Equilibrium folding-unfolding pathways of model proteins: Effect of myoglobin-heme contacts (original) (raw)
Related papers
Most probable intermediates in protein folding-unfolding with a noninteracting globule-coil model
Biochemistry, 1982
Protein conformations are generated with a noninteracting globule-coil model in which each residue is assumed to take only the native or random-coil state, and a protein conformation is regarded to consist of alternating regions of random coil and globules of native conformation. Statistical weights are taken to have two parts, corresponding to intraresidue and interresidue interactions. The intraresidue statistical weight for a residue in its native state is assumed to be proportional to the empirical frequency of the native (&$), from tabulated statistics. Interresidue energies are taken to be proportional to the number of contacts within each native region. The principal adjustable parameter is the contact energy per contact pair. Proteins studied include trypsin inhibitor, ribonuclease A, lysozyme, and apomyoglobin. The number of native residues is employed as a simple one-dimensional representation of the folding-unfolding coordinate to describe probable folding pathways. When conformations are considered at each point on this coordinate, it is possible to obtain detailed descriptions of the conformational characteristics of relatively rare intermediates along the folding pathway. This technique of "trapping" conformational in-'From the Laboratory of Mathematical Biology, Building IO, Room 4B-56, DCBD
Biochemistry, 1993
As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized, and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The behavior of short fragments corresponding to the sequences of the G-and H-helices of myoglobin and to the turn region between these helices has been described in accompanying papers. At the next level of complexity, peptide model compounds have been synthesized to explore the longer-range interactions which may take place in protein folding after initial secondary structure formation has occurred. A series of disulfide-bridged dimeric peptides containing the complete sequences of the G-and H-helices of myoglobin were synthesized and their conformational preferences examined. CD spectra indicate that disulfide-bridged peptides consisting of two H-helix sequences (Mb-HssH) and of one G-and one H-helix (Mb-GssH) are highly helical in water solution, as a result of intermolecular association. A 5 l-residue peptide, Mb-GH5 1, encompassing the entire G-H helical hairpin of myoglobin, including the turn sequence between the two helices, has been successfully synthesized by standard methods. This peptide was designed to be monomeric in aqueous solution. Mb-GH5 1 does not appear from CD spectra to contain any additional helix in water solution above what would be expected froman equimolar mixture of the G-and H-helix peptides. N M R spectra indicate that the turn conformation observed in shorter peptide fragments is retained in Mb-GH5 1 in high population. The addition of TFE results in the formation of some helix, though not as much as might be expected even from a simple combination of the ellipticities of the component helical peptides in TFE. Current experimental and theoretical studies of myoglobin folding implicate the G-H helical hairpin in the earliest stages of folding: the present results imply that other parts of the polypeptide chain may be participating in these early events to a greater extent than heretofore imagined.
Biochemistry - BIOCHEMISTRY-USA, 1993
As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized, and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The behavior of short fragments corresponding to the sequences of the G-and H-helices of myoglobin and to the turn region between these helices has been described in accompanying papers. At the next level of complexity, peptide model compounds have been synthesized to explore the longer-range interactions which may take place in protein folding after initial secondary structure formation has occurred. A series of disulfide-bridged dimeric peptides containing the complete sequences of the G-and H-helices of myoglobin were synthesized and their conformational preferences examined. CD spectra indicate that disulfide-bridged peptides consisting of two H-helix sequences (Mb-HssH) and of one G-and one H-helix (Mb-GssH) are highly helical in water solution, as a result of intermolecular association. A 5 l-residue peptide, Mb-GH5 1, encompassing the entire G-H helical hairpin of myoglobin, including the turn sequence between the two helices, has been successfully synthesized by standard methods. This peptide was designed to be monomeric in aqueous solution. Mb-GH5 1 does not appear from CD spectra to contain any additional helix in water solution above what would be expected froman equimolar mixture of the G-and H-helix peptides. N M R spectra indicate that the turn conformation observed in shorter peptide fragments is retained in Mb-GH5 1 in high population. The addition of TFE results in the formation of some helix, though not as much as might be expected even from a simple combination of the ellipticities of the component helical peptides in TFE. Current experimental and theoretical studies of myoglobin folding implicate the G-H helical hairpin in the earliest stages of folding: the present results imply that other parts of the polypeptide chain may be participating in these early events to a greater extent than heretofore imagined.
Peptide models of protein folding initiation sites. 3. The G-H helical hairpin of myoglobin
Biochemistry, 1993
As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized, and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The behavior of short fragments corresponding to the sequences of the G-and H-helices of myoglobin and to the turn region between these helices has been described in accompanying papers. At the next level of complexity, peptide model compounds have been synthesized to explore the longer-range interactions which may take place in protein folding after initial secondary structure formation has occurred. A series of disulfide-bridged dimeric peptides containing the complete sequences of the G-and H-helices of myoglobin were synthesized and their conformational preferences examined. CD spectra indicate that disulfide-bridged peptides consisting of two H-helix sequences (Mb-HssH) and of one G-and one H-helix (Mb-GssH) are highly helical in water solution, as a result of intermolecular association. A 5 l-residue peptide, Mb-GH5 1, encompassing the entire G-H helical hairpin of myoglobin, including the turn sequence between the two helices, has been successfully synthesized by standard methods. This peptide was designed to be monomeric in aqueous solution. Mb-GH5 1 does not appear from CD spectra to contain any additional helix in water solution above what would be expected froman equimolar mixture of the G-and H-helix peptides. N M R spectra indicate that the turn conformation observed in shorter peptide fragments is retained in Mb-GH5 1 in high population. The addition of TFE results in the formation of some helix, though not as much as might be expected even from a simple combination of the ellipticities of the component helical peptides in TFE. Current experimental and theoretical studies of myoglobin folding implicate the G-H helical hairpin in the earliest stages of folding: the present results imply that other parts of the polypeptide chain may be participating in these early events to a greater extent than heretofore imagined.
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.
Equilibrium folding pathways for model proteins
Journal of Statistical Physics, 1983
Protein conformations have been generated with both a Monte Carlo scheme and a simpler two-state noninteracting globule-coil model. Conformational energies are taken to consist of intraresidue and interresidue terms. Interresidue energies are taken to be proportional to the number of nativelike contacts. To describe probable folding pathways, either energy or the number of native residues are employed as simple one-dimensional folding-unfolding coordinates. By considering only conformations at each point on these coordinates, it is possible to obtain detailed conformational descriptions of relatively rare intermediates on the folding pathway. This technique of "trapping" intermediates and statistically characterizing them is useful for studying conformational transitions. Equilibrium folding-unfolding pathways have been constructed by connecting most probable conformations in order along the folding coordinate. Calculations with the noninteracting globule-coil model have been performed with details chosen to correspond to those in the Monte Carlo calculation for pancreatic trypsin inhibitor. Both pathways are similar. The a helix appears prior to formation of the central beta sheet; beta sheet formation coincides with a large maximum in the free energy because of the attendant loss of conformational entropy. Subsequently the Monte Carlo method indicates two alternative pathways for growth toward either the amino or the carboxyl terminus, followed by completion of the native form. For the globule-coil model, the growth pattern differs somewhat, with the appearance of the single pathway for folding up to the carboxyl terminus prior to completion of folding. This difference may originate in the Monte Carlo sampling procedures or in the simplifications of the globule-coil model.
Proceedings of the National Academy of Sciences, 2004
The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f H) and radius of gyration (R g), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to Ϸ50% of the overall change in R g from the unfolded to native conformation, was observed within 300 s after the pH jump. The collapsed intermediate has a f H of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.
Solvent-induced organization: A physical model of folding myoglobin
Proteins: Structure, Function, and Genetics, 1994
The essential features of the in vitro refolding of myoglobin are expressed in a solvable physical model. Alpha helices are taken as the fundamental collective coordinates of the system, while the refolding is assumed to be mainly driven by solvent-induced hydrophobic forces. A quantitative model of these forces is developed and compared with experimental and theoretical results. The model is then tested by being employed in a simulation scheme designed to mimic solvent effects. Realistic dynamic trajectories of myoglobin are shown as it folds from an extended conformation to a close approximation of the native state. Various suggestive features of the process are discussed. The tenets of the model are further tested by folding the single-chain plant protein leghemoglobin.
Folding/Unfolding Kinetics of Apomyoglobin
Molecular Biology, 2005
The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 ° C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.