Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues

The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain.     

Fluorescence properties of glutamine-binding protein from Escherichia coli and its complex with glutamine

In this work, the fluorescence of glutamine-binding protein (GlnBP) and its complex with glutamine (GlnBP/Gln) in native and unfolded forms was studied. The experimental data were interpreted on the basis of the results of the analysis of Trp and Tyr microenvironments taking into the account the data for GlnBP mutated forms Trp32Phe(Tyr) and Trp220Phe(Tyr), which have been obtained by Axelsen et al. (Biophys. J. 1991, 60, 650-659). This allowed us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of the fluorescence characteristics of GlnBP and GlnBP/Gln, and the uncommon effect of the excess of the fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm respect to the excitation at 280 nm. The last effect is explained by the spectral dependence of the Trp 32 and Trp 220 contributions to the protein absorption. The protein Trp fluorescence dependence on the excitation wavelength must be taken into account for the evaluation of the Tyr residues contribution to the bulk fluorescence of protein, and in principle, it also may be used for the development of an approach for the decomposition of a multicomponent protein fluorescence spectrum.     

The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

Site-directed mutagenesis of conserved C-terminal tyrosine and tryptophan residues of PsbO, the photosystem II manganese-stabilizing protein, alters its activity and fluorescence properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

UV spectroscopic studies of human erythrocyte superoxide dismutase

Using UV absorption spectroscopy, first derivative spectroscopy, and UV difference spectroscopy, the active site of human superoxide dismutase is probed. First derivative spectra (dA/d lambda versus lambda) show the HESOD spectrum to be a composite of Phe and Trp absorbance. The 278 and 288 nm Trp absorbance peaks are sensitive to solvent polarity. A 5-10% decrease in these peaks accompanies copper removal from the active site indicating greater solvent access to Trp in the apoenzyme than the holoenzyme. A Trp UV difference peak at 305-310 nm documents the presence or absence of copper at the active site, and documents also the movement of a nonbridging copper-binding His (His 46 or 120) when HESOD is inhibited by azide or when the copper moiety is reduced. Trp absorbances indicate that neither cyanide nor KCl inhibition affects the Cu(II)-His bonds. Phe UV absorbance is increased by the presence of copper at the active site and increased further by the addition of cyanide or azide. Neither Trp nor Phe responds to the presence of zinc in the active site. A molecular graphics program, FRODO, shows Trp and the four Phe residues lying in an approximate ring around the active site of HESOD and thus excellently placed to report on active site perturbations.     

Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements.

The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit.     

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.     

白茯苓凝集素的荧光光谱研究

白茯苓凝集素（ＳＬＬ）分子中含有４个色氨酸（Ｔｒｐ）残基,ＮＢＳ修饰测得这４个Ｔｒｐ残基位于分子表面。ＳＬＬ在天然状态下荧光发射峰位于３３５ｎｍ处,离子强度和温度对其荧光光谱均无明显的影响。ＮＢＳ修饰后的ＳＬＬ失去凝血活性,相应荧光光谱的强度减弱,荧光发射峰发生蓝移,提示ＳＬＬ的构象发生改变。用ＫＩ·ＣｓＣｌ和丙烯酰胺淬灭剂研究ＳＬＬ分子中Ｔｒｐ残基的微环境,发现丙烯酰胺和ＣｓＣｌ能淬灭分子中１００％和５０％的Ｔｒｐ残基的荧光,而ＫＩ完全不能淬灭ＳＬＬ分子中Ｔｒｐ残基的荧光,因此Ｔｒｐ残基周围存在阴离子区,或者Ｔｒｐ残基处于分子表面的疏水环境中。     

The aromatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine and bovine homologs

The isolated kringle 4 domain of human plasminogen has been compared with homologous structures from bovine and porcine sources, both free and in the presence of the ligand 6-aminohexanoic acid, by two-dimensional 1H-NMR spectroscopies at 300 MHz and 600 MHz. The chemical-shift-correlated, spin-echo-correlated, and double-quantum-correlated aromatic spectra of the three proteins reveal that the globular conformation of the fourth kringle is closely maintained throughout the set of homologs. Direct comparison shows that the three conserved Trp residues (at sites 25, 62 and 72) which exhibit highly non-degenerate subspectra, find themselves in similar intramolecular environments. In particular, proton Overhauser experiments reveal that the close steric interaction between the Trp-II (Trp62 or Trp25) indole group and the aromatic ring at site 74 (Tyr74 or Phe74) is strictly preserved. This feature forces the kringle inner loop, closed by the Cys51-Cys75 link, to fold back onto itself so as to place the site 74 residue proximal to the Cys22-Cys63 bridge. Single-residue substitutions enable unambiguous assignments of His-I to His3, Tyr-III to Tyr41 and Tyr-IV to Tyr74. From this direct evidence, comparison with the kringle 1 spectrum, and the previously reported chemical modification of Tyr-II (Tyr50) [Trexler M., Bányai L., Patthy L., Pluck N. D. & Williams R. J. P. (1985) Eur. J. Biochem. 152, 439-446], Tyr-I and Tyr-V (the latter, an immobile ring on the 600-MHz time scale) could be assigned to Tyr2 and Tyr9, respectively. Since Trp-III has previously been assigned to Trp72 at the lysine-binding site, the present study completes the assignment of 10 out of 12 aromatic spin systems in the kringle 4 1H-NMR spectrum; the only ambiguity which remains concerns the Trp-I and Trp-II indole spin systems, which are totally identified but as yet only tentatively assigned to Trp25 and Trp62, respectively.     

Conformational characteristics of luliberin. Luminescence properties at liquid-nitrogen temperature.

The luminescence properties (fluorescence and phosphorescence) of luliberin have been investigated at liquid-nitrogen temperature (77 K) in 50% ethylene glycolaqueous buffer at various pH's. Calculation of the energy-transfer efficiency between Trp and Tyr residues leads to an evaluation of the distance separating these residues. In alkaline medium, when tyrosine is ionized, a 100% transfer efficiency occurs from Trp to Tyr- at the singlet level and also from Tyr- to Trp at the triplet level indicating that the TRP-Tyr distance is less than about 5A. When luliberin is at pH 7.8 (or 4.5), transfer efficiency from Tyr to Trp at the singlet level is 85-90% which should correspond to a distance between Trp and Tyr of about 10-12 A. These results are discussed with respect to the role played by aromatic amino acids both in the hormone conformation and in the hormone biological potency. Moreover, comparison with the data obtained from fluorescence or circular dichroism studies at room temperature allows us to deduce some characteristics of luliberin conformation. Structure-activity relationships in the aromatic region of luliberin are also discussed.     

Exposure of aromatic residues of Streptomyces subtilisin inhibitor. A photo-CIDNP study

Exposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 8, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55 degrees C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.     

N5-(L-1-carboxyethyl)-L-ornithine synthase: physical and spectral characterization of the enzyme and its unusual low pKa fluorescent tyrosine residues.

N5-(L-1-carboxyethyl)-L-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr approximately equal to 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the alpha,beta family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of approximately 8.5, 11 have pKa approximately 10.75, and the remaining six titrate with pKa approximately 11.3. The two residues with pKa approximately 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the beta alphabeta fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.     

Lipid binding of the exchangeable apolipoprotein apolipophorin III induces major changes in fluorescence properties of tryptophans 115 and 130

The effect of lipid association on the local environment of the two tryptophan residues of Locusta migratoria apolipophorin III (apoLp-III) has been studied. In the lipid-free state, Trp115 in helix 4 is buried in the hydrophobic interior of the helix bundle, while Trp130 is located in a loop connecting helices 4 and 5. Fluorescence spectroscopy of single Trp mutants revealed an emission maximum (lambda(max)) of 321 nm for apoLp-III-W@115 (excitation 280 nm) which red-shifted to 327 nm upon binding to dimyristoylphosphatidylcholine (DMPC). ApoLp-III-W@130 displayed a lambda(max) of 338 nm while interaction with DMPC resulted in a blue shift to 331 nm. Quenching studies with KI and acrylamide revealed decreased accessibility to Trp115 compared to Trp130, while lipid binding induced a decrease in quenching of Trp130. Aromatic circular dichroism (CD) spectra showed that Trp vibronic transitions at 278, 286, and 294 nm for lipid-free apoLp-III were caused by Trp115. Upon lipid association, aromatic extrema are reversed in sign, becoming entirely negative with both Trp residues contributing to the vibronic transitions, implying restriction in side-chain mobility of these residues. Thus, lambda(max), quencher accessibility, and aromatic CD analysis indicate that Trp115 is much less solvent-exposed than Trp130. Differences in fluorescence properties of these residues are minimized in the lipid-bound state, a result of relocation of Trp115 and Trp130 into the lipid milieu. Thus, in addition to the hydrophobic faces of apoLp-III amphipathic alpha-helices, the loop region containing Trp130 comes in close contact with DMPC.     

The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.     

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman,EPR and CD study

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.