Probing the conformational state of apomyoglobin by limited proteolysis 1 1 Edited by P. E. Wright (original) (raw)
Related papers
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, 2004
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.
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.
Probing protein structure by limited proteolysis
2004
Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, a-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to
Conformational substates and motions in myoglobin. External influences on structure and dynamics
Biophysical Journal, 1990
Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, AO, A1, and A3. The entropies and enthalpies of these substates depend on temperature above-180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.
Conformational substates in a protein: structure and dynamics of metmyoglobin at 80 K
Proceedings of the National Academy of Sciences, 1982
The crystal structure ofsperm whale metmyoglobin has been determined at 80 K to a resolution of2 A. The overall structure at 80 K is similar to that at 300 K except that the volume is smaller. Refinement of the structure by the method of restrained least squares (current R = 0.175) permits the assignment ofisotropic atomic mean-square displacements to all nonhydrogen atoms. Comparison with the values obtained earlier at 250-300 K indicates that the protein at 80 K is more rigid. The average experimentally determined Debye-Waller factor, B, for the protein is 14 A at 300 K and 5 A2 at 80 K. Plots of backbone meansquare displacement vs. temperature show a discontinuity of slope for at least one-third ofall residues. This behavior is in good agreement with the temperature dependence of the mean-square displacement of the heme iron as measured by Mossbauer absorption. The magnitudes of the smallest mean-square displacements observed at 80 K indicate that intramolecular motions can be frozen out to a surprisingly large degree. Even at 80 K, however, some atoms in myoglobin still have mean-square displacements
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.
Folding propensities of peptide fragments of myoglobin
Protein Science, 2008
Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthesized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformational preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the a region of (c#J,$) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A-and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate p-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, earlier studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.
Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements
Protein Science, 2004
The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly >> Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.