Arthur G. Szabo

A new intrinsic fluorescent probe for proteins Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin

The tryptophan analog, 5‐hydroxytryptophan (5HW), has a significant absorbance between 310–320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca2+ binding protein, oncomodulin. Fluorescence anisotropy and time‐resolved fluorescence decay measurements of the interaction between anti‐oncomodulin antibodies and the 5HW‐incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use or 5HW as an intrinsic probe in the study of structure and dynamics of protein—protein interactions.

Homology of the D-galactose-specific lectins from Artocarpus integrifolia and Maclura pomifera and the role of an unusual small polypeptide subunit.

The Maclura pomifera agglutinin (MPA) was purified by affinity chromatography from a seed extract and its properties were compared with those of the Artocarpus integrifolia lectin, jacalin. Reverse-phase high-performance liquid chromatography showed both proteins had multiple forms of a small approximately 20-residue polypeptide chain in addition to the major 12,000 Mr subunit. The amino acid sequences of the small chains and the N-terminal sequences of the large subunits showed considerable similarity between the two proteins, approximately 60% identical residues. The homology of the proteins was confirmed by the similarity of their circular dichroism and fluorescence emission spectra. MPA showed much greater spectral changes upon binding methyl alpha-D-galactoside, suggesting it has complete activity rather than the partial activity found for jacalin. The binding of methyl alpha-D-galactoside by MPA was measured by fluorescence titration; the KA was 1.9 X 10(4) M-1 compared to 3.4 X 10(4) M-1 for jacalin. MPA also precipitated human IgA1 in the same manner as jacalin. The spectra indicate the involvement of tryptophan and tyrosine residues in the binding site of these lectins. Since a tryptophan residue is conserved in all the small subunits, they may form part of the binding site.

Tyrosinate fluorescence maxima at 345 nm in proteins lacking tryptophan at pH 7.

Advantage is often taken of the different fluorescence properties of two aromatic amino acids, tyrosine and tryptophan, to study the conformations, environmental properties and interactions of proteins containing these amino acids [ 1,2] . In this work we report the novel fluorescence properties of two peptides, isolated from the Indian Cobra Nujc naja which do not contain any tryptophan yet whose fluorescence spectra include a maximum at 345 nm.

EXCITED STATE pKa BEHAVIOUR OF DAPI. A RATIONALIZATION OF THE FLUORESCENCE ENHANCEMENT OF DAPI IN DAPI‐NUCLEIC ACID COMPLEXES

Abstract— The steady state and time resolved fluorescence of the drug and chromosomal staining agent, 4′,6‐diamidino‐2‐phenylindole dihydrochloride, DAPI, was examined under different solvent conditions. In solutions between pH 3 and pH 9 the fluorescence spectral maximum of DAPI was found at 460 nm. The fluorescence decayed with double exponential kinetics, with decay times of 2.86 and 0.144 ns, at all wavelengths below 550 nm. At 550 nm single exponential decay kinetics with a lifetime of 0.153 ns was observed. The fluorescence spectrum could be resolved into two components, the 2.86 ns component having a spectral maximum near 450 nm and the 0.144 ns component having a spectral maximum near 490 nm. The results are rationalized in terms of there being two different configurations of DAPI, one of which undergoes a rapid protonation of the indole ring by proton transfer from the 6‐amidinium group in the excited singlet state. The 0.144 ns component is assigned as the fluorescence from the excited state of the protonated indole ring. The results provide an explanation of the fluorescence enhancement in DAPI‐nucleic acid complexes.

Fluorescent amino acid analogs

Publisher Summary This chapter describes the fluorescence properties of the fluorinated Trp analogs that are shown to be useful tools in fluorescence studies of proteins. The chapter reviews the use of analogs of tryptophan (Trp) as intrinsic probes of protein structure in protein bimolecular complexes. The use of fluorescence probes as reporters of structure, dynamics, interactions, and local microenvironments is now commonplace in studying biological systems. Among biopolymers, proteins are unique in displaying intrinsic fluorescence chromophores that provide valuable information about their structure, dynamics, and segmental molecular details. To study a particular Trp-containing protein in a complex with another Trp containing protein presents difficulties in resolving and assigning the fluorescence properties to the several Trp components. The biosynthetic approach does, however, have certain disadvantages: (1) 100% incorporation of the Trp analog at a particular site is not ensured, (2) the method is generally restricted to proteins containing only a few Trp residues because all Trps are replaced, and (3) the method is restricted to the few analogs that can successfully replace Trp in the cellular biosynthetic machinery.

Characterization of aminoacyl-adenylates in B. tsubtilis tryptophanyl-tRNA synthetase, by the fluorescence of tryptophan analogs 5-hydroxytryptophan and 7-azatryptophan

The tryptophan analogs 5-hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are capable of being biosynthetically incorporated into bacterial proteins and can be used as intrinsic fluorescence probes of protein structure, function and dynamics. A prerequisite for analog incorporation is their recognition by tryptophanyl-tRNA synthetase (TrpRS) and the formation of the analog aminoacyladenylate in the enzymes active site. The binding of 5HW and 7AW to B. subtilis TrpRS and the stability of the corresponding aminoacyladenylates of 5HW and 7AW were examined using their unique spectroscopic properties. The adenylate of 7AW in the active site of TrpRS exhibited intense fluorescence with a 10.5 ns fluorescence decay time. Enzyme-bound 7AW-adenylate was a long-lived intermediate with a half-life of over 9 hours. Enzyme-bound 5HW-adenylate fluorescence was quenched compared to that of 5HW in solution. The 5HW-adenylate/TrpRS complex was much less stable than that of 7AW, with a half-life of 33 minutes. Rapid hydrolysis of the 5HW-adenylate may explain the apparent proofreading observed which prohibits 5HW incorporation into proteins in the presence of tryptophan. Hydrolysis of the adenylates of both analogs restored the fluorescence parameters towards those of the analogs in solution. Neither 1-methyltryptophan nor 5-methoxytryptophan were capable of forming long-lived aminoacyladenylate intermediates in TrpRS. This study provides perspectives on the usefulness of 5HW and 7AW as intrinsic fluorescence probes of protein structure. The enhanced fluorescence of 7AW suggests its location in a buried hydrophobic environment in the protein. Exposure to water results in significant fluorescence quenching. These studies clearly demonstrate the utility of Trp analogs for the elucidation of molecular details of protein structure and dynamics.

Conformational flexibility of the hormonal peptide bombesin and its interaction with lipids

The conformational flexibility of the tetradecapeptide hormone bombesin has been studied using circular dichroism and fluorescence of its single tryptophan residue. The spectral changes observed indicate that the peptide changed from a random flexible coil in solution to a helical structure in lysolecithin micelles and dimyristoylphosphatidylserine vesicles. The tryptophan residue in the lipid complexes was located in a hydrophobic environment. The interaction with lipids was shown to involve both hydrophobic and electrostatic forces.

Relation between Ca2+ uptake and fluidity of brush-border membranes isolated from rabbit small intestine and incubated with fatty acids and methyl oleate

The rate of incorporation of oleic acid into isolated brush-border membranes was found to be considerably faster than methyl oleate incorporation under similar experimental conditions. The effects of fatty acids and methyl oleate incorporation on Ca2+ uptake and fluidity were monitored. Whereas treatment with 0.01-0.05 mM oleic acid corresponding to incorporations smaller than 90 nmol/mg protein enhanced Ca2+ transport, exposures to higher concentrations of this fatty acid corresponding to incorporations larger than 150 nmol/mg protein, decreased uptake of this cation. On the other hand, treatment with 0.01-0.2 mM methyl oleate corresponding to incorporations of up to 220 nmol/mg protein had only a stimulatory effect on the Ca2+ uptake. Oleic acid, linoleic acid and methyl oleate decreased the fluorescence anisotropy of membranes labelled with diphenylhexatriene in a dose-dependent manner. In contrast, palmitic acid had little or no effect on the diphenylhexatriene-reportable order of the membrane within the range of concentrations used. Monitored as a function of temperature, the anisotropy values showed a gradual melting for both the control and lipid-treated membranes. The results support the concept that saturated and cis-unsaturated fatty acids dissolve in different lipid domains and this in itself appears to be an important factor defining whether the biological function of the membrane is affected by the uptake. Incorporation of cis-unsaturated fatty acids in domains harboring the Ca2+ uptake process increases Ca2+ uptake in concert with increased diphenylhexatriene-monitored fluidity. However, when concentrations of such fatty acids in these domains become sufficiently great, the presence of a largely increased number of free carboxyl groups at the membrane surface causes inhibition of Ca2+ uptake.

A fluorescence decay time study of tryptophan in isolated hemoglobin subunits

The time‐resolved fluorescence behavior of tryptophan residues in isolated human hemoglobin subunits was determined using a sync‐pumped dye laser system and time‐correlated single photon counting detection. Two decay components having values near 80 ps and 2 ns were found in the fluorescence decay of the α‐subunit. The data for the β‐chains were best fitted with 3 decay components of 90 ps, 2.5 ns and 6.4 ns. We propose that the decay times correspond to conformations of the proteins in which the disposition of the tryptophan to the heme residue differs.

Fluorescence decay parameters of tryptophan in a homogeneous preparation of human hemoglobin

Abstract Nanosecond fluorescence lifetime components, observed in hemoglobin samples purified by standard procedures, are removed by a HPLC purification step. The tryptophyl fluorescence of extensively purified human hemoglobin is found to decay as a simple single exponential. The fluorescence lifetime of ≈25 ps is relatively insensitive to protein conformational changes such as those resulting from different ligation states of the heme iron.