Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence

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.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tryptophan phosphorescence of nascent and inactivated actin at the room temperature]

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.     

Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.     

Quenching of room temperature protein phosphorescence by added small molecules

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)     

Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular imprinted ormosils for nafcillin recognition by room temperature phosphorescence optosensing

Nafcillin imprinted sol-gels were used as specific binding materials for the room temperature phosphorescence (RTP) recognition of nafcillin using a flow injection system. Selective and reversible binding of the template to imprinted sol-gels could be observed by tailoring the hydrophobic/hydrophilic balance of the materials. Also, the possibility of minimizing non-specific interactions was evaluated by end-capping with trimethyl silyl groups. Control experiments were performed with non imprinted reference sol-gels. The recognition mechanism and the analytical potential of these materials for developing stable, selective and sensitive approaches for nafcillin recognition in real samples are outlined.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Thermal unfolding and aggregation of actin

Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin-like proteins have recently been identified in bacteria. Actin filament (F-actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat-induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F-actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.     

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

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.     

Assessment of molecularly imprinted sol-gel materials for selective room temperature phosphorescence recognition of nafcillin

Sol-gel imprinted materials were prepared against nafcillin, a semisynthetic beta-lactamic antibiotic employed in the treatment of serious infections caused by penicillinase-producing staphylococci. Two approaches were addressed for preparation of the imprinted materials and the controls: as conventional monoliths, which were ground and sieved to a desired particle size for rebinding analysis, and as films on supporting glass slides. The specific binding sites that are created during the imprinting process are analyzed via selective room temperature phosphorescence (RTP) (sol-gel films) measurements as well as via competitive room temperature phosphorescence ligand assay. Results demonstrated the importance of the physical configuration of the imprinted material for minimizing non-specific binding. The close similarities between the structures of different beta-lactamic antibiotics made it possible to interpret the roles of the template structure on specific molecular recognition. In this article, we introduce the use of room temperature phosphorescence as selective transduction method for the template. The imprinted sol-gel films displayed enhanced specific binding characteristics respect to the monolithic sol-gel and can be envisaged for the use as recognition matrices for the screening and rapid selection of antibiotics from a combinatorial library or for the rapid control of nafcillin in biological samples (e.g. milk, serum, urine).     

Monitoring protein unfolding transitions by NMR-spectroscopy

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

     

p53 unfolding detected by CD but not by tryptophan fluorescence

Full-length human p53 protein was examined using tryptophan fluorescence and circular dichroism spectroscopy (CD) to monitor unfolding. No significant alteration in tryptophan fluorescence for the tetrameric protein was detectable over a wide range of either urea or guanidine hydrochloride concentrations, in contrast to results with the isolated DNA binding domain [Bullock et al. (1997) Proc. Natl. Acad. Sci. USA 94, 14338]. Under similar denaturant conditions, CD demonstrated significant protein unfolding for the full-length wild-type protein, with increased apparent structure loss compared to that detected during thermal denaturation [Nichols and Matthews (2001) Biochemistry 40, 3847]. Examination of X-ray structures containing two of the four tryptophan residues of a p53 monomer suggested local environments consistent with quenched fluorophores. Exploration of p53 fluorescence using potassium iodide as a quencher confirmed that these fluorophores are already substantially quenched in the native structure, and this quenching is not relieved during protein unfolding.     

Comparison of properties on non-protected fluid room temperature phosphorescence of some tetra-ring aromatic hydrocarbons.

A comparative study of the photoluminescence properties of three kinds of tetra-ring aromatic hydrocarbon (1-sodium pyrenesulphonate, benz[alpha]anthracene and chrysene) solution in the absence of any protecting medium is described. It was found that a room temperature phosphorescence signal with different intensities can be induced for these solutions, using only TlNO3 or KI as a heavy atom perturber (HAP) and Na2SO3 as a deoxygenator. An appropriate amount of organic solvent added to the systems of pyrene, benz[alpha]anthracene and chrysene is necessary for increasing the solubility and phosphorescence intensity, and the preferable solvent is acetonitrile. For the pyrene, pyrenesulphonate and chrysene systems, a delayed excimer fluorescence accompanied with the room temperature phosphorescence (RTP) emission can be observed, but that for benz[alpha]anthracene cannot. The ratio of delayed excimer fluorescence and phosphorescence signals for pyrene, pyrenesulphonate and chrysene systems can be controlled by adjusting the concentration of luminophor, kinds and amount of both organic solvents and HAP. Under the optimal conditions, the RTP signals are proportional to the concentration of the four aromatic hydrocarbons, which means that the RTP properties of the four tetra-ring aromatic hydrocarbons can be used for quantitative analysis.     

Sensitive monitoring of the dynamics of a membrane-bound transport protein by tryptophan phosphorescence spectroscopy

This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.     

Intramolecular dynamics of human erythrocyte membrane proteins upon modification of spectrin by tryptophan phosphorescence

The slow (millisecond) protein internal dynamics of isolated human erythrocyte membranes in suspension without treatment, after deleting 95% of spectrin, after spectrin thermal denaturation upon acidification of medium in the pH range 6.0-4.0, and spectrin extracted in solution from membranes has been studied by room-temperature tryptophan phosphorescence. It has been established that integral proteins and spectrin differ in structural and dynamic state. Millisecond movements of structural elements of integral proteins are more restricted compared with those of spectrin. The removal of spectrin from the membrane led to an increase in slow fluctuations of integral protein structure. This indicates that spectrin participates in the control of the structural and dynamic state of erythrocyte membrane proteins. As medium was acidified in the pH range 6.0-4.0, the protein slow internal dynamics of membranes in native state decreased, which was explained by spectrin pH aggregation. After thermal denaturation of spectrin, no pH-induced increase of membrane protein structure rigidity was observed.     

Characterization of tryptophan environments in glutamate dehydrogenases from temperature-dependent phosphorescence

Tryptophan room temperature phosphorescence in solution was detected in glutamic dehydrogenase from bovine liver and Escherichia coli with lifetimes of 1.2 and 0.65 s, respectively. Although these enzymes possess three and five tryptophanyl residues per polypeptide chain, respectively, the temperature dependence of the phosphorescence quantum yield estimates that the room temperature emission is due, in either case, to a single residue. Long triplet-state lifetimes and very small rates of O2 quenching indicate that these tryptophanyl side chains are embedded in a highly inflexible internal region of the macromolecule. Aided by sequence homology with dehydrogenases of known structure and theoretical predictions of secondary structure [Wootton, J.C. (1974) Nature (London) 252, 542-546; Brett, M., Chambers, G.K., Holder, A. A., Fincham, J.R.S., & Wootton, J.C. (1976) J. Mol. Biol. 106, 1-22], the phosphorescing tryptophans have been tentatively placed in the catalytic coenzyme binding domain of each enzyme. The particular sensitivity of the triplet-state lifetime in probing local changes in conformation provides a strong indication that within the time window of phosphorescence measurements the six subunits in the hexameric enzymes are equivalent. Furthermore, while in the bovine enzyme this parameter is markedly affected by the interaction with ligands which have a functional role, the constancy of the phosphorescence lifetime at various degrees of polymerization suggests that the association process is not accompanied by important conformational changes in the macromolecule.     

Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases

Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.