Fluorescence studies of native and modified neurophysins. Effects of peptides and pH.

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by F?rster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.     

Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.

The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to a 120 mOsm bath-to-lumen osmotic gradient in the presence and absence of 250 microU/ml vasopressin. The magnitude of transepithelial volume flow was determined from the self-quenching of FS, or from the ratio of PTSA/FS fluorescence, measured at 380 nm excitation and 420 +/- 10 nm (PTSA) and greater than 530 nm (FS) emission wavelengths. Pf was calculated from the magnitude of transepithelial volume flow, lumen and bath osmolarities, lumen perfusion rate, and tubule geometry. The instrument response time for a change in bath osmolality was less than 3 s. At 37 degrees C, CCT Pf was (in units of cm/s x 10(4] 13 +/- 2 (mean +/- SE, 16 tubules) before, and 227 +/- 10 after addition of vasopressin to the bath. CCT Pf began to increase in 23 +/- 3 s after vasopressin addition and was half-maximal after 186 +/- 20 s. At 23 degrees C, Pf was 9 +/- 1 (seven tubules) before, and 189 +/- 12 after vasopressin addition. Pf began to increase in 40 +/- 4 s and was half-maximal after 195 +/- 35 s. After vasopressin removal from the bath, Pf decreased to its baseline value with a half-time of 14 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism and fluorescence analysis of the interaction of Pf1 gene 5 protein with poly(dT)

Circular dichroism (c.d.) and fluorescence spectroscopy have been used to investigate the interaction of the gene 5 protein of the filamentous bacteriophage Pf1 with single-stranded DNA. The c.d. spectrum of the Pf1 gene 5 protein is consistent with the absence of any significant alpha-helical content. The negative c.d. peak in the region of 210 nm, which arises from the protein, is diminished in the complex with poly(dT). Likewise, the c.d. peak at 265 nm arising from the poly(dT) decreases when the Pf1 gene 5 protein is bound, c.d. titrations of poly(dT) with Pf1 gene 5 protein indicate strong binding with a stoichiometry (n) of four nucleotides per protein subunit. In contrast, when the titrations were done using fluorescence anisotropy or fluorescence spectral shifts to follow binding, apparent stoichiometries between n = 2 and n = 4 were observed, often in the same experiment, depending on precise conditions. The results are interpreted in terms of two distinct modes of binding, in which either one or two subunits of the protein dimer are bound to the polynucleotide lattice, but still retaining the same local interaction with the DNA, with each binding site covering four nucleotides. The apparent stoichiometry of 2 results from the interaction of only one subunit of the dimer with the nucleic acid lattice, when protein is in excess. The second, unfilled, subunit of the dimer is nevertheless incorporated into the complex, resulting in the maximum possible fluorescence change when only half the sites are filled, since the fluorescence properties of the complex arise from protein-protein contacts associated with co-operative binding to the lattice. Further experiments in which the order of addition of components is changed, and the concentration of MgCl2 is varied, show that both of these factors are important in determining the dominant binding mode. In the absence of salt, dissociation and redistribution of the polynucleotide can occur following the addition of excess protein. This transition is suppressed in the presence of greater than 3 mM-MgCl2.     

UV-reactivation,virus production and mutagenesis of SV40 in UV-irradiated monkey kidney cells

The survival of UV-irradiated simian virus 40 (SV40) is higher in UV-irradiated than in non-irradiated monolayers of BSC-1 monkey cells. A similar reactivation is found when cells are infected with SV40-DNA, suggesting that reactivation acts on viral DNA. The enhanced reactivation of UV-irradiated SV40 and SV40-DNA is optimal when infection is delayed for 2–3 days after irradiation of the cells.UV-pretreated cells infected with SV40-DNA produce more virus than infected control cells; the time curve of this process is similar to that found for enhanced virus reactivation and suggests that facilitated virus production in UV-irradiated cells and enhanced virus reactivation might be manifestations of the same process.If the non-irradiated SV40 thermosensitive mutant BC245 is propagated in UV-irradiated BSC-1 cells the rate of back mutation to phenotypically wild-type is increased compared with that of the control. This suggests that an inducible error-prone system is functional in these cells. When the UV-irradiated tsBC245 is propagated in non-irradiated cells the reversion frequency is greatly enhanced, which suggests that either the introduction of UV-irradiated SV40-DNA is sufficient to induce an error-generating system, or that a constitutive error-prone mechanism is operative on this DNA.     

Ultraviolet-resonance raman spectroscopy of the filamentous virus Pf3: interactions of Trp 38 specific to the assembled virion subunit

The class II filamentous virus Pf3 packages a circular single-stranded DNA genome of approximately 5833 [corrected] nucleotides within a cylindrical capsid constructed from approximately 2500 [corrected] copies of a 44 residue alpha-helical subunit. The single tryptophan residue (Trp 38) of the capsid subunit is located within a basic C-terminal sequence (.R(+)WIK(+)AQFF). The local environment of Trp 38 in the native Pf3 assembly has been investigated using 229 nm excited ultraviolet-resonance Raman (UVRR) spectroscopy and fluorescence spectroscopy. Trp 38 exhibits an anomalous UVRR signature in Pf3, including structure-diagnostic Raman bands (763, 1228, 1370, and 1773 cm(-)(1)) that are greatly displaced from corresponding Raman markers observed in either detergent-disassembled Pf3, class I filamentous viruses, most globular proteins, or aqueous L-TRP. An unusual and highly quenched fluorescence spectrum is also observed for Trp 38. These distinctive UVRR and fluorescence signatures together reflect interactions of the Trp 38 side chain that are specific to the native PF3 assembly. The experimental results on PF3 and supporting spectroscopic data from other proteins of known three-dimensional structure favor a model in which pi electrons of the Trp 38 indolyl ring interact specifically with a basic side chain of the subunit C-terminal sequence. Residues Arg 37 AND Lys 40 are plausible candidates for the proposed cation-pi interaction of Trp 38. The present study suggests that raman spectroscopy may be a generally useful probe of interactions between the indolyl pi-electron system of tryptophan and electropositive groups in proteins and their assemblies.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

Histone Hl-DNA interaction. Influence of phosphorylation on the interaction of histone Hl with linear fragmented DNA.

By measuring the fluorescence polarization of fluorescent histone H1 derivatives complexed with DNA, binding of the histone to DNA was studied as a function of ionic strength in the solution prior to and after the H1 phosphorylation on Ser-37 residue. Fluorescent labels were covalently linked either specifically to Tyr-72 residues or unspecifically to lysine residues in the H1 polypeptide chain. The values of the corresponding rotational relaxation times showed that at low ionic strength all the segments of the H1 molecule were immobilized on binding to DNA. The gradual increasing NaC1 concentration in the solution of H1-DNA complex was accompanied at first by additional retardation of the histone mobility in the complex, and then by progressive release of histone H1 from from the complex which was completed at 0.5-0.6 M NaC1 irrespective of phosphorylation. tat the same time the phosphorylation of histone H1 led to removal of the central and, presumably, N-terminal regions of H1 from DNA.     

Antifungal activities of an endophytic <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> strain Pf1TZ harbouring genes from pyoluteorin and phenazine clusters

Pseudomonas fluorescens Pf1TZ was inhibitory in vitro to a number of phytopathogenic fungi and could protect vine plantlets against Botrytis cinerea. Total protection was reached after 3 weeks of bacterial inoculation. The endophytism of Pf1TZ was confirmed by confocal microscopy using its inherent fluorescence. The molecular characterization of Pf1TZ indicated the presence of genes from clusters encoding pyoluteorin and phenazine. The chromatographic purification and LC-MSⁿ analysis revealed that the most active one has a molecular mass of 504 Da.     

Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.     

Nature of the intermediate in the 3-oxo-delta 5-steroid isomerase reaction.

The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.     

Effects of Amino Acid Substitutions at the Active Site in Escherichia Coli β-Galactosidase

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme β-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac(-). Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac(+). The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.     

Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

A sand column adhesion assay was developed which distinguishes the adhesion abilities of a number of pseudomonads isolated from fine sandy loam. Pseudomonas fluorescens Pf0-1 which adhered at >90% of the total cells added was subjected to transposon Tn5 insertion mutagenesis. From 2,500 Pf0-1::Tn5 mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% attachment were isolated. Marker exchange analysis of one of these mutants, Pf0-5, confirmed that the decreased adhesion was linked to the Tn5 insertion in the chromosome. The growth rate of Pf0-5 in enriched media and sterile soil was similar to that of the wild type; in minimal medium, however, Pf0-5 grew faster. In a soil column assay, less Pf0-5 than wild-type bacteria were recovered, suggesting a decreased ability to persist in soil. A 34-kilodalton major outer membrane protein present in the wild type was missing in Pf0-5. Transmission electron microscopy of the cell surface revealed that the wild-type possessed polar flagella which were absent in the mutant.     

Chemical modification of human alpha 1-proteinase inhibitor by tetranitromethane. Structure-function relationship.

Nitration of tyrosine residues of alpha 1-proteinase inhibitor (alpha 1-PI) by tetranitromethane yielded a product that maintained its inhibitory activity against trypsin but lost most of its inhibitory activity against elastase. Chemical analysis of the product showed that four out of the six tyrosine residues in alpha 1-PI had been nitrated to various degrees: Tyr-38 and Tyr-297 were not nitrated, whereas Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were nitrated to extents in the range 40-80%. We interpreted these data to mean that modification of these tyrosine residues decreased the association constant between alpha 1-PI and the proteinases and that the decrease differs from one proteinase to the other. When either alpha 1-PI-trypsin or alpha 1-PI-elastase complex was nitrated, nitration took place only to a very slight extent at these latter four tyrosine residues. On the other hand, Tyr-38 and Tyr-297 underwent nitration to about 20%. We concluded that Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were located on the surface of alpha 1-PI that interacts with either trypsin or elastase in the formation of complexes, and were therefore protected from nitration.     

Two-photon induced fluorescence of Cy5-DNA in buffer solution and on silver island films

We report the observation of a strong two-photon induced fluorescence emission of Cy5-DNA within the tunable range of a Ti:Sapphire laser. The estimated two-photon cross-section for Cy5-DNA of 400GM is about 3.5-fold higher than it was reported for rhodamine B. The fundamental anisotropies of Cy5-DNA are close to the theoretical limits of 2/5 and 4/7 for one- and two-photon excitation, respectively. We also observed an enhanced two-photon induced fluorescence (TPIF) of Cy5-DNA deposited on silver island films (SIFs). In the presence of SIFs, the TPIF is about 100-fold brighter. The brightness increase of Cy5-DNA TPIF near SIFs is mostly due to enhanced local field.     

Dehydration of model membranes induced by lectins from Ricinus communis and Viscum album.

The effects of ribosome-inactivating proteins (RIPs) from Ricinus communis and from Viscum album on the water permeability, Pf, and the surface dielectric constant, epsilon, of model membranes were studied. Pf was calculated from microelectrode measurements of the ion concentration distribution in the immediate vicinity of a planar membrane, and epsilon was obtained from the fluorescence of dansyl phosphatidylethanolamine incorporated into unilamellar vesicles. Pf and epsilon of fully saturated phosphatidylcholine membranes were affected only in the presence of a lectin receptor (monosialoganglioside, GM1) in the bilayer. It is suggested that the membrane area occupied by clustered lectin-receptor complexes is markedly less permeable to water. Protein binding to the receptor was not a prelude for hydrophobic lipid-protein interactions when the membranes were formed from a mixture of natural phospholipids with a high content of unsaturated fatty acids. These membranes, characterized by a high initial water permeability, were found to interact with the RIPs unspecifically. From a decrease of both Pf and epsilon it was concluded that not only water partitioning but also protein adsorption correlates with looser packing of polyunsaturated lipids at the lipid-water interface.     

Molecular dynamics of calmodulin as monitored by fluorescence anisotropy

Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca²⁺. This is diminished in the presence of Ca²⁺.²