Tryptophan Residues In Proteins Research Articles

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes.

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.

Mutational analysis of the role of tryptophan residues in an antimicrobial peptide.

Antimicrobial peptides belonging to the pediocin-like family of bacteriocins (class IIa bacteriocins) produced by lactic acid bacteria contain several tryptophan residues that are highly conserved. Since tryptophan residues in membrane proteins are often positioned in the membrane-water interface, we hypothesized that Trp residues in bacteriocins could be important determinants of the structure of membrane-bound peptides and of anti-microbial activity. To test this hypothesis, the effects of mutating each of the 3 tryptophan residues (Trp18, Trp33, and Trp41) in the 43-residue pediocin-like bacteriocin sakacin P were studied. Trp18 and Trp33 are located at each end of an amphihilic alpha-helix, whereas Trp41 is near the end of an unstructured C-terminal tail. Replacement of Trp33 with the hydrophobic residues Leu and Phe had marginal effects on activity, whereas replacement with the more polar Tyr and Arg reduced activity 10-20 and 500-1000 times, respectively, indicating that Trp33 and the C-terminal part of the helix interact with the hydrophobic core of the membrane. Any mutation of Trp18 and Trp41 reduced activity, indicating that these two residues play unique roles. Substitutions with other aromatic residues were the least deleterious, indicating that both Trp18 and Trp41 interact with the membrane-water interface. The suggested locations of the three Trp residues are compatible with a structural model in which the helix and the C-terminal tail form a hairpin-like structure, bringing Trp18 and Trp41 close to each other in the interface, and placing Trp33 in the hydrophobic core of the membrane. Indeed, the deleterious effect of the W18L and W41L mutations could be overcome by stabilizing the hairpin-like structure by introduction of a disulfide bridge between residues 24 and 44. These results provide a basis for a refined structural model of pediocin-like bacteriocins and highlight the unique role that tryptophan residues can play in membrane-interacting peptides.

Spectroscopic studies on interaction of hemoglobin and serum albumin with nicotine

The interactions of nicotine and Hb/SA were studied in vitro by UV/Vis, fluorescence, 1H NMR and FT-IR spectroscopies. The UV/Vis absorbance of Hb/SA (200 nm) shifted to red and decreased gradually with the addition of nicotine, indicating that the protein conformational change resulted from the chemical interaction. With increasing nicotine concentration, incubation of SA with nicotine caused the quenching of fluorescence typical of protein tryptophan residues, which meant that the vicinity of the tryptophan residues of SA was changed because of nicotine. FT-IR spectra showed that α-helix component of Hb/SA decreased, turn and β-structure components of Hb/SA increased in the presence of nicotine. In the 1H NMR spectra of nicotine, all proton peaks on pyrrolidinyl ring moved to downfield and the resonance emanating from nicotine was preferentially broadened while the concentration of Hb/SA increased. All these results indicate that nicotine and Hb/SA in vitro interact on each other, forming a new complex and inducing the protein conformational change.

On the relationship between the radiative rate constant and the fluorescence intensity in organic fluorophores

In this Letter we explore the relationship between the radiative constant, k r, and the lineshape of an emission spectrum for fluorescent organic molecules, in particular tryptophan. We make a number of judicious approximations to the expression given by Strickler and Berg in conjunction with the log-normal function for the lineshape. Our result is a closed form expression for k r in terms of the inverse maximum wavelength λ max −1 which compares well with recent experimental findings for tryptophan residues in proteins.

Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. III. Correlation between Fluorescence and Microenvironment Parameters of Individual Tryptophan Residues

In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710–1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes.

Incorporation of β-selenolo[3,2- b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography

β-Selenolo[3,2- b]pyrrolyl- l-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno-proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.

On the excited-state energy transfer between tryptophan residues in proteins: the case of penicillin acylase

The problem, whether excited-state energy transfer occurs between Trp residues in a multi-tryptophan proteins and if it does, what kind of changes it induces in different parameters of protein fluorescence, is currently under active investigation. In our previous paper [Biophys. Chem. 72 (1998) 265], the energy transfer was found and studied in detail for Na,K-ATPase. It was shown that this transfer influences all parameters of fluorescence emission, which is detected at site-selective conditions (red-edge of excitation, blue and red edges of emission). Present experiments were performed on unusually tryptophan-rich protein, bacterial penicillin acylase (28 Trp per dimer of 82 kDa) and were aimed to extend these observations. They demonstrate substantial heterogeneity in the environments of tryptophan residues within the protein structure. This suggests, that in the present case, if the energy transfer exists, it should be directed from short-wavelength-emitting to long-wavelength-emitting tryptophan residues and thus could be easily observed by a number of time-resolved and steady-state fluorescence techniques. Unexpectedly, no signature of inter-tryptophan energy transfer was found.

Tryptophan environment in adenosine deaminase: I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues

Adenosine deaminase from bovine cerebral hemisphere (white and gray matter) and spleen was treated with N-bromosuccinimide, a reagent known to oxidize selectively tryptophan residues in proteins. Spectrally observable tryptophan modification was accompanied by enzyme inactivation. Tsow graphics revealed that two Trps are essential for the activity of enzyme from both tissues. Enzyme inhibitors and substrate analogues, derivatives of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine, were able to protect Trp against modification, and this effect correlated in general with the enzyme activity protection. In the presence of adenosine deaza analogues (the noninhibitor tubercidin among them) only two Trps were modified in the fully inactivated enzyme. In the presence of EHNA and its deaza analogues, full inactivation of the enzyme was accompanied by the modification of four Trps. The obtained data confirm the previous hypothesis about the presence on the enzyme of different binding sites for adenosine and EHNA derivatives that are responsible for the different effects on the enzyme conformation elicited by the corresponding derivatives. Moreover, these data allow us to suggest that Trp residues, still unidentified by X-ray analysis, are essential for the functioning of the enzyme.

Probing the Active Site of the Hepatitis C Virus Serine Protease by Fluorescence Resonance Energy Transfer

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide. Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

Functional role of a conserved tryptophan residue of Chromatium vinosum high potential iron protein

A Trp80Asn point mutation of Chromatium vinosum high potential iron protein has provided insight on the functional role of this conserved aromatic residue. Following mutation to Asn, a minimal change in the midpoint potential of the [Fe4S4]3+/2+ cluster is observed (+33 mV). In contrast to other proteins carrying mutations of Tyr and Phe residues, the reduced state of the Trp80Asn derivative is stable in an aerobic environment, but displays a more pronounced temperature-dependent degradation that appears to be mediated by unfolding of the protein prior to degradation of the cluster. Accordingly, Trp80 serves a distinct role from other aromatic residues found in the hydrophobic cluster binding pocket. The rate of electron transfer exchange with ferricyanide is similar to that observed for native protein (knat=0.7 s−1; kTrp80Asn=1.7 s−1), the minor increase in reactivity reflecting the change in driving force from the increase in cluster midpoint potential.

Thermal stability of the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-protein interactions.

Thermal stability of plasma membrane Ca(2+) pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 +/- 12 kJ/mol in the range 33-45 degrees C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca(2+) pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K(LD)) showing that those phospholipids with higher affinity provide greater stability to the Ca(2+) pump. We developed a method for directly measure K(LD) by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K(LD) values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity.

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data.

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.

The In Vivo Fluorescence of Tryptophan Moieties in Human Skin Increases with UV Exposure and is a Marker for Epidermal Proliferation

We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.

Probing local environments of tryptophan residues in proteins: Comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trp14 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J1995;69:20–29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain. Proteins 1999;37:709–716. ©1999 Wiley-Liss, Inc.

Probing local environments of tryptophan residues in proteins: Comparison of19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.

Evaluation of Lipid Exposure of Tryptophan Residues in Membrane Peptides and Proteins

Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b5 and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006–5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.

Packing of aromatic rings against tryptophan residues in proteins.

The geometry of the interaction of the aromatic side chains of phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and histidine (His) with the indole ring of Trp has been analyzed using the structures in the Protein Data Bank in order to understand the dependence of the packing behaviour on the size and chemical nature of the aromatic rings. The Phe ring prefers to interact either perpendicularly, with its edge pointing towards the Trp face, or in an offset-stacked arrangement. The edge-to-face motif is typical of a Trp-Trp pair. While parallel stacking is the dominant feature of Trp-His interaction, Tyr packs in a more uniform manner around Trp with a higher than expected occurrence at the edge and a few cases of possible OH-pi interaction.

Structure, function, and applications of tryptophan tryptophylquinone enzymes.

Tryptophan and tyrosine residues in proteins may be posttranslationally modified to form enzyme cofactors. Tryptophan tryptophylquinone (TTQ), the cofactor of methylamine dehydrogenase (MADH), is formed by covalent cross-linking of two tryptophan residues and incorporation of two oxygen atoms into one of the indole rings to form a quinone. MADH converts primary amines to their corresponding aldehydes plus ammonia. During the catalytic cycle, TTQ mediates electron transfer from substrate to a copper protein, amicyanin. These electrons are transferred to the respiratory chain via a c-type cytochrome. Structural, kinetic and site-directed mutagenesis studies have characterized protein-protein interactions, and mechanisms of catalysis and electron transfer by TTQ. Preliminary results obtained with MADH enzyme-electrodes demonstrate the potential for quinoprotein-based biosensors.

Tyrosine Quenching of Tryptophan Phosphorescence in Glyceraldehyde-3-Phosphate Dehydrogenase from Bacillus stearothermophilus

Tyrosine is known to quench the phosphorescence of free tryptophan derivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demonstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by comparing the phosphorescence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, is replaced with phenylalanine or Y283 is replaced by valine. Phosphorescence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp 84→Phe) mutant proteins, is already quenched in viscous low-temperature solutions, before the onset of major structural fluctuations in the macromolecule, an anomalous quenching that is abolished with the mutation Y283V (Tyr 283→Val). In buffer at ambient temperature, the effect of replacing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 μs to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction with Y283. Tyr quenching of W310 exhibits a strong temperature dependence, with a rate constant k q = 0.1 s −1 at 140 K and 2 × 10 4 s −1 at 293 K. Comparison between thermal quenching profiles of the W84F mutant in solution and in the dry state, where protein flexibility is drastically reduced, shows that the activation energy of the quenching reaction is rather small, E a ≤ 0.17 kcal mol −1, and that, on the contrary, structural fluctuations play an important role on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present results as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an excited-state triplet exciplex.

Effects of Hydrogen Bonding and Hydrophobic Interactions on the Ultraviolet Resonance Raman Intensities of Indole Ring Vibrations

Abstract UV resonance Raman intensities of indole ring vibrations were examined in solutions of different solvent properties to establish the correlation between the intensity and the environment of the indole ring, the main side-chain component of amino acid tryptophan. Each of the Raman bands that are enhanced through resonance with the Bb electronic transition gives the highest intensity when the indole NH site is hydrogen-bonded with a proton acceptor in hydrophobic environments. On the other hand, the Raman bands that gain intensity through resonance with the La transition are most enhanced when the indole ring is not hydrogen-bonded. Although these changes in Raman intensity result from small red and blue shifts of the Bb and La absorptions, respectively, the Raman intensity changes are much greater than the changes in UV absorption spectra. UV resonance Raman intensities are thus expected to be useful markers of hydrogen bonding and hydrophobic interactions of tryptophan residues in proteins.