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1.
Trimethylamine N-oxide (TMAO) is a natural osmolyte accumulated in cells of organisms as they adapt to environmental stresses. In vitro, TMAO increases protein stability and forces partially unfolded structures to refold. Its effects on the native fold are unknown. To investigate the interrelationship between protein stability, internal dynamics and function, the influence of TMAO on the flexibility of the native fold was examined with four different proteins by Trp phosphorescence spectroscopy. Its influence on conformational dynamics was assessed by both the intrinsic phosphorescence lifetime, which reports on the local structure about the triplet probe, and the acrylamide bimolecular quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results demonstrate that for apoazurin, alcohol dehydrogenase, alkaline phosphatase and glyceraldehydes-3-phosphate dehydrogenase 1.8 M TMAO does not perturb the flexibility of these macromolecules in a temperature range between - 10 degreesC and up to near the melting temperature. This unexpected finding contrasts with the dampening effect observed with polyols as well as with the expectations based on the preferential exclusion of the osmolyte from the protein surface. 相似文献
2.
Structural perturbations of azurin deposited on solid matrices as revealed by trp phosphorescence
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The phosphorescence emission of Cd-azurin from Pseudomonas aeruginosa was used as a probe of possible perturbations in the dynamical structure of the protein core that may be induced by protein-sorbent and protein-protein interactions occurring when the macromolecule is deposited into amorphous, thin solid films. Relative to the protein in aqueous solution, the spectrum is unrelaxed and the phosphorescence decay becomes highly heterogeneous, the average lifetime increasing sharply with film thickness and upon its dehydration. According to the lifetime parameter, adsorption of the protein to the substrate is found to produce a multiplicity of partially unfolded structures, an influence that propagates for several protein layers from the surface. Among the substrates used for film deposition, hydrophilic silica, dextran, DEAE-dextran, dextran sulfate, and hydrophobic octodecylamine, the perturbation is smallest with dextran sulfate and largest with octodecylamine. The destabilizing effect of protein-protein interactions, as monitored on 50-layer-thick films, is most evident at a relative humidity of 75%. Stabilizing agents were incorporated to attenuate the deleterious effects of protein aggregation. Among them, the most effective in preserving a more native-like structure are the disaccharides sucrose and trehalose in dry films and the polymer dextran in wet films. Interestingly, the polymer was found to achieve maximum efficacy at sensibly lower additive/protein ratios than the sugars. 相似文献
3.
This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds. 相似文献
4.
After a brief introduction of the potentialities of Trp phosphorescence spectroscopy for probing the conformation and flexibility of protein structure, this presentation summarizes the effects of hydrostatic pressure (up to 3 kbar) on the native fold of monomeric and oligomeric proteins as inferred from the variation of the intrinsic phosphorescence lifetime and the oxygen and acrylamide bimolecular quenching rate constants of buried Trp residues. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of hydration of the polypeptide. The implications of these findings for the thermodynamic stability of proteins and for the determination of subunit dissociation equilibria under high pressure conditions are also discussed. 相似文献
5.
Erythrosin B phosphorescence monitors molecular mobility and dynamic site heterogeneity in amorphous sucrose
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Molecular mobility modulates the chemical and physical stability of amorphous biomaterials. This study used steady-state and time-resolved phosphorescence of erythrosin B to monitor mobility in thin films of amorphous solid sucrose as a function of temperature. The phosphorescence intensity (lifetime), emission energy, and red-edge excitation effect were all sensitive to localized molecular mobility on the microsecond timescale in the glass and to more global modes of mobility activated at the glass transition. Blue shifts in the emission spectrum with time after excitation and systematic variations in the phosphorescence lifetime with wavelength indicated that emission originates from multiple sites ranging from short lifetime species with red-shifted emission spectrum to long lifetime species with blue-shifted emission spectrum; the activation energy for nonradiative decay of the triplet state was considerably larger for the blue-emitting species in both the glass and the melt. This study illustrates that phosphorescence from erythrosin B is sensitive both to local dipolar relaxations in the glass as well as more global relaxations in the sucrose melt and provides evidence of the value of phosphorescence as a probe of dynamic site heterogeneity as well as overall molecular mobility in amorphous biomaterials. 相似文献
6.
D'Auria S Staiano M Varriale A Gonnelli M Marabotti A Rossi M Strambini GB 《Journal of proteome research》2008,7(3):1151-1158
Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs. 相似文献
7.
From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphorescence properties nor the catalytic activity. The variation in the lifetime parameter was found to be a smooth function of the residual volume of liquid water in equilibrium with ice and to depend on the morphology of ice. The addition of cryoprotectants such as glycerol and sucrose profoundly attenuates or even eliminates the perturbation. These results are interpreted in terms of adsorption of protein molecules onto the surface of ice. 相似文献
8.
Gabellieri E Cioni P Balestreri E Morelli E 《European biophysics journal : EBJ》2011,40(11):1237-1245
We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins
involved in interaction with nanoparticles. Characterization of protein–nanoparticle interaction is crucial in assessing biological
hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an
absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular
formula of GSH18S56Cd60. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe,
and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein
interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS.
Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small
size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for
proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated
affinities were comparable to those observed for specific protein–ligand interactions and suggest that protein–nanoparticle
interaction may have a biological impact. 相似文献
9.
Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain
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Bódis E Strambini GB Gonnelli M Málnási-Csizmadia A Somogyi B 《Biophysical journal》2004,87(2):1146-1154
The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique. 相似文献
10.
Gabellieri E Strambini GB Shcharbin D Klajnert B Bryszewska M 《Biochimica et biophysica acta》2006,1764(11):1750-1756
Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers. 相似文献
11.
The fluorescence and phosphorescence properties of the tryptophan residues in glutamate dehydrogenase were utilized to probe the conformation of the macromolecule at various states of aggregation of its subunits (hexamer, trimer, and monomer) in guanidine hydrochloride. According to the phosphorescence lifetime no gross alteration in the conformation of the protein follows from complete dissociation of the hexamer into native monomer, implying that the native fold is stabilized exclusively by intrasubunit bonding. Although modest concentrations of denaturant induce a change in configuration in the enzyme, a comparison with the macromolecule cross-linked into the hexameric form by glutaraldehyde confirms that this alteration in structure is not the result of subunit dissociation. Inhibition of catalysis by the denaturant is found to be considerably smaller than anticipated from the extent of hexamer dissociation. Furthermore, this inhibition is in no way prevented by cross-linking the enzyme in its hexameric form. This finding together with the ability of the trimer to bind the coenzyme and to undergo the characteristic structural changes induced by the effectors ADP and GTP suggests that, contrary to what is generally believed, the smallest functional unit of glutamate dehydrogenase is not the hexameric form. 相似文献
12.
Raymond F. Chen 《Archives of biochemistry and biophysics》1975,166(2):584-591
The phosphorescence of tryptophan and proteins was examined in the presence of silver nitrate in order to obtain information on the mechanisms by which Ag+ quenches fluorescence. The 1:1 Ag+-Trp complex is nonfluorescent both at 77 °C and 296 °C and has 3-fold higher phosphorescence quantum yield than the free amino acid. Silver ion causes loss of vibrational structure in the phosphorescence spectrum, and the lifetime decreases from 7.2 to 0.02. These findings are consistent with an intramolecular heavy-atom effect. A nonsulfhydryl protein, trypsinogen, shows changes in phosphorescence which are qualitatively, but not quantitatively, similar to tryptophan in the presence of silver nitrate. Yeast and liver alcohol dehydrogenases have many sulfhydryl groups and show only phosphorescence quenching on addition of Ag+. In this case, quenching occurs by an energy-transfer mechanism. The phosphorescence yield and spectrum of mercuripapain differed from those of papain and were consistent with a heavy atom effect due to Hg2+.The study was technically much facilitated by the use of aqueous snows containing 10% () methanol. Among the advantages of such aqueous snows is the lack of gross denaturation which has in the past been a major objection to protein phosphorescence studies utilizing glasses of organic solvents. 相似文献
13.
Excited states of tryptophan in cod parvalbumin. Identification of a short-lived emitting triplet state at room temperature.
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The fluorescence and phosphorescence spectra of model indole compounds and of cod parvalbumin III, a protein containing a single tryptophan and no tyrosine, were examined in the time scale ranging from subnanoseconds to milliseconds at 25 degrees C in aqueous buffer. For both Ca- bound and Ca-free parvalbumin and for model indole compounds that contained a proton donor, a phosphorescent species emitting at 450 nm with a lifetime of approximately 20-40 ns could be identified. A longer-lived phosphorescence is also apparent; it has approximately the same absorption and emission spectrum as the short-lived triplet molecule. For Ca parvalbumin, the decay of the long-lived triplet tryptophan is roughly exponential with a lifetime of 4.7 ms at 25 degrees C whereas for N-acetyltryptophanamide in aqueous buffer the decay lifetime was 30 microseconds. In contrast, the lifetime of the long-lived tryptophan species is much shorter in the Ca-free protein compared with Ca parvalbumin, and the decay shows complex nonexponential kinetics over the entire time range from 100 ns to 1 ms. It is concluded that the photochemistry of tryptophan must take into account the existence of two excited triplet species and that there are quenching moieties within the protein matrix that decrease the phosphorescence yield in a dynamic manner for the Ca-depleted parvalbumin. In contrast, for Ca parvalbumin, the tryptophan site is rigid on the time scale of milliseconds. 相似文献
14.
Role of Protein Cavities on Unfolding Volume Change and on Internal Dynamics under Pressure
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Patrizia Cioni 《Biophysical journal》2006,91(9):3390-3396
The effects of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (DeltaV(0)) of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift of the center of mass of the fluorescence spectrum, showed that DeltaV(0) is in the range of 60-70 mL/mol, not significantly different between cavity mutants and compact azurin species such as the wild-type and the mutant C3A/C26A, in which the superficial disulphide has been removed. The lack of extra volume in F110S and I7S proves that the engineered cavities, 40 A(3) in I7S and 100 A(3) in F110S, are filled with water molecules. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)). The application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between compact folds, wild-type and C3A/C26A, and cavity mutants is that the inversion point is powered from approximately 3 kbar to 1.5 kbar for F110S and <0.1 kbar for I7S, meaning that in the latter species pressure-induced internal hydration dominates very early over any compaction of the globular fold resulting from the reduction of internal free volume. The similar response between wild-type and the significantly less-stable C3A/C26A mutant suggests that thermodynamic stability per se is not the dominant factor regulating pressure-induced internal hydration of proteins. 相似文献
15.
A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Tryptophan phosphorescence and the conformation of liver alcohol dehydrogenase in solution and in the crystalline state 总被引:2,自引:0,他引:2
Information on the effects of crystallization upon the structure of liver alcohol dehydrogenase from horse is obtained from a comparison of the phosphorescence properties of its tryptophan residues in solution and in the crystalline state. In the crystalline state the red shift in the phosphorescence spectrum of the solvent-exposed Trp-15 attests to a decreased polarity of its environment consistent with its shielding away from the aqueous solvent probably through its involvement in an intermolecular contact. On the other hand, the triplet-state lifetime of Trp-314 which is buried deeply in the coenzyme-binding domain demonstrates that the flexibility of this region of the macromolecule is unaffected by crystallization; a conclusion supported also by the similarity in the rate of oxygen quenching of its phosphorescence. Given that lattice constraints strongly inhibit large-scale conformational changes these results allow us to identify the average solution structure with the 'open' conformer determined crystallographically. 相似文献
17.
The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold. 相似文献
18.
Tryptophan luminescence from liver alcohol dehydrogenase in its complexes with coenzyme. A comparative study of protein conformation in solution 总被引:2,自引:0,他引:2
The extent of fluorescence quenching and that of phosphorescence quenching of Trp-15 and Trp-314 in alcohol dehydrogenase from horse liver as well as the intrinsic phosphorescence lifetime of Trp-314 in fluid solution have been utilized as structural probes of the macromolecule in binary and ternary complexes formed with coenzyme, analogous, and various substrate/inhibitors. Luminescence quenching by the coenzyme reveals that (1) while the reduced form quenches Trp emission exclusively from the fluorescent state, the oxidized form is very effective on the phosphorescent state as well and that (2) among the series of NADH binary and ternary complexes known by crystallographic studies to attain the closed form, distinct nicotinamide/indole geometrical arrangements are inferred from a variable degree of fluorescence quenching. Information of the dynamic structure of the coenzyme-binding domain derived from the phosphorescence lifetime of Trp-314 points out that within the series of closed NADH complexes there is considerable conformational heterogeneity. In solution, the variability in dynamical structure among the various protein complexes emphasizes that the closed/open forms identified by crystallographic studies are not two well-defined macrostates of the enzyme. 相似文献
19.
Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans. 总被引:2,自引:2,他引:0
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The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed. 相似文献
20.
D'Auria S Staiano M Varriale A Gonnelli M Marabotti A Rossi M Strambini GB 《Proteins》2008,71(2):743-750
Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel beta-strands. The large domain is similar to the small domain but it contains two additional alpha-helices and three more short antiparallel beta-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called "closed form" structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP. 相似文献