Dynamics of the aromatic amino acid residues in the globular conformation of the basic pancreatic trypsin inhibitor (BPTI). I. 1H NMR studies.

The basic pancreatic trypsin inhibitor (BPTI) was investigated by high resolution 1H NMR techniques at 360 MHz. Observation of the amide proton resonances of the polypeptide backbone showed that the globular conformation of BPTI determined by X-ray studies in single crystals is maintained in aqueous solution over the temperature range from 4 degrees to 87 degrees. NMR studies over this temperature range of the aromatic amino acid residues of BPTI. i.e. 4 tyrosines and 4 phenylalanines, led to complete assignments of all the aromatic spin systems in the protein. From this, information was obtained on the rotational motions about the C beta--Cv bond axis of the aromatic rings in the globular form of PBTI. At 25 degrees, two tyrosine rings and one phenylalanine ring are rotating rapidly on the NMR time scale. For the other rings the transitions from slow to rapid rotational motions were investigated at variable temperatures and energy barriers for these intramolecular rate processes determined. The studies of the tyrosine resonances had been described in detail in a previous publication. The present paper describes the identification of the phenylalanine resonances and comments on some technical aspects which might be of quite general interest for the analysis of highly resolved 1H NMR spectra of proteins. Data for the tyrosines and the phenylalanines are compiled in three tables, i.e. the pK alpha-values for the tyrosines, the NMR parameters for all eight aromatics, and the parameters delta G not equal to, and, where available, delta H not equal to and delta S not equal to for the rotational motions of the rings.     

Labeling proteins via hole burning of their aromatic amino acids: pressure tuning spectroscopy of BPTI

We demonstrate hole burning on a protein by using an intrinsic aromatic amino acid as a probe. The protein is bovine pancreatic trypsin inhibitor (BPTI), the labeled amino acid is tyrosine. Only one of the four tyrosines could be burned. As an application we present pressure tuning experiments from which the local compressibility around the burned tyrosine probe is determined.     

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens.

A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.     

Second derivative fluorescence spectroscopy of tryptophan in proteins

The second derivatives of N-acetyl- -tryptophan amide (AcTrpNH₂) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH₂ fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase–PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.     

Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb³⁺ as a fluorescence probe, that EoCen has four calcium-binding sites. The sensitized emission arises from nonradiative energy transfer between the three tyrosine residues (Tyr46, Tyr72, and Tyr79) of the N-terminal half and the bound Tb³⁺ ions. To determine the most critical of the three tyrosine residues for the process of fluorescence resonance energy transfer, six mutants of the N-terminal domain of EoCen, which contain one (N-Tyr46/N-Tyr72/N-Tyr79) or two (N-Y46F/N-Y72F/N-Y79F) tyrosine residues, were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb³⁺ fluorescence of N-Y79F was most affected, displaying a 50% reduction compared with wild-type N-EoCen. Among the tyrosines, Tyr79 is the shortest mean distance from the protein-bound Tb³⁺ (at sites I/II), as calculated via the Förster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr79, which exists in a hydrophobic environment, has the highest quantum yield and a relatively long average lifetime. The decay of Tyr79 is the least heterogeneous among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr79 and the oxygen from the side chains of the residue Asn39. Kinetic experiments on tyrosine and Tb³⁺ fluorescence demonstrate that tyrosine fluorescence quenching is largely due to the self-assembly of EoCen, and that the quenching degrees of the mutants differ. Resonance light scattering and crosslinking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr79 also plays the most important role in the Tb³⁺-dependent self-assembly of EoCen among the three tyrosines.     

Analysis of internal motion of single tryptophan in Streptomyces subtilisin inhibitor from its picosecond time-resolved fluorescence.

A mode of internal motion of single tryptophan, Trp 86, of Streptomyces subtilisin inhibitor, was analyzed from its time-resolved fluorescence. The intensity and anisotropy decays of Trp 86 were measured in the picosecond range. These decays were analyzed with theoretical expressions derived assuming that the indole ring of tryptophan as an asymmetric rotor rotates around covalent bonds connecting indole with the peptide chain and an effective quencher of fluorescence of Trp 86 is the nearby SS bond of Cys 35-Cys 50. First, the intensity decays at 6 degrees, 20 degrees, and 40 degrees C were analyzed, and then the both decays of the intensity and anisotropy at 20 degrees C were simultaneously simulated with common parameters. Constants concerning geometrical structures of the protein used for the analysis were obtained from x-ray crystallographic data. Best fit between the observed and calculated decay curves was obtained by a nonlinear least squares method by adjusting a quenching constant averaged over the rotational angles, koq height of the potential energy, p, and three of six diffusion coefficients, Dxx, Dyy, Dzz, Dxy, Dyz, and Dzx, as variable parameters. The obtained results revealed that the internal motion of the indole ring became faster, the quenching rate of the fluorescence of Trp 86 was enhanced and the height of potential energy became lower at higher temperatures, and suggested that Trp 86 was wobbling around the long axis of the indole ring in the protein.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5-13C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degrees C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in slow exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters: delta H++ = 28 kcal/mol for both I and III; delta S++ = 42 cal/(mol.K) for I, and delta S++ = 41 cal/(mol.K) for III. The remaining tyrosine (V) has a larger enthalpy and entropy of activation: delta H++ - 36 kcal/mol, delta S++ = 72 cal/(mol.K). Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.     

Molecular mobility of a fluorescent probe in binding sites of an albumin molecule

The molecular mobility of the fluorescent probe, N-(carboxymethyl)imide of 4-(dimethylamino)naphthalic acid (K-35), in three types of binding site on a human serum albumin (HSA) molecule has been studied. Study of the time-resolved decay of K-35 polarized fluorescence in HSA has shown that probe molecules bound to different sites have different fluorescence decay times, which poses problems in interpreting the polarization curves. However, it has been found that, in the case of rather slow thermal rotation of the probe, the decay of the vertical and the horizontal components of polarized fluorescence can each be approximated with three exponentials corresponding to three types of binding site. The mobility of the probe in different sites was estimated. The mobility was different but in all cases hindered by tens of times relative to the rotation of K-35 in water. The slowest motion occurred in the sites of the first type localized in the region of the well known drug site I: there the rotational correlation time was at least 72 ns. In the sites of the second type, this time was about 40 ns, and in the sites of the third type, about 10 ns. The faster was the rotation, the higher was the fluorescence quenching rate. Probably, it is this motion that is responsible for different fluorescence decay times in different HSA sites.     

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.     

A spectroscopic and conformational study of pertussis toxin

The conformation of native pertussis toxin has been investigated by secondary structure prediction and by circular dichroism, fluorescence and second-derivative ultraviolet absorption spectroscopy. The far-ultraviolet circular dichroic spectrum is characteristic of a protein of high beta-sheet and low alpha-helix content. This is also shown by an analysis of the circular dichroic spectrum with the Contin programme which indicates that the toxin possesses 53% beta-sheet, 10% alpha-helix and 37% beta-turn/loop secondary structure. Second-derivative ultraviolet absorption spectroscopy suggests that 34 tyrosine residues are solvent-exposed and quenching of tryptophan fluorescence emission has shown that 4 tryptophan residues are accessible to iodide ions. One of these tryptophans appears to be in close proximity to a positively charged side-chain, since only 3 tryptophans are accessible to caesium ion fluorescence quenching. When excited at 280 nm, the emission spectrum contains a significant contribution from tyrosine fluorescence, which may be a consequence of the high proportion (55%) of surface-exposed tyrosines. No changes in the circular dichroic spectra of the toxin were found in the presence of the substrate NAD. However, NAD did quench both tyrosine and tryptophan fluorescence emission but did not change the shape of the emission spectrum, or the accessibility of the tryptophans to either the ionic fluorescence quenchers or the neutral quencher acrylamide.     

Solution structural studies of the Saccharomyces cerevisiae TATA binding protein (TBP)

The intrinsic fluorescence of the six tyrosines located within the C-terminal domain of the Saccharomyces cerevisiae TATA binding protein (TBP) and the single tryptophan located in the N-terminal domain has been used to separately probe the structural changes associated with each domain upon DNA binding or oligomerization of the protein. The unusually short-wavelength maximum of TBP fluorescence is shown to reflect the unusually high quantum yield of the tyrosine residues in TBP and not to result from unusual tryptophan fluorescence. The anisotropy of the C-terminal tyrosines is very high in monomeric, octameric, and DNA-complexed TBP and comparable to that observed in much larger proteins. The tyrosines have low accessibility to an external fluorescence quencher. The anisotropy of the single tryptophan located within the N-terminal domain of TBP is much lower than that of the tyrosines and is accessible to an external fluorescence quencher. Tyrosine, but not tryptophan, fluorescence is quenched upon TBP-DNA complex formation. Only the tryptophan fluorescence is shifted to longer wavelengths in the protein-DNA complex. In addition, the accessibility of the tryptophan residue to the external quencher and the internal motion of the tryptophan residue increase upon DNA binding by TBP. These results show the following: (i) The structure of the C-terminal domain structure is unchanged upon TBP oligomerization, in contrast to the N-terminal domain [Daugherty, M. A., Brenowitz, M., and Fried, M. G. (2000) Biochemistry 39, 4869-4880]. (ii) The environment of the tyrosine residues within the C-terminal domain of TBP is structurally rigid and unaffected by oligomerization or DNA binding. (iii) The C-terminal domain of TBP is uniformly in close proximity to bound DNA. (iv) While the N-terminal domain unfolds upon DNA binding by TBP, its increased correlation time shows that the overall structure of the protein is more rigid when complexed to DNA. A model that reconciles these results is proposed.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Time-Resolved fluorescence studies on the internal motion of chlorophyll a of light-harvesting chlorophyll a/b-protein complex in lipid membranes

By analyzing the steady state and time-resolved fluorescence anisotropy, the internal motions of chlorophyll a of light-harvesting chlorophyll a/b-protein complex (LHCII) were characterized in a dimyristoylphosphatidylcholine (DMPC) liposome. Corresponding to the thermotropic phase of the membrane, chlorophyll a showed an unique internal motion in LHCII. At the gel phase, two motional components, one fast and the other slow, were observed, which would originate in the heterogeneity of the mutual orientation and the binding site of the chlorophyll a in LHCII. Interestingly, the faster motion was suppressed and only the slower segmental rotation with the larger motional amplitude was allowed on the phase transition to a liquid crystalline phase.     

Motion of aromatic side chains, picosecond fluorescence, and internal energy transfer in Escherichia coli thioredoxin studied by site-directed mutagenesis, time-resolved fluorescence spectroscopy, and molecular dynamics simulations

We have determined the picosecond fluorescence of the four aromatic amino acid residues (W28, W31, Y49, and Y70) in wild-type Escherichia coli thioredoxin (wt Trx) and a mutant Trx with W31 replaced by phenylalanine, Trx-W28-W31F. The internal motions of the four aromatic side chains were also analyzed. We examined the possibility of using internal energy transfer from tyrosine to tryptophan as a measure of long-range distances. The major features of the lifetime distribution of tryptophan fluorescence were unchanged in the W31F mutation, indicating that the environment of W28 is similar in both wt Trx and Trx-W28-W31F. However, the mutation of W31F changed the mobility of W28, situated close to the active-site disulfide/dithiol, but not the mobility of two tyrosines, Y49 and Y70, situated on the other side of the molecule. The mobility of the two tyrosine residues increased upon reduction of the active-site disulfide, indicating a looser structure with reduction. This increased motion could also be seen from molecular dynamics simulations. The change in energy transfer rates, as judged by tyrosine fluorescence lifetimes, was in agreement with energy transfer rates calculated from the molecular dynamics simulations. The anisotropy of tryptophan and tyrosine fluorescence could be separated in three parts: (I) overall rotation of the protein (10(-9)s), (II) internal mobility of side chains (10(-10)s), and (III) a very fast relaxation (10(-12)s). We can only experimentally detect this very fast relaxation when the internal motion is not present.     

Reinvestigation of the aromatic side-chains in the basic pancreatic trypsin inhibitor by heteronuclear two-dimensional nuclear magnetic resonance

The 1H nuclear magnetic resonance (n.m.r.) assignments for the aromatic spin systems of the four tyrosines and four phenylalanines in the basic pancreatic trypsin inhibitor (BPTI) were reinvestigated using novel 13C-1H heteronuclear two-dimensional experiments. Resonance lines which are degenerate in homonuclear 1H n.m.r. spectra could thus be resolved. Based on this new evidence the previous assignments for Phe22 and Phe33 had to be corrected. This affects the earlier conclusions on aromatic ring flips in BPTI in that Phe22 is rotating rapidly on the n.m.r. time scale at 36 degrees C, rather than being immobilized up to 80 degrees C.