Chain Length Dependence of Apomyoglobin Folding: Structural Evolution from Misfolded Sheets to Native Helices †

Department of Chemistry, University of Washington Seattle, Seattle, Washington, United States
Biochemistry (Impact Factor: 3.02). 07/2003; 42(23):7090-9. DOI: 10.1021/bi0273056
Source: PubMed


Very little is known about how protein structure evolves during the polypeptide chain elongation that accompanies cotranslational protein folding. This in vitro model study is aimed at probing how conformational space evolves for purified N-terminal polypeptides of increasing length. These peptides are derived from the sequence of an all-alpha-helical single domain protein, Sperm whale apomyoglobin (apoMb). Even at short chain lengths, ordered structure is found. The nature of this structure is strongly chain length dependent. At relatively short lengths, a predominantly non-native beta-sheet conformation is present, and self-associated amyloid-like species are generated. As chain length increases, alpha-helix progressively takes over, and it replaces the beta-strand. The observed trends correlate with the specific fraction of solvent-accessible nonpolar surface area present at different chain lengths. The C-terminal portion of the chain plays an important role by promoting a large and cooperative overall increase in helical content and by consolidating the monomeric association state of the full-length protein. Thus, a native-like energy landscape develops late during apoMb chain elongation. This effect may provide an important driving force for chain expulsion from the ribosome and promote nearly-posttranslational folding of single domain proteins in the cell. Nature has been able to overcome the above intrinsic misfolding trends by modulating the composition of the intracellular environment. An imbalance or improper functioning by the above modulating factors during translation may play a role in misfolding-driven intracellular disorders.

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    • "The lower α-helical content of both the native and partially folded state of single tryptophan-containing apomyoglobins compared to that of wild-type protein clearly indicates that each single-tryptophanyl substitution at either position 7 or 14 affects the protein secondary structure. In particular, the folding intermediate of W7F mutant, very similar to that of the amyloidogenic mutant, contains less α-helical structure and more β-content than wild type, thus indicating that the secondary structural organization of the folded portion (AGH subdomain) of the compact intermediate changes because of the substitution of the indole residue at position 7. Chow et al. [75] reported that the 1–36 N-terminal fragment of wild-type apomyoglobin displays a high level of β-structure and forms macroscopic aggregates when the pH becomes closer to neutrality. Both observations suggest that the tryptophanyl substitution at position 7 could cause an increased propensity of the N-terminal region to form a β-structure in the intact protein, confirm this suggestion. "
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    ABSTRACT: Apomyoglobin is an excellent example of a monomeric all α-helical globular protein whose folding pathway has been extensively studied and well characterized. Structural perturbation induced by denaturants or high temperature as well as amino acid substitution have been described to induce misfolding and, in some cases, aggregation. In this article, we review the molecular mechanism of the aggregation process through which a misfolded form of a mutated apomyoglobin aggregates at physiological pH and room temperature forming an amyloid fibril. The results are compared with data showing that either amyloid or aggregate formation occurs under particular denaturing conditions or upon cleavage of the residues corresponding to the C-terminal helix of apomyoglobin. The results are discussed in terms of the sequence regions that are more important than others in determining the amyloid aggregation process.
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    • "Furthermore, the non-native beta-sheet content of the misfolded PfAQP species in Brij35 fits well into this scenario. Chow et al. [16] found that nascent alpha-helical proteins can undergo a conversion from initially present beta-sheets to alpha-helices with increasing peptide chain length. The respective CD spectra documenting this process depict a change in shape from a singlet to a doublet minimum in the 210–220 nm range, which is highly reminiscent of the spectra obtained with PfAQP in Brij35 vs. Brij78 (Fig. 2A, left panel). "
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    • "The CD spectrum of W7F mutant at pH 4.0 is very similar to that of the amyloidforming mutant and characterized by an increased amount of b-structure. Chow et al. (2003) reported that the 1–36 Fig. 9 Prediction of apomyoglobin b-aggregation propensity based on PASTA aggregation scores of wild type and W7F, W14F, and W7FW14F mutants Fig. 10 Camp prediction of wild-type and W7F, W14F, and W7FW14F mutant apomyoglobin. lnp \ 5 nonprotected residues, lnp [ 5 protected residues Eur Biophys J 123 Author's personal copy N-terminal fragment of wild-type apomyoglobin displays a high level of b-structure and forms macroscopic aggregates when the pH gets closer to neutrality. "
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    ABSTRACT: Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation.
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