Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure-the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS-protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe-protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS-apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS-apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U(A). At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS-apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S(1,CT) of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S(1,np) of ANS stabilized by the strong ANS-apomyoglobin interaction. Lowering the pH diminishes the contribution from the S(1,np) state. Meanwhile, more protein molecules become populated at the U(A) state, which exhibits a short lifetime that is not distinguishable from the S(1,np) state. At pH 3.4, when the population of the U(A) becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U(A). Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U(A) below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.     

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.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Photothermal studies of pH induced unfolding of apomyoglobin

Conformational dynamic and enthalpy changes associated with pH induced unfolding of apomyoglobin were studied using photoacoustic calorimetry and photothermal beam deflection methods. The transition between the native state and the I intermediate was induced by a nanosecond pH jump from o-nitrobenzaldehyde photolysis. Deconvolution of photoacoustic waves indicates two kinetic processes. The fast phase ( < 50ns) is characterized by a volume expansion of 8.8 ml mol^–1. This process is followed by a volume contraction of about –22 ml mol^–1 ( 500 ns). Photothermal beam deflection measurements do not reveal any volume changes on the time scale between 100 s and 5 ms. We associate the volume contraction with structural changes occurring during the transition between the native state and the I intermediate. The lack of any processes on the ms time scale may indicate the absence of structural events involving larger conformational changes of apomyoglobin after the pH jump.     

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     

Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding.

Structural characterization of protein unfolding intermediates [Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318], which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent [5, 5'-dithiobis (2-nitrobenzoic acid)] with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.     

Structural details,pathways, and energetics of unfolding apomyoglobin

Protein folding is often difficult to characterize experimentally because of the transience of intermediate states, and the complexity of the protein-solvent system. Atomistic simulations, which could provide more detailed information, have had to employ highly simplified models or high temperatures, to cope with the long time scales of unfolding; direct simulation of folding is even more problematic. We report a fully atomistic simulation of the acid-induced unfolding of apomyoglobin in which the protonation of acidic side-chains to simulate low pH is sufficient to induce unfolding at room temperature with no added biasing forces or other unusual conditions; and the trajectory is validated by comparison to experimental characterization of intermediate states. Novel insights provided by their analysis include: characterization of a dry swollen globule state forming a barrier to initial unfolding or final folding; observation of cooperativity in secondary and tertiary structure formation and its explanation in terms of dielectric environments; and structural details of the intermediate and the completely unfolded states. These insights involve time scales and levels of structural detail that are presently beyond the range of experiment, but come within reach through the simulation methods described here. An implicit solvation model is used to analyze the energetics of protein folding at various pH and ionic strength values, and a reasonable estimate of folding free energy is obtained. Electrostatic interactions are found to disfavor folding.     

Ultraviolet resonance Raman examination of horse apomyoglobin acid unfolding intermediates.

We have used UV resonance Raman spectroscopy to study the acid-induced denaturation of horse apomyoglobin (apoMb) between pH 7. 0 and 1.8. The 206.5 nm excited Raman spectra are dominated by amide vibrations, which are used to quantitatively determine the apoMb secondary structure. The 229 nm excited Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes of Tyr and Trp environments and solvent exposures. We observe two partially unfolded apoMb intermediates at pH 4 and pH 2, while we observe only one partially unfolded holoMb intermediate at 2, in which the G and H helices are mainly intact, while the rest of protein is unfolded. This partially unfolded holoMb intermediate at pH 2 is essentially identical to the pH 2 apoMb intermediate. The partially unfolded pH 4 apoMb intermediate is composed of the three folded A, G, and H helices and contains 38% helical structure. The changes in the Trp Raman cross sections during the acid-induced denaturation indicates that Trp 7 is likely to be fully exposed in the apoMb pH 4 intermediate and that the A helix melts with a pKa approximately 3.5.     

Similarity of force-induced unfolding of apomyoglobin to its chemical-induced unfolding: an atomistic molecular dynamics simulation approach

We have compared force-induced unfolding with traditional unfolding methods using apomyoglobin as a model protein. Using molecular dynamics simulation, we have investigated the structural stability as a function of the degree of mechanical perturbation. Both anisotropic perturbation by stretching two terminal atoms and isotropic perturbation by increasing the radius of gyration of the protein show the same key event of force-induced unfolding. Our primary results show that the native structure of apomyoglobin becomes destabilized against the mechanical perturbation as soon as the interhelical packing between the G and H helices is broken, suggesting that our simulation results share a common feature with the experimental observation that the interhelical contact is more important for the folding of apomyoglobin than the stability of individual helices. This finding is further confirmed by simulating both helix destabilizing and interhelical packing destabilizing mutants.     

Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.     

Viscosity dependence of the solute quenching of the tryptophanyl fluorescence of proteins

We have studied the viscosity dependence of the acrylamide quenching of the fluorescence on the internal tryptophan residues in cod parvalbumin and ribonuclease T1, as well as the model systems. N-acetyl-L-tryptophanamide and glucagon. For the latter systems, the apparent rate constant, kq(app), for acrylamide quenching shows a typical diffusion-limited behavior. For parvalbumin and ribonuclease T1, however, the viscosity dependence of kq(app) is quite different. There is little change in the kq(app) values on increasing the bulk viscosity from 1 to 10 cP (by addition of glycerol), but a further increase from 10 to 100 cP results in a significant reduction in the kq(app). Both an unfolding mechanism and a quencher penetration mechanism are considered to explain the results. Only the penetration mechanism is found to be consistent, and our data are interpreted as indicating that the rate-limiting step for quenching goes from being that of diffusion through the protein matrix, at low viscosity, to diffusion through the bulk solvent, at high viscosity. By also considering the Kramers' relationship in fitting our data, we are able to obtain insight regarding the coupling between internal fluctuations in the structure of the protein and motion of the bulk solvent. For parvalbumin and ribonuclease T1, the internal dynamics are found to be very weakly coupled to the bulk.     

Effect of halide anions on the binding of FAD to D-amino acid oxidase and the tryptophanyl fluorescence of the apoenzyme.

The quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25 degrees C. All of the tryptophanyl fluorescence of the apoenzyme in 6 M guanidine hydrochloride was quenched. The tryptophanyl fluorescence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism. By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution containing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.     

Early events in apomyoglobin unfolding probed by laser T-jump/UV resonance Raman spectroscopy

Early events in the unfolding of apomyoglobin are studied with time-resolved ultraviolet resonance Raman (UVRR) spectroscopy coupled to a laser-induced temperature jump (T-jump). The UVRR spectra provide simultaneous probes of the aromatic side-chain environment and the amide backbone conformation. The amide bands reveal helix melting, with relaxation times of 70 and 16 micros at pH 5.5 and 4, respectively, in reasonable agreement with previously reported amide I' FTIR/T-jump relaxations (132 and 14 micros at pD 5.5 and 3). The acceleration at pH 4 is consistent with destabilization of the hydrophobic AGH core of the protein via protonation of a pair of buried histidines. The same relaxation times are found for intensity loss by the phenylalanine F12 band, signaling solvent exposure of the phenyl rings. There are seven Phe residues, distributed throughout the protein; they produce a global response, parallel to helix melting. Relaxation of the tryptophan W16 intensity also parallels helix melting at pH 5.5 but is twice as fast, 7 micros, at pH 4. The pH 5.5 signal arises from Trp 7, which is partially solvent-exposed, while the pH 4 signal arises from the buried Trp 14. Thus, Trp 14 is exposed to the solvent prior to helix melting of the AGH core, suggesting initial displacement of the A helix, upon which Trp 14 resides. All of the UVRR signals show a prompt response, within the instrument resolution (approximately 60 ns), which accounts for half of the total relaxation amplitude. This response is attributed to solvent penetration into the protein, possibly convoluted with melting of hydrated helix segments.     

Effect of cations on the quantum yield and the lifetime of the chloroplast fluorescence]

The interrelationship between the cation-induced fluorescence changes and the state of the photosystem 2 (PS-2) reaction centers for pea chloroplasts and their osmotic fragments was studied. The effects of K+ and Mg2+ on the fluorescence quantum yield (phi f1) under varying light intensities as well as on the fluorescence lifetime (tau f1) in the saturating light were demonstrated. K+ induces the decrease in tau f1; Mg2+ exerts an opposite effect. The effects were more pronounced when the reaction centers of PS-2 were converted into an inactive state by illuminating the sample with a saturating light or by adding DCMU. Under these conditions the cations' effect on tau f1 was accompanied by proportional changes in tau f1. It was concluded that in Mg-deficient chloroplasts an efficient channel of the excitation quenching appears in antenna chlorophyll of PS-2 with the rate constant of 7 . 10(8) s-1. The simultaneous measurements of tau f1 by phase and modulation type techniques allowed to reveal the emission heterogeneity within the nanosecond time interval and the DCMU-sensitive delayed fluorescence with the lifetime exceeding 10(-7) s and the overall quantum yield approximately equal to 2 . 10(-3).     

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.     

Diffusive motions control the folding and unfolding kinetics of the apomyoglobin pH 4 molten globule intermediate

The sperm whale apomyoglobin pH 4 folding intermediate exists in two forms, Ia and Ib, that mimic transient kinetic intermediates in the folding of the native protein at pH 6. To characterize the nature of the kinetic barrier that controls the formation of the earliest intermediate Ia, we have investigated the effects of small viscogenic cosolvents on its folding and unfolding kinetics. The kinetics are measurable by stopped-flow fluorescence and follow a cooperative two-state model in the absence and presence of cosolvents. Small cosolvents stabilize Ia, but, by applying the isostability test to separate the viscogenic effect of the cosolvent from its stabilizing effect, we found that, in both folding and unfolding conditions, the apparent rate constant decreases when solvent viscosity increases. The unitary inverse dependence of the apparent rate constant on solvent viscosity indicates a diffusion-controlled reaction. This result is consistent with the hypothesis that folding of the apomyoglobin pH 4 intermediate obeys a diffusion-collision model. Additionally, the temperature dependence of the reaction rate at constant viscosity indicates that the formation of Ia is also controlled by an energy barrier. Linear free energy relationships show that the transition state of the U <==> Ia reaction is compact and buries 45% of the surface area that is buried in native apomyoglobin. We conclude that the transition state of the U <==> Ia reaction resembles that for the formation of native proteins; namely, it is dry and its compactness is closer to that of the folded (Ia) form than of the unfolded form.