime distributions of parinaroyl phospholipids in choline plasmalogen and phosphatidylcholine bilayers containing different amounts of cholesterol

The fluorescence decay of alkenylparinaroyl- and palmitoylparinaroyl glycerophosphocholines in vesicles of the unlabeled alkenyloleoyl and palmitoyloleoyl analogs was determined by multifrequency phase and modulation fluorometry. The measured phase angles and demodulations could be equally well fitted to a biexponential decay, as well as unimodal or bimodal continuous lifetime distributions. The latter model was applied to study the influence of cholesterol on parinaroyl phospholipid fluorescence in vesicles. The long-living component of a bimodal lifetime distribution was sensitive toward the presence of the sterol. Upon increasing cholesterol concentrations, its lifetime center increased and its distribution widths decreased. Lifetime distribution widths in vesicles of alkenyloleoyl- or palmitoyloleoyl-glycerophosphocholine (choline plasmalogen and phosphatidylcholine, respectively) were reduced by the sterol to the same extent. We interprete the sterol-induced lifetime distribution narrowing as an effect due to an increase of membrane homogeneity in cholesterol-phospholipid membranes.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.     

Fluorescence lifetime distributions in human superoxide dismutase. Effect of temperature and denaturation.

The internal dynamics of human superoxide dismutase has been studied using time-resolved fluorescence. The fluorescence decay has been analyzed using continuous distribution of lifetime values. The effect of temperature and conformational state on the lifetime distribution has been investigated. The emission of the single tryptophan residue depends on the nature and dynamics of the protein matrix. Conformational changes have been induced by increased concentration of guanidinium hydrochloride. We found that both temperature and conformation strongly effect the width of the lifetime distribution.     

Capillary affinophoresis as a versatile tool for the study of biomolecular interactions: a mini-review

Combination of capillary electrophoresis and bioaffinity interaction gave rise to powerful research tools for analyzing molecular recognition. They take advantages of the electrophoretic behavior of the complex formed between a target biomolecule and a specially designed mobile ligand molecule (affinophore or affinity probe), and enable detection of complex formation, determination of the equilibrium constants and stoichiometry, etc.     

A solid-state NMR study of phospholipid-cholesterol interactions: sphingomyelin-cholesterol binary systems

We used solid-state NMR techniques to probe the interactions of cholesterol (Chol) with bovine brain sphingomyelin (SM) and for comparison of the interactions of Chol with dipalmitoylphosphatidylcholine (DPPC), which has a similar gel-to-liquid crystalline transition temperature. (1)H-, (31)P-, and (13)C-MASNMR yielded high-resolution spectra from multilamellar dispersions of unlabeled brain SM and Chol for analysis of chemical shifts and linewidths. In addition, (2)H-NMR spectra of oriented lipid membranes with specific deuterium labels gave information about membrane ordering and mobility. Chol disrupted the gel-phase of pure SM and increased acyl chain ordering in the liquid crystalline phase. As inferred from (13)C chemical shifts, the boundaries between the ordered and disordered liquid crystalline phases (L and L) were similar for SM and DPPC. The solubility limit of Chol in SM was ~50 mol %, the same value as previously reported for DPPC membranes. We found no evidence for specific H-bonding between Chol and the amide group of SM. The order parameters of a probe molecule, d31-sn1-DPPC, in SM were slightly higher than in DPPC for all carbons except the terminal groups at 30 mol % but were not significantly different at 5 and 60 mol % Chol. These studies show a general similarity with some subtle differences in the way Chol interacts with DPPC and SM. In the environment of a typical biomembrane, the higher proportion of saturated fatty acyl chains in SM compared to other phospholipids may be the most significant factor influencing interactions with Chol.     

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbon-13 as a tool for the study of carbohydrate structures,conformations and interactions

The application of ¹³C-NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both ¹³C-enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon-carbon and carbon-proton coupling constants obtained from 1-¹³ C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C-3 couples to C-1 only in the β-anomers while C-5 couples to C-1 only in the α-anomers. C-6 and C-2 always couple to C-1 in both anomeric species. The alkaline degradation of glucose [1-¹³ C] to saccharinic acids was followed by ¹³C-NMR. The conversion of glucose [1-¹³ C] to fructose-1,6-bisphosphate [1,6-¹³ C] by enzymes of the glycolytic pathway was shown as an example of the use of ¹³C-enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the ³¹P to H-1 and ³¹P to C-2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O-5 and the H-1 of the pyranose ring. The influence of paramagnetic Mn²⁺ ions on the proton decoupled ¹³C-NMR spectra of uridine diphosphate N-acetylglucosamine indicates that the Mn²⁺ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N-acetyl groups.     

Microspectrofluorometry as a tool for investigation of non-calcium interactions of Indo-1.

Indo-1 is a fluorescent calcium probe used to measure intracellular free calcium concentrations. These measurements are often performed by comparing the fluorescence intensities of Indo-1-treated cells at two selected wavelengths corresponding to the maxima of the fluorescence spectra of the calcium-bound and calcium-free forms. In this study, we used an optical multichannel analyser to numerise the fluorescence emitted by a single cell. A computerised resolution of numerised spectra was used on intracellular Indo-1 fluorescence. Calculation of numerical and graphic estimators allows us to evaluate the fit of the resolution. Different sets of characteristic spectra were compared using this method. It appeared that no linear combination of the two known forms of Indo-1 and of the cell autofluorescence can fit with spectra of Indo-1-treated cells. In addition, a study of the physico-chemical properties of Indo-1 shows the existence of two other forms of the molecule: a protonated form (maximum emission at 455 nm) and a form in interaction with proteins (maximum emission at 438 nm). Taking into account the contribution of these two new forms leads to an improved spectral resolution of the fluorescence of Indo-1-treated living cells and, therefore, improves calcium measurements. Moreover, quantification of the amount of the protonated form of Indo-1 allows a measurement of intracellular pH at the same time as calcium determination.     

Allele-specific suppression as a tool to study protein-protein interactions in bacteria

Suppression analysis is well suited to study the interactions of gene products. It offers the advantage of simplicity for any organism for which a convenient genetic system has been developed, which holds for a wide spectrum of bacteria and an ever-increasing number of unicellular as well as complex eukaryotes. No other method provides as much information about the functional relationships of biological macromolecules. The intrinsic value of suppression analysis is enhanced by advances in genomics and in biophysical techniques for investigating the properties of nucleic acids and proteins, such as X-ray crystallography, liquid and solid-state nuclear magnetic resonance, electron spin labeling, and isothermal calorimetry. These approaches confirm and complement whatever is revealed by genetics. Despite these sterling qualities, suppression analysis has its dangers, less in execution than in conceptualization of experiments and interpretation of data. A consistent nomenclature is essential for a uniform and widespread understanding of the results. Familiarity with the genetic background and idiosyncracies of the organism studied is critical in avoiding extraneous phenomena that can affect the outcome. Finally, it is imperative not to underestimate potentially bizarre and improbable consequences that can transpire when rigorous genetic selection is maintained for an appreciable length of time. The article begins with a somewhat pedagogical discussion of genetic terminology. It then moves on to the necessary precautions to observe while planning and conducting suppression analysis. The remainder of the article considers different manifestations of suppression: bypass suppression; gradients of suppression; suppression by relaxed specificity; allele-specific "suppression at a distance"; and true conformational suppression. The treatment is not exhaustive, but representative examples have been gleaned from the recent bacterial literature.     

Analytical ultracentrifugation as a tool for studying protein interactions

The last two decades have led to significant progress in the field of analytical ultracentrifugation driven by instrumental, theoretical, and computational methods. This review will highlight key developments in sedimentation equilibrium (SE) and sedimentation velocity (SV) analysis. For SE, this includes the analysis of tracer sedimentation equilibrium at high concentrations with strong thermodynamic non-ideality, and for ideally interacting systems, the development of strategies for the analysis of heterogeneous interactions towards global multi-signal and multi-speed SE analysis with implicit mass conservation. For SV, this includes the development and applications of numerical solutions of the Lamm equation, noise decomposition techniques enabling direct boundary fitting, diffusion deconvoluted sedimentation coefficient distributions, and multi-signal sedimentation coefficient distributions. Recently, effective particle theory has uncovered simple physical rules for the co-migration of rapidly exchanging systems of interacting components in SV. This has opened new possibilities for the robust interpretation of the boundary patterns of heterogeneous interacting systems. Together, these SE and SV techniques have led to new approaches to study macromolecular interactions across the entire spectrum of affinities, including both attractive and repulsive interactions, in both dilute and highly concentrated solutions, which can be applied to single-component solutions of self-associating proteins as well as the study of multi-protein complex formation in multi-component solutions.     

Treatment with lithium as a tool for the study of animal-vegetal interactions in sea urchin embryos

Summary The question about the nature of the effect of lithium on early sea urchin development is reexamined. Essential features of the morphogenetic changes of lithium treated embryos are followed with continuous comparisons with control embryos. The key to the lithium effect is the greater cytoplasmic susceptibility in the animal polar region as compared with the vegetal one. This is diagrammed in Fig. 23a-c on the basis of the double gradient concept. There is a decline of the animal gradient with increasing lithium concentration. As a consequence the level of differentiation of the terminal region becomes more and more vegetal, seean/veg-values in Fig. 23. The region suppressed by a prolonged exposure to lithium, e.g. 9–16 hours, cannot be restored. Nevertheless, there are data from previous research supporting the view that the primary effects of lithium are reversible within certain limits. However, when the normal balance is disturbed by decline of the animal gradient and particularly by suppression of its higher levels, there is a compensatory enhancement of the vegetal gradient system which stabilizes the suppression. As a consequence of the suppression of the higher animal levels, a certain accumulation of cells in an anterior direction has taken place in the blastula stage. The degree of accumulation reflects the degree of vegetalization. Later there is to varying extent a backflow of cells in the vegetal direction. It was shown how a great part of the blastula wall may have the aspect of an attachment zone (Fig. 7). The primary mesenchyme cells attach themselves only to a certain level of the ectoderm in which the relativean/veg-value is around 0.7 according to the conventions behind Fig. 23.Sections of Carnoy fixed embryos were exposed to trypsin. It proved that the external cytoplasm of blastodermic cells in lithium treated embryos was more strongly attacked than the internal one. The latter showed a strong resistance to tryptic action. On the other hand, in the control embryos the inner part of blastodermic cells was completely digested with exception of the vegetal region including the attachment zone. The trypsin resistant structure may be preformed and may be responsible for the higher rigidity of the cytoplasm in lithium treated embryos (section IIIf).It is proposed that in the period of lithium susceptibility, the colloidal state is most affected in the animal region, thereby creating a block to the diffusion of the animalizing substances which results in the shifts diagrammed in Fig. 23.     

Biomass size distributions as a tool for characterizing lake fish communities

Biomass size distributions (BSDs) can be useful tools to (1) summarize complex information about fish community structure in a condensed graphical form, facilitating the characterization of freshwater fish communities, (2) compare the position of fish communities along environmental gradients and (3) elucidate major trophic interactions in freshwater fish communities. Biomass size distributions are presented by taxonomic and trophic group, for a selection of fish communities from 35 Scandinavian and eight Dutch lakes. They were used for the analysis of taxonomic and trophic shifts in the fish communities along a large environmental gradient, with productivity (expressed as total phosphorus concentration, TP) as its most important component. Regression analysis of fish community variables (such as proportion of cyprinids, or biomass of benthivores) were consistent with the semi‐quantitative conclusions drawn from BSDs, regarding taxonomic and trophic shifts with changes in TP in both Scandinavian and Dutch lakes, especially an increase in the amount and size of benthivorous fishes with increasing TP‐levels. In addition, differences in mortality and growth rates were shown to partly explain differences in BSDs. Biomass size distributions thus provide an integrative tool for qualitative and quantitative comparisons among fish communities.     

Protein-protein interactions as a tool for site-specific labeling of proteins

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.     

Fluorescence resonance energy transfer as a structural tool for nucleic acids

Fluorescence resonance energy transfer is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-80 A. It is particularly suited to the analysis of the global structure of nucleic acids because the long-range distance information provides constraints when modelling these important structures. The application of fluorescence resonance energy transfer to nucleic acid structure has seen a resurgence of interest in the past decade, which continues to increase. An especially exciting development is the recent extension to single-molecule studies.     

Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

Fluorescence lifetime distributions of DNA-4',6-diamidino-2-phenylindole complex

Time-resolved fluorescence of 4',6-diamidino-2-phenylindole (DAPI) complexes show that for a homogeneous polymer (polyd(AT) or polyd(A).polyd(T)) at high P/D (phosphate/dye) ratio, a single exponential component adequately describes the fluorescence decay. For the AT polymers at low P/D ratio or for native DNA, the decay cannot be described by a single-exponential term. A continuous distribution of lifetime values of Gaussian shape gives a good fit to the decay data. We propose that the lifetime distribution method for the analysis of the fluorescence decay of DNA-DAPI complexes provides a useful method of characterizing the microheterogeneity of site binding.     

Protein microarrays as a discovery tool for studying protein-protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein-protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein-protein interaction studies.