Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis

The individual emission properties of the two tryptophanyl residues of sperm-whale apomyoglobin have been resolved by examining the fluorescence variations induced by denaturants, i.e., acid and guanidine, on apomyoglobin mutants W7F and W14F. The fluorescence changes have been correlated to the conformational transitions undergone by apomyoglobin on increasing denaturant concentration. The results indicate that the fluorescence decrease, observed for sperm-whale apomyoglobin on going from pH 8.0 to pH 6.0, cannot be ascribed to the formation of a charge transfer complex between a nearby histidine residue and W14 as reported in earlier papers but rather to minor structural changes affecting the microenvironments of both residues. The formation of the acidic partly folded state around pH 4.0 determines an increase of the fluorescence yield and a small red shift (5 nm) of W7 due to removal of sterically interacting K79, which is able to attenuate the emission of this residue in the native state. The fluorescence intensity of the other residue, i.e., W14, is not affected by the acidic transition. Guanidine denaturation experiments revealed an increase of fluorescence yield of W14 upon the intermediate formation, whereas the fluorescence of the other residue remained constant. The results suggest that the unfolding pathway may be different depending on the chemical nature of the denaturant used.     

Single tryptophanyl substitutions affect the structure of apomyoglobin

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions A-5 (W7) and A-12 (W14) in the N-terminal region (A helix) of the protein molecule. To determine the contribution of each tryptophanyl residue to the structure and stability of myoglobin, recombinant proteins with single indole residue, i.e., W7 or W14, were obtained by site-directed mutagenesis. The mutant proteins, expressed in Escherichia coli, were found correctly folded, the far ultraviolet circular dichroism of both mutants as well as the Soret absorption being superimposed to that of wild type protein. The removal of the prosthetic group from mutant proteins determined a loss of helical content much larger than that observed in the case of wild type myoglobin. These results suggest that tryptophanyl residues can play a crucial role on globin folding and structure.     

Role of tryptophanyl residues in driving myoglobin folding

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The simultaneous substitution of both tryptophanyl residues causes an incorrect folding with subsequent loss of heme binding. The introduction of a indolic residue in different molecular regions, i.e. G, E, and C helix resulted in a not correctly folded protein, suggesting that the tryptophanyl residues are strong structural determinants.     

Resolution of the effects induced by W?→?F substitutions on the conformation and dynamics of the amyloid-forming apomyoglobin mutant W7FW14F

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.     

Tryptophanyl substitutions in apomyoglobin determine protein aggregation and amyloid-like fibril formation at physiological pH

Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.     

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The double W/F replacement renders apomyoglobin highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions. In this work we analyze the early stage of W7FW14F apomyoglobin aggregation following the time dependence of the process by far-UV CD, Fourier-transform infrared (FTIR) spectroscopy, and heme-binding properties. The results show that the aggregation of W7FW14F apomyoglobin starts from a native-like globin state able to bind the prosthetic group with spectroscopic properties similar to those observed for wild-type apoprotein. Nevertheless, it rapidly aggregates, forming amyloid fibrils. However, when the prosthetic group is added before the beginning of aggregation, amyloid fibrillization is inhibited, although the aggregation process is not prevented. Moreover, the apomyoglobin aggregates formed in these conditions are not cytotoxic differently from what is observed for all amyloidogenic proteins. These results open new insights into the relationship between the structure adopted by the protein into the aggregates and their ability to trigger the impairment of cell viability.     

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

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

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.     

Near-ultraviolet circular dichroic activity of apomyoglobin: resolution of the individual tryptophanyl contributions by site-directed mutagenesis

The individual tryptophanyl contributions to the near-ultraviolet circular dichroic activity of apomyoglobin in its native conformation have been resolved by studying recombinant proteins with single tryptophanyl substitutions. Site-directed mutagenesis of sperm whale apomyoglobin was performed in order to obtain proteins containing only Trp A-5 or Trp A-12. These amino acid substitutions have very little effect on the overall globin fold as indicated by comparing the spectroscopic properties of the mutants with those of the wild type protein. The circular dichroism spectra of the two apomyoglobin mutants in the near ultraviolet were found to be significantly different, both indole residues having significant activity but of opposite sign. In particular, Trp A-5 shows the presence of a main positive peak centered near 294 – 295 nm with a marked shoulder at 285 nm, ascribed to the ¹L_Btransition. The spectrum of the mutant protein containing only Trp A-12 shows a large negative contribution with a minimum near 283 nm and a marked shoulder at 293 nm. The broadness of the negative contribution exhibited by Trp A-12 suggests that it may originate mainly from the ¹L_A transition. Received: 17 February 1997 / Accepted: 14 August 1997     

Effect of unfolding on the tryptophanyl fluorescence lifetime distribution in apomyoglobin

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 degrees C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments.     

Glycation Accelerates Fibrillization of the Amyloidogenic W7FW14F Apomyoglobin

Neurodegenerative diseases are associated with misfolding and deposition of specific proteins, either intra or extracellularly in the nervous system. Advanced glycation end products (AGEs) originate from different molecular species that become glycated after exposure to sugars. Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. To this concern, in the present study we have investigated the effect of glycation on the aggregation pathway of the amyloidogenic W7FW14F apomyoglobin. Although this protein has not been related to any amyloid disease, it represents a good model to resemble proteins that intrinsically evolve toward the formation of amyloid aggregates in physiological conditions. We show that D-ribose, but not D-glucose, rapidly induces the W7FW14F apomyoglobin to generate AGEs in a time-dependent manner and protein ribosylation is likely to involve lysine residues on the polypeptide chain. Ribosylation of the W7FW14F apomyoglobin strongly affects its aggregation kinetics producing amyloid fibrils within few days. Cytotoxicity of the glycated aggregates has also been tested using a cell viability assay. We propose that ribosylation in the W7FW14F apomyoglobin induces the formation of a cross-link that strongly reduces the flexibility of the H helix and/or induce a conformational change that favor fibril formation. These results open new perspectives for AGEs biological role as they can be considered not only a triggering factor in amyloidosis but also a player in later stages of the aggregation process.     

Elucidation of information encoded in tryptophan 140 of staphylococcal nuclease

We investigated the role of W140 in the folding of Staphylococcal nuclease. For this purpose, we constructed the 19 possible substitution mutations at residue 140. Only three mutants, W140F, W140H, and W140Y, adopted native-like structures under physiological conditions and showed native-like enzymatic activities. In contrast, the other 16 mutants took on compact unfolded structures under physiological conditions and the enzymatic activities of these mutants were decreased to approximately 70% of wild-type levels. These 16 mutants maintained substrate-induced foldability. These results strongly indicate that the side-chain information encoded by residue 140 is essential to maintain a stable native structure, and that this residue must be an aromatic side chain. The order of thermal stability was wild type > W140H > W140F = W140Y. Therefore, the five-membered nitrogen-containing ring of the indole is thought to bear the essential information. In the crystal structure of staphylococcal nuclease, the five-membered ring is at the local center of the C-terminal cluster through hydrophobic interactions. This cluster plays a key role in the interaction connecting the C-terminal region and the N-terminal beta-core. Mutants other than W140H, W140F, and W140Y lost the ability to form the local core, which caused the loss of the long-range interactions between the C-terminal and N-terminal regions. Inhibitor or substrate binding to these mutants compensates for the lack of long-range interactions generated by W140.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Identification of Tyr504 as an alternative tyrosyl radical site in human prostaglandin H synthase-2

Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000) Prost. Lipid Med. 62, 231-254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr --> Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.     

Origin of the anomalous circular dichroism spectra of many apomyoglobin mutants

Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins.     

Structural characterization of partially folded intermediates of apomyoglobin H64F

We present a detailed investigation of unfolded and partially folded states of a mutant apomyoglobin (apoMb) where the distal histidine has been replaced by phenylalanine (H64F). Previous studies have shown that substitution of His64, located in the E helix of the native protein, stabilizes the equilibrium molten globule and native states and leads to an increase in folding rate and a change in the folding pathway. Analysis of changes in chemical shift and in backbone flexibility, detected via [1H]-15N heteronuclear nuclear Overhauser effect measurements, indicates that the phenylalanine substitution has only minor effects on the conformational ensemble in the acid- and urea-unfolded states, but has a substantial effect on the structure, dynamics, and stability of the equilibrium molten globule intermediate formed near pH 4. In H64F apomyoglobin, additional regions of the polypeptide chain are recruited into the compact core of the molten globule. Since the phenylalanine substitution has negligible effect on the unfolded ensemble, its influence on folding rate and stability comes entirely from interactions within the compact folded or partly folded states. Replacement of His64 with Phe leads to favorable hydrophobic packing between the helix E region and the molten globule core and leads to stabilization of helix E secondary structure and overall thermodynamic stabilization of the molten globule. The secondary structure of the equilibrium molten globule parallels that of the burst phase kinetic intermediate; both intermediates contain significant helical structure in regions of the polypeptide that comprise the A, B, E, G, and H helices of the fully folded protein.     

Native protein sequences are designed to destabilize folding intermediates

Hydrophobic core mutants of sperm whale apomyoglobin were constructed to investigate the amino acid sequence features that determine the folding properties. Replacements of all of the Ile residues with Leu and of all of the Ile and Val residues with Leu decreased the thermodynamic stability of the folded states against the unfolded states but increased the stability of the folding intermediates against the unfolded states, indicating that the amino acid composition of the protein core is important for the protein stability and folding cooperativity. To examine the effect of the arrangement of these hydrophobic residues, mutant proteins were further constructed: 12 sites out of the 18 Leu, 9 Ile, and 8 Val residues of the wild-type myoglobin were randomly replaced with each other so that the amino acid compositions were similar to that of the wild-type protein. Four mutant proteins were obtained without selection of the protein properties. These residue replacements similarly resulted in the stabilization of both the intermediate and folded states against the unfolded states, as compared to the wild-type protein. Thus, the arrangements of the hydrophobic residues in the native amino acid sequence are selected to destabilize the folding intermediate rather than to stabilize the folded state. The present results suggest that the two-state transition of protein folding or the transient formation of the unstable intermediate, which seems to be required for effective production of the functional proteins, has been a major driving force in the molecular evolution of natural globular proteins.     

The plane of cleavage in human ferrihemoglobin. I. Ultraviolet difference spectroscopy.

X-ray diffraction studies have shown that hemoglobin has two predominant interfaces in the tetramer at which dissociation to dimers could occur. These interfaces have been designed as alpha1-beta1 and alpha1-beta2. There are 2 tyrosyl residues and 1 tryptophanyl residue in the alpha1-beta2- interface but only 1 tyrosyl residue in the alpha1-beta1 interface exposed to the solvent are perturbed. The ultraviolet difference spectrum between ferrihemoglobin dissociated in 1 M NaClO4 and undissociated hemoglobin revealed two negative peaks, one at 292.5 nm and another at 285 nm. This difference spectrum is due to tyrosyl and tryptophanyl residues which reside on the plane of cleavage and were exposed to 1 M NaClO4 upon dissociation. Hence, dissociation must have occurred along the alpha1-beta2 interface to yield alpha1 beta1 dimers. The deltaF degrees value extrapolated to zero salt concentration calculated on the basis of difference spectroscopy and sedimentation velocity experiments is 8.6 plus or minus 0.7 kcal per mol at pH 7.1 (K equals 4.5 times 10-7.     

How strong are side chain interactions in the folding intermediate?

Influence of 12 nonpolar amino acids residues from the hydrophobic core of apomyoglobin on stability of its native state and folding intermediate was studied. Six of the selected residues are from the A, G and H helices; these are conserved in structure of the globin family, although nonfunctional, that is, not involved in heme binding. The rest are nonconserved hydrophobic residues that belong to the B, C, D, and E helices. Each residue was substituted by alanine, and equilibrium pH‐induced transitions in apomyoglobin and its mutants were studied by circular dichroism and fluorescent spectroscopy. The obtained results allowed estimating changes in their free energy during formation of the intermediate state. It was first shown that the strength of side chain interactions in the apomyoglobin intermediate state amounts to 15–50% of that in its native state for conserved residues, and practically to 0% for nonconserved residues. These results allow a better understanding of interactions occurring in the intermediate state and shed light on involvement of certain residues in protein folding at different stages.     

Conformational changes in pantetheine hydrolase as a function of guanidinium chloride concentration

The denaturation of pantetheinase (pantetheine hydrolase, EC 3.5.1.-) was followed in guanidinium chloride using tyrosyl and tryptophanyl residues as probes in connection with change in enzymatic activity. Movements of tryptophanyl and tyrosyl residues during denaturation were studied by second-derivative and fluorescence spectroscopy and the number of these amino acids present in the protein was calculated from spectroscopic data. Pantetheinase shows a very high resistance to denaturation, being completely unfolded at guanidinium chloride concentration higher than 6.5 M. Monitoring enzymatic activity shows that inactivation of the enzyme occurred before noticeable conformational changes were detected and it is suggested that the conformation of the active site is flexible and easily perturbable compared to the protein as a whole. This inactivation is reversible, as shown by renaturation experiments. Second-derivative and fluorescence spectra showed also that tyrosyl and tryptophanyl residues are largely exposed in the native protein, confirming its hydrophobic behavior.