Solvent accessibility of the heme pocket in tuna myoglobin

The accessibility of the heme binding site of two apomyoglobins, i.e. tuna and sperm whale apomyoglobin, has been evaluated by quenching the fluorescence of their ANS-conjugates. The quenching pattern obtained by using charged and uncharged quenchers revealed that the heme pocket of tuna apomyoglobin is more accessible than that of sperm whale. Moreover, a larger number of positively charged groups is present in the heme pocket of tuna apomyoglobin as indicated by comparing the extent of quenching produced by iodide and cesium ion. The relaxation time of ANS bound to tuna apomyoglobin is lower than that of the same chromophore bound to sperm whale globin thus indicating that there is some localized flexibility in the tuna globin.     

Dynamic fluorescence of extrinsic fluorophores as a tool for studying protein conformational substates

Summary The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.     

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.     

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.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

Proof of nanosecond timescale relaxation in apomyoglobin by phase fluorometry

Apomyoglobin was labeled with the fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS). Apparent phase shift and demodulation lifetimes of bound TNS were measured at various emission wavelengths. The lifetimes increased with increasing wavelength. Similar results were obtained for TNS in the viscous solvent glycerol at 10°C but not for TNS in vitrified or fluid solvent. The wavelength-dependent lifetimes suggest apomyoglobin is relaxing around the TNS molecule during its fluorescent lifetime. Importantly, the apparent phase lifetimes exceeded the apparent modulation lifetimes on the long wavelength side of the emission for TNS in apomyoglobin at 3°C and for TNS in glycerol at 10°C. This result proves the increasing lifetimes are a result of an excited state reaction during the lifetime of the excited state and are not a result of heterogeneity in the fluorescence emission. From the lifetimes on the short wavelength side of the emission the relaxation time of apomyoglobin was estimated to be 18 nsec.     

The effect of molecular confinement on the conformational dynamics of the native and partly folded state of apomyoglobin.

Inclusion in agarose gel significantly affects the conformational dynamics of native and acidic partly folded states of tuna apomyoglobin, a single tryptophan containing protein, as documented by frequency domain fluorometry investigations. The heterogeneity of the tryptophanyl emission decay increases on gel inclusion compared to that observed for free-in-solvent protein at both neutral and acidic pH, thus suggesting that the interconversion rate among conformational substates is somewhat reduced. The observation that this effect is much more pronounced for the partly folded state suggests that confined environments such as those existing in the living cells might favor the sequential folding process avoiding that structured intermediates rapidly convert into less structured ones.     

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.     

Pressure-induced perturbation of ANS-apomyoglobin complex: frequency domain fluorescence studies on native and acidic compact states.

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.     

Intermediate states of apomyoglobin: Are they parts of the same area of conformations diagram?

Several research teams have reported detection and characterization of various apomyoglobin intermediate states different in their accumulation mode, thus putting a natural question as to proportions of these intermediates. The current report presents spectral properties of sperm whale apomyoglobin studied over a wide range of conditions with the use of circular dichroism and fluorescence techniques. Based on the experimental data, a diagram of apomyoglobin conformational states has been constructed. It shows that though induced by various denaturants, all the observed intermediates belong to one and the same area in the diagram.     

Molecular dynamics simulation of the acidic compact state of apomyoglobin from yellowfin tuna

A molecular model of the acidic compact state of apomyoglobin (A-state) from yellowfin tuna was obtained using molecular dynamics simulations (MD) by calculating multiple trajectories. To cause partial unfolding within a reasonable amount of CPU time, both an acidic environment (pH 3 and 0.15M NaCl) and a temperature jump to 500 K were needed. Twenty-five acidic structures of apomyoglobin were generated by MD, 10 of them can be clustered by RMSD in an average structure having a common hydrophobic core as was reported for acidic sperm whale apomyoglobin, with shortened helices A,G,E, and H (the helix A appears to be translated along the sequence). Prolonging the MD runs at 500 K did not cause further substantial unfolding, suggesting that the ensemble of generated structures is indicative of a region of the conformational space accessible to the apoprotein at acidic pH corresponding to a local energy minimum. The comparison of experimentally determined values of specific spectroscopic properties of the apomyoglobin in acidic salt conditions with the expected ones on the basis of the MD generated structures shows a reasonable agreement considering the characteristic uncertainties of both experimental and simulation techniques. We used frequency domain fluorometry, acrylamide fluorescence quenching, and fluorescence correlation spectroscopy together with far UV circular dichroism to estimate the helical content, the Stern-Volmer quenching constant and the radius of gyration of the protein. Tuna apomyoglobin is a single tryptophan protein and thus, interpretation of its intrinsic fluorescence is simpler than for other proteins. The high sensitivity of the applied fluorescence techniques enabled experiments to be performed under very dilute conditions, that is, at concentrations of subnanomolar for the FCS measurements and 6 muM for the other fluorescence measurements. As high concentrations of proteins can strongly affect the association equilibrium among partially unfolded states, fluorescence techniques can provide complementary information with respect to other techniques requiring higher sample concentrations, such as NMR. The analysis of exposed hydrophobic regions in each of the MD-generated acidic structures reveals potential candidates involved in the aggregation processes of apomyoglobin in the acidic compact state. Our investigation represents an effective model system for studying amyloid fibril formation found in important diseases that are believed to proceed via aggregation of protein in the molten globule state.     

Probing Substates in Sperm Whale Myoglobin Using High-Pressure Crystallography

Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.     

The stabilities of mammalian apomyoglobins vary over a 600-fold range and can be enhanced by comparative mutagenesis

Apomyoglobins from 13 different mammals were examined for resistance to denaturation by guanidinium chloride. Unfolding was followed by circular dichroism and tryptophan fluorescence and analyzed globally using the two-step, three-state mechanism first described by Barrick and Baldwin (Barrick, D., and Baldwin, R. L. (1993) Biochemistry 32, 3790-3796). With one exception, the rise and fall of Trp fluorescence intensity correlates quantitatively with the native to intermediate to unfolded steps seen in the CD curves. Although the O(2) binding properties of the holoproteins are nearly identical, the unfolding transitions of the apomyoglobins show 600-fold differences in resistance to guanidinium chloride denaturation. Apomyoglobins from diving mammals, particularly from sperm whales, are the most stable, whereas the apoproteins from pig, horse, and sheep are the least stable, indicating selective pressure for resistance to denaturation in the whale proteins. Sequence comparisons suggest that the key stabilizing residues in whale globins are Ala(5), His(12), Ile(28), Thr(51), Ala(53), Ala(74), Lys(87), Lys(140), and Ile(142). Combinations of these residues were substituted into pig myoglobin. The resultant multiple mutants showed stabilities approaching that of recombinant sperm whale apomyoglobin. Thus, comparative mutagenesis can be used to increase heme protein stability and improve expression yields in bacteria without compromising function.     

Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins

In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common intermediate in the folding of all members of the globin family, we have extensively studied a site-directed mutant of the myoglobin from Aplysia limacina, distantly related to the mammalian counterpart, in which one of the two tryptophans in the A-G-H cluster [i.e., Trp(H8)130] has been mutated to tyrosine. The results presented here show that this mutation destabilizes both the native state and the acid intermediate I(A) but exerts little or no effect on the thermally stable core of an intermediate species (called I(T)) peculiar to Aplysia apomyoglobin. Dynamic quenching of Trp emission by acrylamide provides information on the accessibility of the chromophores at the native and the intermediate states of wild-type and mutant Aplysia apomyoglobin, consistent with the thermodynamics. Our results agree well with those obtained for the corresponding topological position of apomyoglobin from sperm whale and clearly show that the H8 position is involved in the stabilization of the main intermediate in both apoproteins. This residue thus plays a role which is evolutionarily conserved in the globin family from invertebrates to mammals; our results support the contention that the A-G-H cluster is important in the folding pathway of different globins.     

Structural characterization of non-native states of sperm whale myoglobin in aqueous ethanol or 2,2,2-trifluoroethanol media

The effects of aqueous ethanol or 2,2,2-trifluoroethanol media on the structure of sperm whale myoglobin have been investigated by absorption, CD, and NMR spectra. The structural properties of myoglobin such as heme environments, helix contents, protein folding, and interactions between heme and the protein moiety have been sharply manifested in these spectra. The characterization demonstrated that alcohol-induced conformational change of myoglobin depends on the nature of alcohol and its concentration. It was shown for the first time that, upon the alcohol-induced denaturation of myoglobin, heme is released from partially denatured protein of which helix contents is altered by only about 20% relative to that of native state. Myoglobin has shown to unfold and refold reversibly by controlling the alcohol concentration. Novel methods for the preparation of apomyoglobin and in situ reconstitution of apomyoglobin with heme, based on the alcohol-induced denaturation of the protein, were presented.     

Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry

We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational correlation times of TNS bound to apomyoglobin are consistent with those expected for this probe rigidly bound to the protein. TNS in propylene glycol also displayed a single exponential decay of anisotropy. These results, in conjunction with the previous successful resolution of multiexponential decays of fluorescence intensity (Lakowicz, J. R., Gratton, E., Laczko, G., Cherek, H., and Limkeman, M. (1984) Biophys. J., in press; Gratton, E., Lakowicz, J. R., Maliwal, B. P., Cherek, H., Laczko, G., and Limkeman, M. (1984) Biophys. J., in press) demonstrate that frequency-domain measurements provide information which is, at a minimum, equivalent to that obtainable from time-domain measurements.