Spectroscopic study and evaluation of red-absorbing fluorescent dyes

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.     

Oxazine dye-conjugated dna oligonucleotides: Förster resonance energy transfer in view of molecular dye-DNA interactions

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive F?rster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large F?rster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.     

Intrinsic fluorescence of the P-glycoprotein multidrug transporter: sensitivity of tryptophan residues to binding of drugs and nucleotides

P-glycoprotein is a member of the ATP binding cassette family of membrane proteins, and acts as an ATP-driven efflux pump for a diverse group of hydrophobic drugs, natural products, and peptides. The side chains of aromatic amino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluorescence techniques were used to probe the environment of the 11 tryptophan residues within purified functional P-glycoprotein, and their response to binding of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environment, and time-resolved experiments showed the existence of at least two lifetimes. Quenching studies with acrylamide and iodide indicated that those tryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguing against large changes in tryptophan accessibility following substrate binding. P-glycoprotein fluorescence was highly quenched on binding of fluorescent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggesting that the site for nucleotide binding is located relatively close to tryptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowing estimation of dissociation constants. Many compounds exhibited biphasic quenching, suggesting the existence of multiple drug binding sites. The quenching observed for many substrates was attributable largely to resonance energy transfer, indicating that these compounds may be located close to tryptophan residues within, or adjacent to, the membrane-bound domains. Thus, the regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hydrolysis to drug transport.     

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.     

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.     

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.     

Location of tryptophans in membrane-bound annexins

The annexins are a novel class of calcium-dependent membrane binding proteins with highly homologous sequences and similar binding characteristics. In order to better define structural parameters of the membrane-bound form, the localization of tryptophan residues in several of these proteins was studied by use of quenchers of their intrinsic fluorescence. Lipocortin I contains a single tryptophan located near its N-terminus, while the single tryptophan in lipocortin V is located in a repeated consensus sequence. Calcium-dependent binding to vesicles composed of 50% egg phosphatidylcholine and 50% bovine brain phosphatidylserine was accompanied by an increase in emission intensity resulting from a relief of internal quenching. The tryptophan fluorescence of bound lipocortin I was nearly unaffected by substituting the quencher 1-palmitoyl-2-(5-doxylstearoyl)-sn-glycero-3-phosphocholine (5-PC) for egg phosphatidylcholine, while that of the lipocortin V tryptophan was quenched significantly. With the quenching doxyl spin-label located deeper in the bilayer at the 12- and 16-positions of the acyl chain, the quenching was progressively weaker, suggesting an interfacial location for the tryptophan of lipocortin V. The same experiments with lipocortin I show almost no quenching in any case, suggesting that this tryptophan near the amino terminus is protected or oriented away from the membrane surface. Data on the bovine liver calelectrins are also presented showing that endonexin also has a tryptophan residue that interacts strongly with phospholipids.     

Conformational changes of FtsZ reported by tryptophan mutants

E. coli FtsZ has no native tryptophan. We showed previously that the mutant FtsZ L68W gave a 2.5-fold increase in trp fluorescence when assembly was induced by GTP. L68 is probably buried in the protofilament interface upon assembly, causing the fluorescence increase. In the present study we introduced trp residues at several other locations and examined them for assembly-induced fluorescence changes. L189W, located on helix H7 and buried between the N- and C-terminal subdomains, showed a large fluorescence increase, comparable to L68W. This may reflect a shift or rotation of the two subdomains relative to each other. L160W showed a smaller increase in fluorescence, and Y222W a decrease in fluorescence, upon assembly. These two are located on the surface of the N and C subdomains, near the domain boundary. The changes in fluorescence may reflect movements of the domains or of nearby side chains. We prepared a double mutant Y222W/S151C and coupled ATTO-655 to the cys. The Cα of trp in the C-terminal subdomain was 10 ? away from that of the cys in the N-terminal subdomain, permitting the ATTO to make van der Waals contact with the trp. The ATTO fluorescence showed strong tryptophan-induced quenching. The quenching was reduced following assembly, consistent with a movement apart of the two subdomains. Movements of one to several angstroms are probably sufficient to account for the changes in trp fluorescence and trp-induced quenching of ATTO. Assembly in GDP plus DEAE dextran produces tubular polymers that are related to the highly curved, mini-ring conformation. No change in trp fluorescence was observed upon assembly of these tubes, suggesting that the mini-ring conformation is the same as that of a relaxed, monomeric FtsZ.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies

Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that the parinaric acid microenvironment in the fatty acid-binding site of I-FABP was much less polar than that of L-FABP. Parinaric acids displayed similar rotational correlation time and limiting anisotropy when bound to I-FABP and to L-FABP. These results are consistent with a close proximity of bound fatty acids to the tyrosine and tryptophan residues and with immobilization of the polyene fatty acids in the fatty acid-binding site(s) of L-FABP and I-FABP. The two proteins differ in that only L-FABP has two fatty acid-binding sites and appears to undergo significant conformational change upon fatty acid binding.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

A quenched fluorescent dipeptide for assaying dispase- and thermolysin-like proteases

Metalloproteases such as dispase and thermolysin play a crucial role in the life cycle of bacteria. Commonly, they prefer hydrophobic amino acids at P1' of substrate proteins, thereby cleaving the peptide bond at the alpha amino group. Activity of such proteases has been measured by the use of tailor-made oligopeptides provided with fluorescence resonance energy transfer dyes. We can now show that the short dipeptide Dabcyl-Ser-Phe-EDANS is an appropriate substrate of dispase and thermolysin. It was cleaved by both enzymes at the single peptide bond accompanied by a steep increase in fluorescence. Substantial quenching effects of the formed products were observed only when more than 80microM substrate was hydrolyzed. High affinity of the proteases for the dipeptide resulted in low K(m) values of 91+/-9 and 104+/-18microM, which are comparable to those measured for longer peptides. Dabcyl-Ser-Phe-EDANS was also used to determine the pH and optimal temperature of dispase, which were found at pH 7.0 and 50 degrees C. Buffer substances such as acetate, citrate, and tris(hydroxymethyl)aminomethane had no significant effect on enzyme activity. Measurements up to 100 degrees C revealed that hydrolysis of the quenched fluorescent dipeptide took place only in the presence of active dispase.     

Fluorescence quenching due to mercuric ion interaction with aromatic amino acids and proteins

Mercuric ion interacts with indoles, including tryptophan, to produce complexes whose absorption spectra are broader, less structured, and red-shifted as compared with those of the parent compound. Fluorescence and phosphorescence are totally quenched. In a survey of the effect of transition metal ions on tryptophan fluorescence, the strong quenching by Hg²⁺ was unique among the uncolored ions. Mercuric nitrate quenched the fluorescence of practically every protein tested, but the sensitivity to quenching varied with the protein. Ovalbumin was the most sensitive to quenching by Hg²⁺, over 70% of the intrinsic fluorescence being quenched by 2 moles of mercuric ion. Difference absorption spectra show that sulfhydryl groups are attacked by these reagents and Hg²⁺ is, in addition, perturbing the environment near some tryptophans. In contrast to Hg²⁺, Zn²⁺ had negligible effect on protein fluorescence. The emission spectra of proteins which were partly quenched by mercuric ion showed shifts in their maxima to higher or lower wavelengths. This suggests that mercuric ion quenched certain tryptophans more than others, and supports the idea that protein fluorescence is heterogeneous and arises from tryptophans in different microenvironments.     

Molecular conformation and fluorescence properties of lactalbumin from four animal species

The quantum yield of tryptophan fluorescence of guinea-pig α lactalbumin is more than twice as large as found for the bovine, goat, or human proteins. This difference is due to the absence of Trp 60 from the guinea-pig amino acid sequence. It is proposed that the presence of this residue results in marked quenching of a second tryptophan residue for the other three proteins. Reference to the “lysozyme analogy model” suggests that the quenched residue is most likely Trp 104, which lies within 7Å of Trp 60, the latter lying within 6Å of two disulfide bridges. It is suggested that transfer of the excited state energy takes place from Trp 104 to 60 with subsequent quenching by the two vicinal disulfide bridges. These observations provide support for the validity of the “lysozyme analogy model”.     

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.     

Spatial orientation and dynamics of the U1A proteins in the U1A-UTR complex

The human U1A protein contains three distinct domains: the N-terminal RBD1 (amino acids 1-101), the C-terminal RBD2 (amino acids 195-282), and the linker region (amino acids 102-194). The RBD1 domains of two U1A proteins bind specifically to two internal loops in the 3' untranslated region (3' UTR) of its own pre-mRNA. Tryptophan fluorescence and fluorescence resonance energy transfer data show that the two RBD2 domains do not interact with any regions of the UTR complex and display an overall tumbling that is uncorrelated from the core of the complex (formed by RBD1-UTR), indicating that the linker regions of the two U1A proteins remain flexible. The two RBD2 domains are separated by an apparent distance greater than 74 A in the UTR complex. The linker region adjacent to the RBD1 domain (103-ERDRKREKRKPKSQETP-119) is supposedly involved in protein-protein interactions (12). A single cysteine, introduced at position 101 or 121 of the U1A protein, was used as a specific attachment site for the fluorophore pair IAEDANS [N'-iodoacetyl-N'-(1-sulfo-5-n-naphthyl)ethylenediamine]/DABMI [4-(dimethylamino)-phenylazophenyl-4'-maleimide]. In the U1A-UTR complex (2:1), the dyes at the 101 position are separated by = approximately 51 A, while the dyes at the 121 position are at an apparent distance = approximately 58 A. The 101-121 crossed distance on adjacent U1A proteins averages to = 55 A. These results suggest that the amino acid sequence 101-121 of the two U1A proteins in the complex are held in proximity to each other in a compact conformation.     

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.     

Bacterial expression and characterization of the ligand-binding domain of the vitamin D receptor

The ligand-binding domain of the rat vitamin D receptor (amino acids 115-423) was expressed as an amino-terminal His-tagged protein in a bacterial expression system and purified over Ni-nitrilotriacetic acid resin and a Mono S column. The purified protein bound its ligand, 1,25-dihydroxyvitamin D3, with high affinity, similar to that of the full-length protein. Saturation of the protein with ligand quenched 90% of the tryptophan fluorescence, consistent with the purified protein being uniformly able to bind ligand. Addition of ligand produced no change in the tryptophan fluorescence lifetime, suggesting static quenching as the mechanism of fluorescence decrease. The near-UV circular dichroism spectrum showed a large increase in signal following the addition of ligand, consistent with a change in the environment of aromatic amino acid side chains. The far-UV circular dichroism spectrum was consistent with a protein of high alpha-helical content. Sedimentation equilibrium experiments demonstrated that the protein formed higher-order complexes, and the distribution of the protein among these complexes was significantly shifted by addition of ligand.     

Rotational freedom of tryptophan residues in proteins and peptides

We studied the rotational motions of tryptophan residues in proteins and peptides by measurement of steady-state fluorescence anisotropies under conditions of oxygen quenching. By fluorescence quenching we can shorten the fluorescence lifetime and thereby decrease the average time for rotational diffusion prior to fluorescence emission. This method allowed measurement of rotational correlation times ranging from 0.03 to 50 ns, when the unquenched fuorescence lifetimes are near 4 ns. A wide range of proteins and peptides were investigated with molecular weights ranging from 200 to 80 000. Many of the chosen substances possessed a single tryptophan residue to minimize the uncertainties arising from a heterogeneous population of fluorophores. In addition, we also studied a number of multi-tryptophan proteins. Proteins were studied at various temperatures, under conditions of self-association, and in the presence of denaturants. A wide variety of rotational correlation times were found. As examples we note that the single tryptophan residue of myelin basic protein was highly mobile relative to overall protein rotation whereas tryptophan residues in human serum albumin, RNase T1, aldolase, and horse liver alcohol dehydrogenase were found to be immobile relative to the protein matrix. These results indicate that one cannot generalize about the extent of segmental mobility of the tryptophan residues in proteins. This physical property of proteins is highly variable between proteins and probably between different regions of the same protein.