Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, λ_max, does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at λ_max, I_F, differs significantly for the five Trp containing variants of RNase Sa; 3), the I_F differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I_F differences in the peptides are also present in the proteins; 4) the I_F values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.     

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.     

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)     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Unraveling the mechanisms of tryptophan fluorescence quenching in the triosephosphate isomerase from Giardia lamblia

In the native state several proteins exhibit a quenching of fluorescence of their tryptophans. We studied triosephosphate isomerase from Giardia lamblia (GlTIM) to dissect the mechanisms that account for the quenching of fluorescence of its Trp. GlTIM contains four Trp per monomer (Trp75, Trp162, Trp173, and Trp196) distributed throughout the 3D structure. The fluorescence of the denatured enzyme is 3-fold higher than that of native GlTIM. To ascertain the origin of this phenomenon, single and triple mutants of Trp per Phe were made. The intrinsic fluorescence was determined, and the data were interpreted on the basis of the crystal structure of the enzyme. Our data show that the fluorescence of all Trp residues is quenched through two different mechanisms. In one, fluorescence is quenched by aromatic-aromatic interactions due to the proximity and orientation of the indole groups of Trp196 and Trp162. The magnitude of the quenching of fluorescence in Trp162 is higher than in the other three Trp. Fluorescence quenching is also due to energy transfer to the charged residues that surround Trp 75, 173 and 196. Further analysis of the fluorescence of GlTIM showed that, among TIMs from other parasites, Trp at position 12 exhibits rather unique properties.     

酶切菌紫质C端引起的紫膜蛋白的发光性质变化

用木瓜蛋白酶切去bRC端尾巴约20—25个氨基酸之后发现:在KCl盐浓度作用下,其蛋白荧光光谱中的色氨酸荧光成份有明显的增强;用Cs~ 对bR蛋白荧光的猝灭实验表明此色氨酸荧光成份的增加是由于在KCl作用下bR中某些色氨酸残基暴露的结果;磷光光谱实验表明在KCl作用下色氨酸磷光的增加也是由于色氨酸残基暴露的结果。本文讨论了这种构象变化可能影响正常bR分子中菌蛋白光循环的进行,从而使质子泵效率降低;并且此构象变化可能与其C端尾巴的构象有关。     

Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa

Gaining a better understanding of the denatured state ensemble of proteins is important for understanding protein stability and the mechanism of protein folding. We studied the folding kinetics of ribonuclease Sa (RNase Sa) and a charge-reversal variant (D17R). The refolding kinetics are similar, but the unfolding rate constant is 10-fold greater for the variant. This suggests that charge-charge interactions in the denatured state and the transition state ensembles are more favorable in the variant than in RNase Sa, and shows that charge-charge interactions can influence the kinetics and mechanism of protein folding.     

Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Trp scanning analysis of Tet repressor reveals conformational changes associated with operator and anhydrotetracycline binding.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.     

Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions.

The interaction of several tryptophan (Trp)-rich cationic antimicrobial peptides with membranes was investigated. These peptides included tritrpticin, indolicidin, lactoferricin B (Lfcin B), and a shorter fragment of lactoferricin (LfcinB4-9). The average environment of the Trp residues of these peptides was assessed from their fluorescence properties, both the wavelength of maximal emission as well as the red edge effect. The insertion of the peptides into vesicles of differing composition was examined using quenching of the Trp fluorescence, with both soluble acrylamide and nitroxide-labelled phospholipids as well as by chemical modification of the Trp residues with N-bromosuccinimide. The results were consistent with the Trp side chains positioned mostly near the membrane-water interface. The extent of burial of the Trp side chains appears to be greater in vesicles containing phospholipids with the anionic phosphatidylglycerol headgroup. Leakage of the aqueous contents of liposomes was also measured using the 8-aminonaphthalene-1,3,6-trisulfonic acid--p-xylene-bis-pyridinium bromide assay. Tritrpticin, which demonstrated the greatest red edge shift, also displayed the largest amount of leakage from liposomes. Taken together, the results illustrate that cationic Trp-rich antimicrobial peptides preferentially disrupt large unilamellar vesicles with a net negative charge following their insertion into the interfacial region of the phospholipid bilayer.     

Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue can reduce bimane fluorescence intensity by up to 500% and induce complicated fluorescence decay kinetics. Replacing the neighboring Trp residue with phenylalanine removes these spectral perturbations. The advantages of using the Trp quenching of bimane fluorescence for protein structural studies include the low amount of protein required and the substantial simplification of labeling strategies. We anticipate this method will prove suitable for a wide array of high-throughput protein studies such as protein folding, the detection of protein-protein interactions, and, most importantly, the dynamic monitoring of conformational changes.     

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I⁻, Cs⁺ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I⁻, was proven to be an ineffective quencher with Stern-Volmer constants, K_sv, of 0.60 and 0.63 M⁻¹, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs⁺, showed about a 2-fold difference in the K_sv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25–37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs⁺ showed more than 40% increase in the K_sv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.     

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.     

Location and dynamics of tryptophan in transmembrane alpha-helix peptides: a fluorescence and circular dichroism study

Amphiphilic and hydrophobic peptides play a key role in many biological processes. We have developed a reference system for evaluating the insertion of such peptides bearing Trp fluorescent reporter groups into membrane mimetic systems. This system involves a set of six 25-amino acid synthetic peptides that are models of transmembrane alpha-helices. They are Lys-flanked polyLeu sequences, each containing a single Trp residue at a different position (P i, with i=3, 5, 7, 9, 11 and 13). These peptides were inserted into micelles of a non-ionic detergent, dodecylmaltoside (DM). We analyzed this system by use of circular dichroism and steady-state and time-resolved fluorescence in combination with Trp quenching with two brominated DM analogs. We found significant variations in the Trp emission maximum according to its position in each peptide (from 327 to 313 nm). This is consistent with the radial insertion of the peptides within DM micelles. We observed characteristic patterns of fluorescence quenching of these peptides in mixed micelles of DM, with either 7,8-dibromododecylmaltoside (BrDM) or 10,11-dibromoundecanoylmaltoside (BrUM), that reflect differences in the accessibility of the Trp residue to the bromine atoms located on the detergent acyl chain. In the isotropic reference solvent, methanol, the alpha-helix content was high and identical (approximately 76%) for all peptides. In DM micelles, the alpha-helix content for P9 to P13 was similar to that in methanol, but slightly lower for P3 to P7. The fluorescence intensity decays were heterogeneous and depended upon the position of the Trp. The Trp dynamics of each peptide are described by sub-nanosecond and nanosecond rotational motions that were significantly lower than those observed in methanol. These results, which precisely describe structural, dynamic and microenvironment parameters of peptide Trp in micelles according to its depth, should be useful for describing the interactions of peptides of biological interest with micelles.     

Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes

A novel fluorescence method for determining the depth of Trp residues in membrane-inserted polypeptides is introduced. Quenching of Trp by acrylamide and 10-doxylnonadecane (10-DN) was used to measure Trp depth. Transmembrane helices with Trp residues at varying positions (and thus locating at different depths in lipid bilayers) were used to calibrate the method. It was found that acrylamide quenches Trp close to the bilayer surface more strongly than it quenches deeply buried Trp, while 10-DN quenches Trp close to the center of the bilayer more strongly than Trp close to the surface. The ratio of acrylamide quenching to that of 10-DN was found to be nearly linearly dependent on the depth of Trp in a membrane. It was also found that it was possible to detect coexisting shallowly and deeply inserted populations of Trp-containing polypeptides using these quenchers. In the presence of such mixed populations, acrylamide induced large blue shifts in fluorescence emission lambda(max) whereas 10-DN induced large red shifts. In a more homogeneous population quencher-induced shifts were found to be minimal. Dual quencher analysis can be used to distinguish hydrophobic helices with a transmembrane orientation from those located close to the bilayer surface and, when applied to a number of different peptides, revealed novel aspects of hydrophobic helix behavior.