Conformational properties of native sperm whale apomyoglobin in solution

Lecomte, Juliette T. J.; Sukits, Steven F.; Bhattacharya, Shibani; Falzone, Christopher J.

doi:10.1110/ps.8.7.1484

Cited by 63 publications

(81 citation statements)

References 69 publications

(26 reference statements)

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“…In the native form of apoMb, the side chain of Met-131 has nuclear Overhauser effects to the A and G helices consistent with the holoMb structure (3). Further, nuclear Overhauser effect-based structure determination suggests a holoMb-like fold in this region, in which Met-131 is docked, along with more N-terminal residues of the H helix, against the A and G helices (4,6). Position 131 was one of the three sites at which an underpacking substitution was found to decrease the stability of I (14).…”

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confidence: 94%

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Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Bertagna

Barrick

2004

Proc. Natl. Acad. Sci. U.S.A.

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Acid-induced unfolding of apomyoglobin (apoMb) proceeds in a multistate process involving at least one equilibrium intermediate (I) at pH 4.2. The structure of the I form has been investigated thoroughly, with significant effort devoted to identifying potentially stabilizing native contacts. Here, we test whether rigid side-chain packing interactions like those in holomyoglobin persist at a buried position, Met-131, within the low-pH apoMb intermediate. We have measured the urea-induced unfolding transitions of overpacking, underpacking, and polar substitutions of Met-131 to determine the effect on the stability of the native and intermediate states of apoMb. Whereas underpacking substitutions should destabilize the I form irrespective of the degree of native side-chainpacking interactions, we anticipate that overpacking replacements might show opposite effects in a tightly packed environment, compared with a region lacking native side-chain packing interactions. We observe that, whereas underpacking and polar substitutions destabilize the I form, overpacking substitutions are stabilizing, implying that I is structurally plastic. We also report a strong correlation between the I state unfolding free energies and side-chain transfer free energies from water to octanol. Our results suggest that, whereas side-chain hydrophobicity is important for the stability of the I form, specific side-chain packing interactions are not.

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confidence: 94%

“…However, apoMb has a more dynamic structure than holoMb, showing a slight decrease in helix content and protection from hydrogen exchange (2)(3)(4). Multidimensional NMR studies suggest that the F helix, which contacts the heme in holoMb, is structurally disordered and that the N terminus of the G helix and the C terminus of the H helix are frayed (2,5,6).…”

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confidence: 99%

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Bertagna

Barrick

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The structure of apomyoglobin is similar to that of the holoprotein except that residues in the F helix and the C terminus of the H helix are disordered (8)(9)(10). During refolding, apomyoglobin forms an on pathway kinetic intermediate, in which major portions of the A, G, and H helices and part of the B helix are folded, within the 6-ms burst phase of conventional quench-flow H/D exchange experiments (11)(12)(13)(14).…”

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confidence: 94%

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

Uzawa

Nishimura

Akiyama

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

172

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The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to timeresolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.protein folding ͉ pulse labeling ͉ rapid mixing M ost proteins fold rapidly from the highly heterogeneous conformational ensemble of the unfolded state into their well defined native conformations. For proteins with Ͼ100 residues, collapsed, partially folded intermediates are formed within hundreds of microseconds after the initiation of folding (1-4). Quench-flow pulse-labeling experiments have yielded considerable information about the development of secondary structure in such intermediates, on a time scale of milliseconds (5, 6). However, little is known about the processes that occur within the dead time (Ϸ6 ms) of the conventional quench flow apparatus, nor about the dynamic behavior of the kinetic folding intermediates. To gain insights into the early folding processes for sperm whale apomyoglobin, we have performed pulsed hydrogen-deuterium (H/D) exchange experiments with submillisecond time resolution.Apomyoglobin has been studied extensively by kinetic and equilibrium methods as a paradigm for understanding protein folding pathways and the structure of folding intermediates (7). The structure of apomyoglobin is similar to that of the holoprotein except that residues in the F helix and the C terminus of the H helix are disordered (8-10). During refolding, apomyoglobin forms an on pathway kinetic intermediate, in which major portions of the A, G, and H helices and part of the B helix are folded, within the 6-ms burst phase of conventional quench-flow H/D exchange experiments (11)(12)(13)(14). These same regions adopt stable secondary structure in the equilibrium molten globule intermediate formed at pH 4.2 (10,(15)(16)(17). Recent H/D exchange experiments under a variety of conditions detected heterogeneity in the apomyogl...

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“…13 -16 The heme-containing holoprotein adopts a compact structure containing eight a-helices (designated A to H); although the apoprotein is less stable, it retains an extensive hydrophobic core and most of the secondary structure, as well as specific tertiary interactions within the hydrophobic clusters. 17,18 Acid denaturation of apoMb occurs in two distinct stages, 19,20 forming a compact molten globule at pH 4.1 with a helix content of , 35%, and then a more highly denatured state at pH 2.3 with a smaller content of helix. The transient helical secondary structure formed in the pH 2.3 state 10 is disrupted in the presence of 8 M urea.…”

Section: Introductionmentioning

confidence: 99%

Structural Characterization of Unfolded States of Apomyoglobin using Residual Dipolar Couplings

Mohana‐Borges

Goto

Kroon

et al. 2004

Journal of Molecular Biology

172

202

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The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between 15 N and 1 H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with f and c dihedral angles favoring the b or P II regions. Each statistical segment has a highly anisotropic shape, with the N -H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the aciddenatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.

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Conformational properties of native sperm whale apomyoglobin in solution

Cited by 63 publications

References 69 publications

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR

Structural Characterization of Unfolded States of Apomyoglobin using Residual Dipolar Couplings

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