Unfolding pathway of myoglobin: Molecular properties of intermediate forms (original) (raw)
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Complexities in the denaturation of horse metmyoglobin by guanidine hydrochloride
Journal of Biological Chemistry, 1985
The denaturation of horse metmyoglobin by guanidine hydrochloride was studied at pH 6.4 and 25 OC. Measurements of both the peptide circular dichroism and the absorbance in the Soret region suggest that the extent of renaturation strongly depends on the time interval during which the protein is exposed to concentrated solutions of the denaturant. From the equilibrium measurements of the absorption in the Soret region, it is concluded that the unfolding of metmyoglobin is complex. This is further supported by kinetic studies of denaturation which suggest the occurrence of the least four species in the reaction.
Stability of Myoglobin: A Model for the Folding of Heme Proteins
Biochemistry, 1994
Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E1 l ) , and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein) -I(intermediate) -U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both
Determining globular protein stability: guanidine hydrochloride denaturation of myoglobin
Biochemistry, 1979
The guanidine hydrochloride (Gdn-HCI) denaturation of horse myoglobin has been investigated a t several pH values using absorbancy measurements at 409 nm. From these data the free energy of denaturation, AGD, can be calculated and AGD values have been measured to zero concentrations of denaturant. The dependence of AGD on Gdn-HC1 concentration, d(AGD)/d(Gdn.HC1), increases markedly as the denaturant concentration decreases. This indicates that an increase in the number of Gdn-HCI binding sites on unfolding is the major driving force for denaturation by Gdn.HCI. An equation based on denaturant binding which fits the experimental data for myoglobin a t p H 7 is AGD = AGDHIo-A n R T In (1 + ka,), where AGDHzo (= 10.1 G u a n i d i n e hydrochloride (Gdn.HC1),l urea, and sodium dodecyl sulfate are the three most popular protein denaturants. Gdn-HC1 and urea produce a randomly coiled denatured state (Tanford, 1968) which, for many purposes, is a more useful denatured state than the helical, rod-like denatured state (Reynolds & Tanford, 1970) produced by sodium dodecyl sulfate. The use of Gdn.HC1 as a denaturant has increased substantially over the past I O years. Gdn-HC1 is a more potent denaturant than urea, unfolding proteins at two to three times lower concentrations (Greene & Pace, 1974), and Gdn.HC1 is chemically stable, while urea slowly decomposes to form cyanate and ammonia. One of the interesting results from a study of the Gdn.HC1 denaturation of a globular protein is an estimate of the stability of the protein; i.e., it is possible to estimate the free energy change, AGD, for the reaction, globular conformation randomly coiled conformation, in the absence of denaturant, AGDH2'. This is done by measuring AGD as a function of Gdn.HC1 concentration and extrapolating to zero concentration. Several different approaches have been used for making this extrapolation, but, unfortunately, they lead to estimates of ACDH20 which differ by as much as 20-50??.
Probing the conformational state of apomyoglobin by limited proteolysis 1 1 Edited by P. E. Wright
Journal of Molecular Biology, 1997
We show here that limited proteolysis can probe the structural and dynamic differences between the holo and apo form of horse myoglobin (Mb). Initial nicking of the polypeptide chain of apoMb (153 amino acid residues, no disul®de bonds) by several proteases (subtilisin, thermolysin, chymotrypsin and trypsin) occurs at the level of chain segment 89-96. In contrast, holoMb is resistant to proteolytic digestion when reacted under identical experimental conditions. Such selective proteolysis implies that the F-helix of native holoMb (residues 82 to 97) is disordered in apoMb, thus enabling binding and adaptation of this chain segment at the active site of the proteolytic enzymes for an ef®cient peptide bond ®ssion. That essentially only the F-helix in apoMb is largely disrupted was earlier inferred from spectroscopic measurements and molecular dynamics simulations. The results of this study provide direct experimental evidence for this and emphasize therefore that limited proteolysis is a useful and reliable method for probing structure and dynamics of proteins, complementing other experimental techniques such as NMR and X-ray crystallography.
Changes in the Apomyoglobin Folding Pathway Caused by Mutation of the Distal Histidine Residue
Biochemistry, 2000
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.
Biochemistry, 2005
A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.0 and pH 7.8 toward glutamic acid (Glu), was used as a proteolytic enzyme. Using this method enabled us to compare the proteolytic cleavages of native apoMb (at pH 8.0) and molten globule (at pH 4.2) directly. Only the first cleavage event in each molecule was considered as reflecting original structural information since the original structure of the protein can be altered after the fist cleavage. Structural changes of apoMb in various pH conditions were studied here to elucidate the local helicity of molten globule apoMb. Among 13 Glu sites, only Glu83 and Glu85 in the F-helix were cleaved at pH 8.0, which confirms that only helix F is frayed upon removal of heme group. At acidic molten globule state, rapid cleavages at Glu38, Glu52, Glu54, Glu85, and Glu148 were detected, while the remaining eight sites were protected. Glu6 and Glu18 in the A-helix, and Glu105 in the G-helix were protected due to the helicity of the secondary structures. The cleavage at Glu38 and the protection at Glu41 in the C-helix indicate that the first half of the C-helix is frayed and the second half of the C-helix is structured. Cleavage at both Glu52 and Glu54 in the D-helix proves that the D-helix is disordered. The N-terminal end of the E-helix at Glu59 was protected, and the beginning of the F-helix was protected by aid of the pH-induced C-cap of the E-helix. The cleavage at Glu148 in H suggests that the C-terminal end of the H-helix is disordered. The A-helix and the first half of the B-helix were highly stable.
Molecular Biology, 2009
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.
Denaturation properties and folding transition States of leghemoglobin and other heme proteins
Biochemistry. Biokhimii͡a, 2015
This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant ...
Probing the conformational state of apomyoglobin by limited proteolysis
1997
We show here that limited proteolysis can probe the structural and dynamic differences between the holo and apo form of horse myoglobin (Mb). Initial nicking of the polypeptide chain of apoMb (153 amino acid residues, no disul®de bonds) by several proteases (subtilisin, thermolysin, chymotrypsin and trypsin) occurs at the level of chain segment 89-96. In contrast, holoMb is resistant to proteolytic digestion when reacted under identical experimental conditions. Such selective proteolysis implies that the F-helix of native holoMb (residues 82 to 97) is disordered in apoMb, thus enabling binding and adaptation of this chain segment at the active site of the proteolytic enzymes for an ef®cient peptide bond ®ssion. That essentially only the F-helix in apoMb is largely disrupted was earlier inferred from spectroscopic measurements and molecular dynamics simulations. The results of this study provide direct experimental evidence for this and emphasize therefore that limited proteolysis is a useful and reliable method for probing structure and dynamics of proteins, complementing other experimental techniques such as NMR and X-ray crystallography.
Biochemistry, 2005
We studied the effect of deleted and circularly permuted mutations in sperm whale myoglobin and present here results on three classes of mutants: (i) a deletion mutant, Mb 1-99 , in which the C-terminal helices, G and H, were removed; (ii) two circular permutations, Mb-B_GHA, in which helix B is N-terminal and helix A is C-terminal, and Mb-C_GHAB, in which helix C is N-terminal and helices A and B are C-terminal; and (iii) a deleted circular permutation, Mb-HAB_F, in which helix H is N-terminal, helix F is C-terminal, and helix G is deleted. The conformational characteristics of the apo and holo forms of these mutants were determined at neutral pH, by spectroscopic and hydrodynamic methods. The apo form of the deleted and permuted mutants exhibited a stronger tendency to aggregate and had lower ellipticity than the wild type. The mutants retained the ability to bind heme, but only the circularly permuted holoproteins had native-like heme binding and folding. These results agree with the theory that myoglobin has a central core that is able to bind heme, but also indicate that the presence of N-and C-terminal helices is necessary for native-like heme pocket formation. Because the holopermuteins were less stable than the wild-type protein and aggregated, we propose that the native position of the N-terminus is important for the precise structural architecture of myoglobin. Abbreviations: Mb, myoglobin; WT, wild type; Mb1-123, Mb with residues 124-153 deleted (helix H); Mb1-99, Mb with residues 100-153 deleted (helices G and H); Mb-C_GHAB, circularly permuted protein that starts at residue S35 (using WT residue numbering) and ends at residue K34; Mb-B_GHA, circularly permuted protein that starts at residue A19 and ends at residue E18; Mb-HAB_F, circularly permuted and deleted protein that starts at residue G124 and ends at residue I99; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; T m, temperature at the midpoint of the thermal transition; A, absorbance; CD, circular dichroism; AUC, analytical ultracentrifugation; NMR, nuclear magnetic resonance; Bis-ANS, bis-1-anilino-8-naphthalenesulfonate.