Accessibility of tryptophan residues in Na,K-ATPase

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.     

Fluorescence properties and conformation of human milk bile salt-activated lipase

The fluorescence properties of human milk bile salt-activated lipase (BAL) in aqueous solution at various pH and in the presence of denaturing reagents and bile salts have been studied by measuring the accessibility of tryptophan side chains to the iodide ion. The fluoresence quenching studies of BAL demonstrated that the BAL conformation was pH sensitive. At pH 7.5, in the presence of denaturing reagents, all of the BAL tryptophan became accessible to iodide, suggesting the presence of random conformation in this medium. The decrease in tryptophan accessibility to iodide with various bile salt activators was found to correlate with the corresponding activity of BAL with long chain triacylglycerol substrate.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ricin structure: the study by the fluorescence quenching method

To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.     

Fluorescence studies on the dissociation and denaturation of pigeon liver malic enzyme

Exposure of pigeon liver malic enzyme [S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) in medium concentrations of guanidine-HCl at 25 degrees C and pH 7.45 caused biphasic conformational changes of the enzyme molecule. Molecular weight determination confirmed that the enzyme tetramers were dissociated to monomers in phase I transition. Enzymatic activity was completely lost in this phase. Recovery of the enzyme activity was only possible in the early stages of the phase I transition. Phase II was due to enzyme unfolding, as judged by circular dichroism and the fluorescence parameters of the enzyme. The steps of the transformation of native malic enzyme into a completely denatured state were in the following sequence: tetramer----monomer----random coil. Extensive denaturation of the enzyme molecule resulted in irreversible aggregation. Dissociation and denaturation were accompanied by a red-shift of the fluorescence spectrum (328----368 nm). Fluorescence quenching studies indicated that tryptophan residues of the enzyme molecule were buried deeply in the interior of the molecule. The tryptophan residues were only partially accessible by acrylamide and almost inaccessible by KI. Dissociation and denaturation were accompanied by exposure of the tryptophan residues, as manifested by the accessibility of the enzyme molecule toward KI or acrylamide.     

Probing the renin active site by collisional quenching of endogenous fluorescence

The structural and enzymatic aspects of renin are of great interest in hypertension research. In this paper, we examine the solution accessibility of the three tryptophan (Trp) residues of mouse submaxillary gland renin by solute collisional fluorescence quenching. Our studies indicate that there are two "classes" of Trp residues in renin: class I, a class of Trp residues which are at or near the surface of renin and fully accessible to the fluorescence quencher iodide; and class II, a class of Trp residues which are, for practical experimental conditions, totally inaccessible to the aqueous solution. The former class contains 2 Trp residues, while only a single Trp is identified in the latter class. The presence of a tetradecapeptide substrate or a nonhydrolyzable substrate analogue (peptide H-77) lowers the accessibility of iodide to the class I Trp residues. These data indicate that the class I Trp residues are at or near the peptide-binding site of renin. In addition, the finding that the class I Trp residues are quantitatively quenched more efficiently than the Trp model compound indole suggests that the environment of the class I tryptophans may be positively charged, and thus have a higher "local" concentration of iodide. These data, taken together with the available sequence and computer-generated three-dimensional structure of renin, permit us to speculate that the class I Trp residues are Trp-39 and Trp-300. This solution study of renin structure is discussed in light of the known information about renin catalysis and physiology.     

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.     

Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers

The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

Structure and stability of gamma-crystallins: tryptophan, tyrosine, and cysteine accessibility

The solute perturbation techniques of fluorescence of tryptophan (Trp) and dye-labeled thiol groups of cysteine as well as phosphorescence of tyrosine (Tyr) were utilized to obtain information on the relative solvent exposure and accessibility of these residues in gamma-crystallins. Both acrylamide and iodide quenchers were used to evaluate the quenching parameters in terms of accessibility and charge characteristics of the proteins. Stern-Volmer plots reveal the presence of more than one class of Trp residues in gamma-III and gamma-IV, and these residues in gamma-II are least accessible compared to the other two. Both steady-state and lifetime quenching studies of the dye-labeled fluorescence indicate that distinct differences also exist among these crystallins in cysteine (Cys) accessibilities. All three proteins, gamma-II, gamma-III, and gamma-IV, show two distinct lifetime components of the dye-labeled Cys residues. Both components of gamma-II undergo dynamic quenching, whereas only the major component of the other two crystallins is affected by the quenchers. Addition of acrylamide causes a decrease in Tyr phosphorescence of gamma-III and gamma-IV, but no change in the emission of gamma-II. The decrease is attributed to the formation of a nonemittive ground-state complex between the acrylamide and Tyr of the proteins; the association constant, Ka, calculated from the emission data, has been considered as a measure of Tyr accessibility. Ka values indicate that Tyr residues in gamma-III are most exposed and accessible compared to those in the other two proteins. Results of quenching by iodide ion reveal significant differences in the surface charge of the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lifetime and quenching of tryptophan fluorescence in whiting parvalbumin

The fluorescence lifetime of the single tryptophan in whiting parvalbumin has been measured by time-correlated single-photon counting. In the presence of saturating calcium, greater than 2 mol/mol of protein, the decay of fluorescence is accurately single exponential with a lifetime of 4.6 ns (0.1 M KCl, 20 mM borate, 1 mM dithiothreitol, 20 degrees C, pH 9). Upon complete removal of calcium from parvalbumin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid the emission decay becomes biphasic, and a second more rapid decay process with a lifetime of 1.3 ns comprising approximately 18% of the fluorescence emission at 350 nm is observed. The fluorescence emission of the calcium-saturated form is not measurably quenched by iodide. In contrast, upon complete removal of calcium, the fluorescence is completely quenchable as shown by extrapolation of the data to infinite iodide concentration. These results indicate that there is a large increase in the accessibility of the tryptophan residue in the protein to solvent upon removal of calcium. Stern-Volmer plots of the quenching data are nonlinear and indicate that there is more than one quenchable conformation of the calcium-free protein. The lifetime and quenching results are consistent with the presence of significant concentrations of only two stoichiometric species, apoparvalbumin and parvalbumin--Ca2, at partial occupancy of the calcium binding sites.     

Intrinsic fluorescence of the P-glycoprotein multidrug transporter: sensitivity of tryptophan residues to binding of drugs and nucleotides

P-glycoprotein is a member of the ATP binding cassette family of membrane proteins, and acts as an ATP-driven efflux pump for a diverse group of hydrophobic drugs, natural products, and peptides. The side chains of aromatic amino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluorescence techniques were used to probe the environment of the 11 tryptophan residues within purified functional P-glycoprotein, and their response to binding of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environment, and time-resolved experiments showed the existence of at least two lifetimes. Quenching studies with acrylamide and iodide indicated that those tryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguing against large changes in tryptophan accessibility following substrate binding. P-glycoprotein fluorescence was highly quenched on binding of fluorescent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggesting that the site for nucleotide binding is located relatively close to tryptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowing estimation of dissociation constants. Many compounds exhibited biphasic quenching, suggesting the existence of multiple drug binding sites. The quenching observed for many substrates was attributable largely to resonance energy transfer, indicating that these compounds may be located close to tryptophan residues within, or adjacent to, the membrane-bound domains. Thus, the regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hydrolysis to drug transport.     

pH-dependent quenching of the fluorescence of tryptophan residues in class A beta-lactamase from E. coli (TEM-1)

We performed an investigation of the pH-dependent quenching of the fluorescence of tryptophan residues of TEM-1 beta-lactamase from E. coli by uncharged and charged quenchers. pH-dependent Stern-Volmer constants (Ksv/pH) of tryptophan residues allowed us to determine subtle but discrete structurally and functionally important processes.     

Distribution of tryptophan groups in molecules of troponin T, troponin I-troponin T complex, and alpha-actinin

The method of fluorescence quenching was used to experimentally determine the distribution of tryptophan residues in molecules of troponin T, troponin T-troponin I complexes, and alpha-actinin. Iodide and cesium ions, and acrylamide were used as quenchers. It was shown that cesium ions decrease the fluorescence intensity of troponin T and its complex with troponin I by the mode of dynamic quenching. For alpha-actinin such a dynamic quencher is anionic iodide. By using the modified Stern-Volmer equation, the quenching was found to be about 90% of total fluorescence intensity for troponin T, approximately 70% for the troponin T-troponin I complexes, and 20% for alpha-actinin. The penetration of cesium ions to tryptophan 206 (tryptophan 204) in the troponin T-troponin I complex is hindered, probably due to the participation of this tryptophan in the formation of bonds between troponin subunits.     

Fluorimetric studies of the binding of Momordica charantia (bitter gourd) lectin with ligands.

The association constants for the binding of a series of ligands with a galactose-specific lectin from Momordica charantia (bitter gourd) has been determined through the ligand-induced quenching of protein fluorescence. Analysis of the iodide quenching suggested that there is a slight increase in the accessibility of tryptophan residues of the lectin on binding lactose.     

Fluorescence of human liver alanine aminopeptidase.

Fluorescence of human liver alanine aminopeptidase has been attributed to tryptophan fluorescence. The fluorescence maximum is at 330 nm, 20 nm lower than that for free tryptophan, suggesting that most of the enzyme tryptophans are in a nonpolar environment and are shielded from solvent. Quenching of enzyme fluorescence by iodide, pyridine, and N-methyl nicotinamide also demonstrates that enzyme tryptophan residues are largely buried and inaccessible to solvent. Those accessible are in negatively charged environments. 8-(1'-dimethylaminonaphthalene-5'-sulfonylamido-octanoic acid (8-DNS-octanoic acid) and epsilon-DNS-L-Lys inhibit aminopeptidase. One molecule of inhibitor when bound to the enzyme quenched 57% and 63% of enzyme fluorescence, respectively. Such efficient quenching may indicate a degree of segregation of tryptophan toward the active center.     

Inaccessibility of tryptophan residues of recombinant human renin to quenching agents

The fluorescence quenching of the three tryptophan residues of recombinant human renin was determined using ionic and penetrating quenchers. Tryptophans 44,200, and 312 of recombinant human renin were found to be totally inaccessible to the ionic quenchers cesium and iodide and only partially accessible to the penetrating quencher acrylamide. The renin had a fluorescence emission maximum at 325 nm which was made up of three separate components as determined by second derivative spectroscopy. These data are in accord with solvent accessibility calculations from three-dimensional models of human renin but differ from findings published previously from similar analysis of mouse submandibular gland renin (Quay, S. C., Heropoulous, A., Commes, K., Dzau, V. J. (1985) J. Biol. Chem. 260, 15055-15058), which is 68% identical in sequence to human renin.     

Tryptophanyl substitutions in apomyoglobin affect conformation and dynamic properties of AGH subdomain

The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W --> F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule.     

Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein

D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.     

Fluorescence quenching studies of bovine growth hormone in several conformational states

The use of steady-state fluorescence quenching methods is reported as a probe of the accessibility of the single fluorescent tryptophan residue of bovine growth hormone (bGH, bovine somatotropin, bSt) in four solution-state conformations. Different bGH conformations were prepared by using previous knowledge of the multi-state nature of the equilibrium unfolding pathway for bGH: alterations in denaturant and protein concentration yielded different bGH conformations (native, monomeric intermediate, associated intermediate and unfolded). Because the intramolecular fluorescence quenching which occurs in the native state is reduced when the protein unfolds to any of the other conformations, steady-state fluorescence intensity measurements can be used to monitor bGH unfolding as well as the formation of the associated intermediate. These steady-state intensity changes have been confirmed with fluorescence lifetime measurements for the different conformational states of bGH. Fluorescence quenching results were obtained using the quenchers iodide (ionic), acrylamide (polar) and trichloroethanol (non-polar). Analysis of the results for native-state bGH reveals that the tryptophan environment is slightly non-polar (in agreement with the emission maximum of 335 nm) and the tryptophan is more exposed to acrylamide than most native-state tryptophan residues which have been studied. The tryptophan is most accessible to all quenchers in the unfolded state, because no steric restrictions inhibit quencher interaction with the tryptophan residue. The iodide quenching results indicate that the associated intermediate tryptophan is not accessible to iodide, probably due to negative charges inhibiting iodide penetration. The associated intermediate tryptophan is less accessible to all three quenchers than the monomeric intermediate tryptophan, due to tight packing of molecules in the associated intermediate state.