Circular dichroism and difference spectra of members of the troponin-tropomyosin system in cetyltrimethylammonium bromide.

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The delta epsilonM values at their difference maxima were found to be 1300 at 292 nm for Ac-Trp-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studies by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.     

Difference spectroscopic study of the interaction between soybean beta-amylase and substrate or substrate analogues

1. In order to investigate the interactions between soybean beta-amylase [EC 3.2.1.2] and ligands (maltotriose as substrate, and maltose and alpha- and beta-cyclodextrins as inhibitors for the hydrolysis of maltoheptaose), the difference spectra were measured at 25 degrees C and pH 5.4, in 0.05 M acetate buffer. Each difference spectrum produced by these ligands showed a clear peak at 292-293 nm due to a tryptophan residue. In addition to this peak, the spectra of alpha- and beta-cyclodextrins showed a specific peak at 298-299 nm, and that of maltotriose showed a shoulder at 298 nm. 2. From the concentration dependency of the difference molar extinction delta epsilon, at 292-293 nm or at 298-299 nm, the dissociation constant of the enzyme-ligand complex, Kd, was evaluated for maltotriose, and alpha- and beta-cyclodextrins. For each ligand, the Kd values obtained at these two wavelengths were in good agreement with Michaelis constant, Km, or the inhibitor constant, Ki. The Kd value for maltose obtained from the titration of delta epsilon at 292 nm was also in good agreement with Ki. 3. Maltose produced a hydrophobic change in the environment of the tryptophan residue, while the interactions of maltotriose, and alpha- and beta-cyclodextrins with this enzyme caused an electrostatic change in the vicinity of the tryptophan residue in addition to the hydrophobic change. Since the signal at 298-299 nm was not found in the difference spectrum of maltose, this signal may be due to a tryptophan residue different from that which produces the signal at 292-293 nm. If both the signals are due to the same tryptophan residue, we must conclude that some conformational change is caused in the enzyme active site by the ligand binding.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Diffuse reflectance spectroscopy of fibrous proteins

UV-visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700-400?nm) than the other proteins and only those above 450?nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450-400?nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV-visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)(∞). The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290?nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400?nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid L: -cystine in the DR spectrum at 290?nm is significantly red shifted by ~40?nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20?nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV-visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.     

Tryptophan contributions to the unusual circular dichroism of fd bacteriophage.

The circular dichroism (CD) spectrum of fd bacteriophage has a deep minimum at 222 nm characteristic of highly alpha-helical protein, but there is a shoulder at 208 nm rather than a minimum, with a 222/208-nm amplitude ratio near 1.5 rather than near 1. Oxidation of fd phage with the tryptophan reagent N-bromosuccinimide (NBS) changes the ratio. In this report, the NBS titration of fd is followed by CD and three other spectroscopies, the results of which yield an explanation of the unusual CD spectrum. Absorbance, fluorescence, and Raman data show the oxidation to have two phases, the first of which involves the destruction of tryptophan and the second, tryptophan and tyrosine. Raman spectra reveal the invariance of an environmentally-sensitive tyrosine Fermi resonance doublet during the first oxidative phase. Raman spectra also show that little or no change of alpha-helicity occurs in the first or second oxidation phase, although very slight changes in the helix parameters might be occurring. Concurrent with the destruction of tryptophan during the first phase is the appearance in CD difference spectra ([theta]NBS-treated fd - [theta]native fd) of positive maxima at 208-210 nm and negative maxima at 224 nm, with crossovers at 217 nm. Enormous difference ellipticities, per oxidized subunit of 50 amino acids, of +490,000 +/- 80,000 deg cm2 dmol-1 at 208 nm and -520,000 +/- 110,000 deg cm2 dmol-1 at 224 nm have been derived from the data.(ABSTRACT TRUNCATED AT 250 WORDS)     

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.     

Evidence that cysteine 298 is in the active site of tryptophan indole-lyase

Escherichia coli tryptophan indole-lyase (tryptophanase) mutants, with cysteine residues 294 and 298 selectively replaced by serines, have been prepared by site-directed mutagenesis. Both mutant enzymes are highly active for beta-elimination reactions measured with both L-tryptophan and S-(o-nitrophenyl)-L-cysteine. The Cys-294----Ser mutant enzyme is virtually identical to the wild type with respect to pyridoxal phosphate binding (KCO = 2 microM), cofactor absorption spectrum (lambda max = 420 and 337 nm) and pH dependence (pK alpha = 7.3), pH profile for catalysis, and rate of bromopyruvic acid inactivation. In contrast, the Cys-298----Ser mutant enzyme exhibits a reduced affinity for pyridoxal phosphate (KCO = 6 microM), a shift in the cofactor absorption spectrum to 414 nm and an altered pK alpha = 8.5, an alkaline shift in the pH profile for catalysis, and resistance to inactivation of the apoenzyme by bromopyruvic acid. The C298S mutant enzyme (wherein cysteine 298 is altered to serine) also undergoes an isomerization to an unreactive state upon storage at 4 degrees C. These results demonstrate that the sulfhydryl groups of Cys-294 and Cys-298 are catalytically nonessential. However, these data suggest that Cys-298 is located within or very near the active site of the enzyme and is the reactive cysteine residue previously observed by others.     

Ricin D-saccharide interaction as studied by ultraviolet difference spectroscopy

The interaction of ricin D with specific saccharides was investigated by ultraviolet difference spectroscopy. Upon binding to saccharides, ricin D displayed ultraviolet difference spectra with maxima at 280 nm and 288 nm. Such difference spectra suggest that the environment of a tyrosine residue(s) located at or near the saccharide-binding site is changed by the binding of saccharide. In addition to the two positive peaks, a small trough was observed around 300 nm in the complexes with galactose-containing saccharides but not in the complex with N-acetylgalactosamine or galactosamine, suggesting the participation of tryptophan in the binding with galactose-containing saccharides. The magnitude of the difference maxima increased with increasing concentration of saccharides until the binding site was saturated. From the variation of the maximum at 288 nm as a function of saccharide concentration, the association constants were obtained for the binding of saccharides to ricin D at various temperatures and pH's. The saccharide binding of ricin D decreased with increasing temperature and with decreasing pH below pH 6.0. It was suggested that difference maximum at 288 nm observed in the ricin D-saccharide interaction reflects the binding of saccharides to the high-affinity saccharide-binding site of ricin D.     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

The ultraviolet fluorescence spectra of DPN-linked isocitrate dehydrogenase from bovine heart.

The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.     

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism.

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Individual tyrosine side-chain contributions to circular dichroism of ribonuclease

Experimental and theoretical studies using site-directed mutants of ribonuclease A (RNase A) offer more extensive information on the tyrosine side-chain contributions to the circular dichroism (CD) of the enzyme. Bovine pancreatic RNase A has three exposed tyrosine residues (Tyr73, Tyr76, and Tyr115) and three buried tyrosine residues (Tyr25, Tyr92 and Tyr97). The difference CD spectra between the wild type and the mutants at pH 7.0 (Deltaepsilon(277,wt) - Deltaepsilon(277,mut)) show bands with more negative DeltaDeltaepsilon(277) values for Y73F and Y115F than those for Y25F and Y92F and bands with positive DeltaDeltaepsilon(277) values for Y76F and Y97F. The theoretical calculations are in good semiquantitative agreement for all the mutants. The pH difference spectrum (pH 11.3-7.0) for the wild type shows a negative band at 295 nm and an enhanced positive band at 245 nm. The three mutants at buried tyrosine sites and one mutant at an exposed tyrosine site (Y76F) exhibit pH-difference spectra that are similar to that of the wild type. In contrast, two mutants at exposed tyrosine sites (Y73F and Y115F) exhibit diminished 295-nm negative bands and, instead of positive bands at 245 nm, negative bands are observed. Our results indicate that Tyr73 and Tyr115, two of the exposed tyrosine residues, are the largest contributors to the 277- and 245-nm CD bands of RNaseA, but the buried tyrosine residues and the one remaining exposed residue also contribute to these bands. Disulfide contributions to the 277- and 240-nm bands and the peptide contribution to the 240-nm band are confirmed theoretically.     

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

Fluorescence and circular-dichroism properties of pig intestinal calcium-binding protein (Mr=9000), a protein with a single tyrosine residue.

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.     

Conformational changes in pennisetin using intrinsic fluorescence spectroscopy

Fluorescence spectra of native pennisetin resulted in a single emission peak at 335 nm at excitation wavelength of 274 and 295 nm with quantum yield values for tyrosine and tryptophan as 0.086 and 0.097, respectively. These results indicate the presence of tryptophan residues in a polar environment and quenching of tyrosine residues in the native state of pennisetin. In the presence of an increasing concentration of guanidine hydrochloride (Gdn · HCl), changes such as red shift in emission peak from 335 to 344 nm, decrease in relative fluorescence intensity and increase in quantum yield value were observed, suggesting unfolding of the pennisetin molecule during denaturation. The quenching of tryptophanyl fluorescence by acrylamide and iodide further showed the presence of a single kind of tryptophanyl residue and its polar environment in pennisetin molecule.     

Ligand-induced isomerizations of Escherichia coli ornithine transcarbamoylase. An ultraviolet difference analysis

Ligand-induced ultraviolet difference spectra have been determined for Escherichia coli ornithine transcarbamoylase. The most prominent feature of the spectra is an absorbance difference which resembles a single period of a sine wave spanning the 245-320 nm region with a maximum at approximately 270 nm and a minimum at around 295-300 nm. This broad absorbance difference is typical of a blue-shift 1La band of tryptophan. Superimposed on the broad band in the 275-310 nm region is a series of smaller, narrow peaks resulted from red-shifted 1Lb bands of tryptophan and tyrosine residues. At pH 8.5, only carbamoyl phosphate and its analog phosphonacetamide yield a large ultraviolet difference absorbance (approximately 1800 M-1 cm-1) when bound to the enzyme. The spectra obtained are essentially the same in lineshape to and 80% in intensity of that produced by the bisubstrate analogy, N-(phosphonacetyl)-L-ornithine. In contrast, inorganic phosphate, a product of the reaction, induces small protein absorbance changes (approximately 300 M-1 cm-1) mainly in the 275-310 nm range. When complexed to the free enzyme, L-ornithine yields a marginally discernible ultraviolet difference spectrum in the 275-310 nm region, and its analogs L-norvaline and L-citrulline provide no absorbance change. However, inorganic phosphate in combination with any of the L-amino acids produces a difference spectrum similar to that given by carbamoyl phosphate alone. Collectively, these spectra suggest that carbamoyl phosphate elicits an isomerization required for the formation of the ternary complex and are consistent with the compulsory ordered mechanism of the enzyme at pH 8.5 with carbamoyl phosphate being the first substrate bound. Below pH 8, there is a kinetically discernible amount of random binding, but ordered addition is still the preferred pathway (Wargnies B., Legrain, C., and Stalon, V. (1978) Eur J. Biochem. 89, 203-212). Reflecting this change, the difference absorbance of the enzyme bound with carbamoyl phosphate is also pH dependent. The 1La band in the carbamoyl phosphate difference spectrum diminishes by approximately 20% at low pH. The PALO-induced changes, however, are pH invariant suggesting that full extent of the induced-fit isomerization is always reached in the ternary complex.     

Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.     

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.     

Proximity relations between tyrosine and tryptophan residues in fd phage determined by luminescence measurements

Resonance energy transfer from tyrosine to tryptophan residues was detected in the phage fd. The magnitude of the transfer efficiency was estimated by both a traditional and an alternative method. The latter involved comparison of the indole acceptor excitation spectrum full-width half-maximum with a set of standard values differing in the amount of absorbance contributed by tyrosine donor. Both methods lead to the same conclusion: essentially all the tyrosine residues of the viral coat are within 0.9 nm of a tryptophan residue. Also, fluorescence lifetime measurements provide additional support for the hypothesis that there are at least two different environments for the coat protein's sole indole side-chain. Little if any DNA phosphorescence was seen, consistent with the nucleic acid bases being stacked in the DNA core.