High-pressure denaturation of apomyoglobin

The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.     

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.     

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.     

Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue

Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.     

Consequences of stabilizing the natively disordered f helix for the folding pathway of apomyoglobin

The F helix region of sperm whale apomyoglobin is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. To examine the effects of F helix stabilization on the folding pathway of apomyoglobin, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared: a double mutant, P88K/S92K (F2), and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst-phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate.     

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.     

Equilibrium unfolding of mutant apomyoglobins carrying substitutions of conserved nonfunctional residues with alanine

Protein aggregation or misfolding in the cell is connected with many genetic diseases and can result from substitutions in proteins. Substitutions can influence the protein stability and folding rates in both intermediate and native states. The equilibrium urea-induced unfolding was studied for mutant apomyoglobins carrying substitutions of the conserved nonfunctional residues Val10, Trp14, Ile111, Leu115, Met131, and Leu135 with Ala. Conformational transitions were monitored by intrinsic Trp fluorescence and far-UV circular dichroism. Free energy changes upon transition from the native to the intermediate state and from the intermediate to the unfolded state were determined. All substitutions considerably decreased the stability of native apomyoglobin, whereas the effect on the stability of the intermediate state was essentially smaller.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

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     

Equilibrium unfolding of mutant apomyoglobins with substitutions of conserved nonfunctional residues by alanine

The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.     

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the ?-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I→N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I→N transition.     

Effect of Substitutions in Surface Amino Acid on Energy Profile of Apomyoglobin

Studies on the process of spontaneous protein folding into a unique native state are an important issue of molecular biology. Apomyoglobin from the sperm whale is a convenient model for these studies in vitro. Here, we present the results of equilibrium and kinetic experiments carried out in a study on the folding and unfolding of eight mutant apomyoglobin forms of with hydrophobic amino acid substitutions on the protein surface. Calculated values of apparent constants of folding/unfolding rates, as well as the data on equilibrium conformational transitions in the urea concentration range of 0–6 M at 11°C are given. Based on the obtained information on the kinetic properties of the studied proteins, a Φ-value analysis of the transition state has been performed and values of urea concentrations corresponding to the midpoint of the transition from the native to intermediate state have been determined for the given forms of mutant apomyoglobin. It has been found that a significant increase in the stability of the native state can be achieved by a small number of amino acid substitutions on the protein surface. It has been shown that the substitution of only one amino acid residue exclusively affects the height of the energy barrier that separates different states of apomyoglobin.     

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.     

Use of site-directed mutagenesis to destabilize native apomyoglobin relative to folding intermediates

Site-directed mutagenesis has been used to study the effect on the stability of human apomyoglobin (apoMb) of modifying the size, hydrophobicity, and charge of a central residue in the G.B helix-helix packing interface. Some stability measurements have also been made on the corresponding holomyoglobins (heme present). Cys-110, a central helix pairing residue in the G helix, has been changed to Ala, Ser, Asp, and Leu. Stability to low-pH-induced unfolding has been measured for both native apoMb and the compact folding intermediate discovered by Griko et al. [Griko, Y. V., Privalov, P. L., Venyaminov, S. Y., & Kutyshenko, V. P. (1988) J. Mol. Biol. 202, 127-138]. As judged by its circular dichroism spectrum, this intermediate has a substantial helix content (about 35%). Whether or not this inferred helical structure is closely related to the myoglobin structure is not yet known. The mutational evidence shows that integrity of G.B helix pairing is important for the stability of apoMb as well as of myoglobin and that this helix pairing site is very sensitive to both steric and electrostatic disruption. Our results also suggest that G.B helix pairing does not stabilize the compact intermediate; hence, disrupting this site destabilizes the native protein relative to the compact intermediate. Such selective destabilization of the native state relative to equilibrium folding intermediates is not restricted to acid denaturation: urea denaturation of the Leu mutant appears to display at least one stable intermediate, while wild-type and the remaining mutant apoMbs undergo two-state urea unfolding transitions.     

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.     

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.     

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.     

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.     

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent ΔG°_UN(w) = − 3.1 ± 0.3 kcal mol^− 1; m_UN = − 1.7 kcal mol^− 1 M^− 1] and predominant α-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (β_I = 0.9 ± 0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.