Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

The Conformational State of Apomyoglobin in the Presence of Phospholipid Vesicles at Neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Unfolding pathway of myoglobin: effect of denaturants on solvent accessibility to tyrosyl residues detected by second-derivative spectroscopy

The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.     

Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.     

The stacking tryptophan of galactose oxidase: a second-coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis

The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower in the W290 variants. The Km for D-galactose for W290H is similar to that of the wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in the W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes are also closely similar to the wild-type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

Evidence of conformational switch in Streptococcus pneumoniae FtsZ during polymerization

FtsZ, the master coordinator of bacterial cell division, assembles into filaments in the presence of nucleotide. FtsZ from Streptococcus pneumoniae bears two tryptophan residues (W294 and W378) in its amino acid sequence. The tryptophan fluorescence of FtsZ increases during the assembly of FtsZ. We hypothesized that this increase in the fluorescence intensity was due to the change in the environment of one or both tryptophan residues. To examine this, we constructed two mutants (W294F and W378F) of FtsZ by individually replacing tryptophan with phenylalanine. The mutants displayed similar secondary structures, GTPase activity, and polymerization ability as the wild type FtsZ. During the polymerization, only one tryptophan (W294) showed an increase in its fluorescence intensity. Using time‐correlated single‐photon counting, the fluorescence lifetime of W294 was found to be significantly higher than W378, indicating that W294 was more buried in the structure than W378. The lifetime of W294 further increased during polymer formation, while that of W378 remained unchanged. Fluorescence quenching experiment suggested that the solvent exposure of W294 reduced during the polymerization of FtsZ. W294 is located near the T‐7 loop of the protein, a region important for the monomer‐monomer interaction during the formation of a protofilament. The results indicated that the region around W294 of S. pneumoniae FtsZ undergoes a conformational switch during polymerization as seen for FtsZ from other bacteria.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Pressure effect on the conformational fluctuation of apomyoglobin in the native state

We have investigated the effect of pressure on fluctuations of the native state of sperm whale apomyoglobin (apoMb) by H/D exchange, fluorescence, and limited proteolysis. The results from intrinsic fluorescence showed that a large fraction of apoMb molecules is in the native conformation in the pressure range from 0.1 to 150 MPa at 293 K and pH 6.0. The H/D exchange of protons of the individual backbone amino acids in this pressure range was monitored by NMR. The rate of H/D exchange was enhanced at high pressure, with the protection factors for some residues decreasing by factors of more than 100 compared to the values at 0.1 MPa. The amplitude of the decrease of the protection factor varied among the individual amino acids on the same secondary structure unit. This result suggests that H/D exchange in apoMb is explained best by the penetration model, in which solvent penetrates into the protein matrix via small motions. The result from limited proteolysis under high pressure showed that a pressure increase does not induce local unfolding of the secondary structure units of apoMb. Conformational fluctuations much smaller than local unfolding evidently provide pathways for water to diffuse into the protein interior, and are enhanced by an increase of pressure.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

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.     

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.     

High-resolution study of the three-dimensional structure of horse heart metmyoglobin

The three-dimensional structure of horse heart metmyoglobin has been refined to a final R-factor of 15.5% for all observed data in the 6.0 to 1.9 A resolution range. The final model consists of 1242 non-hydrogen protein atoms, 154 water molecules and one sulfate ion. This structure has nearly ideal bonding and bond angle geometry. A Luzzati plot of the variation in R-factor with resolution yields an estimated mean co-ordinate error of 0.18 A. An extensive analysis of the pattern of hydrogen bonds formed in horse heart metmyoglobin has been completed. Over 80% of the polypeptide chain is involved in eight helical segments, of which seven are composed mainly of alpha-helical (3.6(13))-type hydrogen bonds; the remaining helix is composed entirely of 3(10) hydrogen bonds. Altogether, of 102 hydrogen bonds between main-chain atoms only six are not involved in helical structures, and four of these six occur within beta-turns. The majority of water molecules in horse heart metmyoglobin are found in solvent networks that range in size from two to 35 members. The size of water molecule networks can be rationalized on the basis of three factors: the number of hydrogen bonds to the protein surface, the presence of charged side-chain atoms, and the ability to bridge to neighboring molecules in the crystal lattice. Bridging water networks form the dominant intermolecular interactions. The backbone conformation of horse heart metmyoglobin is very similar to sperm whale metmyoglobin, with significant differences in secondary structure occurring only near residues 119 and 120, where residues 120 to 123 in sperm whale form a distorted type I reverse turn and the horse heart protein has a type II turn at residues 119 to 122. Nearly all of the hydrogen bonds between main-chain atoms (occurring mainly in helical regions) are common to both proteins, and more than half of the hydrogen bonds involving side-chain atoms observed in horse heart are also found in sperm whale metmyoglobin. Unlike sperm whale metmyoglobin, the heme iron atom in horse heart metmyoglobin is not significantly displaced from the plane of the heme group.     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Lipid and signal peptide-induced conformational changes within the C-domain of Escherichia coli SecA protein

SecA ATPase is an essential component of the Sec-dependent protein translocation machinery. Upon interaction with the plasma membrane containing SecYE, preprotein, and ATP, SecA undergoes cycles of membrane insertion and retraction resulting in the translocation of segments of the preprotein to the trans side of the membrane. To study the structural basis of SecA function, we employed fluorescence spectroscopy along with collisional quenchers with a set of SecA proteins containing single tryptophan substitutions. Our data show that among the seven naturally occurring tryptophan residues of Escherichia coli SecA, only the three tryptophan residues contained within the C-domain contributed significantly to the fluorescence signal, and they occupied distinct local environments in solution: Trp723 and Trp775 were found to be relatively solvent accessible and inaccessible, respectively, while Trp701 displayed an intermediate level of solvent exposure. Exposure to increased temperature or interaction with model membranes or signal peptide elicited a similar conformational response from SecA based upon the fluorescence signals of the SecA-W775F and SecA-W723F mutant proteins. Specifically, Trp775 became more solvent exposed, while Trp723 became less solvent accessible under these conditions, indicating similarities in the overall conformational change of the C-domain promoted by temperature or translocation ligands. Only Trp701 did not respond in parallel to the different conditions, since its solvent accessibility changed only in the presence of signal peptide. These results provide the first detailed structural information about the C-domain of SecA and its response to translocation ligands, and they provide insight into the conformational changes within SecA that drive protein translocation.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.     

Steady-state and time-resolved fluorescence studies on wild type and mutant chromatium vinosum high potential iron proteins: holo- and apo-forms

Detailed circular dichroism (CD), steady-state and time-resolved tryptophan fluorescence studies on the holo- and apo- forms of high potential iron protein (HiPIP) from Chromatium vinosum and its mutant protein have been carried out to investigate conformational properties of the protein. CD studies showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster does not have any effect on formation of secondary structure in the protein. Steady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe(4)S(4)](3+)) cluster from the protein leads to an increase in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. CD studies on the holo- and apo- HiPIP also showed that removal of the metal prosthetic group drastically affects the tertiary structure of the protein. Time-resolved fluorescence decay of the wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fluorescence decay was also analyzed by maximum entropy method (MEM). The results of the MEM analysis agreed with those obtained from discrete exponentials model analysis. Studies on the wild type and mutants helped to assign the fast picosecond lifetime component to the W80 residue, which exhibits fast fluorescence energy transfer to the [Fe(4)S(4)](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calculated from the time-resolved fluorescence results at different emission wavelengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solvent.     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

Fibrillogenesis of apomyoglobin facilitated by aggregation sequence of yeast Sup35 in various regions

To examine the effect of aggregation sequence QGGYQQQYNP from yeast Sup35 on fibril formation of sperm whale apomyoglobin (apoMb), we constructed several mutants via substitution. Urea-induced unfolding of apoMb confirms that the substitution of the aggregation sequence does not significantly affect the stability of the mutants compared to wild type (WT) at pH 4.2. Under this condition, however, despite the difference in rate most apoMb mutants form fibrils more readily than WT with distinct morphology. These results suggest that the aggregation sequence facilitates fibril assembly of apoMb at acidic pH in vitro and this facilitation depends on the regions replaced.