Chain length dependence of apomyoglobin folding: structural evolution from misfolded sheets to native helices.

Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Biochemistry (Impact Factor: 3.38). 07/2003; 42(23):7090-9. DOI: 10.1021/bi0273056
Source: PubMed

ABSTRACT 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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    ABSTRACT: Most proteins require appropriate folding in order to perform their respective functions, whereas misfolding can lead to pathological conditions. Thus, protein folding presents a complex problem which requires extensive study from both physical and biological perspectives. While significant progress has been made towards a solution to the protein folding problem, a quantitative and predictive understanding of how proteins fold is yet to be reached. This is partly due to the fact that protein molecules are complex, and that many (weak) interactions work together to define a protein's native structure. To reduce the complexity of the problem, we have taken a bottom-up approach and focused on studying the folding dynamics and mechanism of small peptides that exhibit folding characteristics of large proteins. These studies not only yield insights into the early steps of protein folding, including the theoretical folding speed limit, but they also provide ideal models for computer simulations. The systems described in this thesis include simple protein secondary structural elements and miniproteins (i.e., trp-cage, GCN4-p1, and villin headpiece subdomain). Since these peptides fold on the sub-millisecond timescale, we have employed a laser-induced temperature jump infrared technique to measure their folding kinetics. Whenever necessary, we have also made use of sequence and structure perturbing methods to systematically assess the mechanistic role of various native-state properties, including protein length, hydrophobic and electrostatic interactions, and backbone-backbone hydrogen bonding, in the formation of the transition state ensemble. For example, our study of Trp-cage folding indicates that, contrary to molecular dynamics simulation, the formation of a solvent exposed salt-bridge is not required for achieving fast folding; instead, a well-placed aromatic interaction lowers the folding free energy barrier. Also, through investigation of the folding dynamics of a GCN4 coiled-coil variant, we show that the folding of such dihelical structural motifs is likely initiated by contacts throughout the sequence rather than those localized in a previously identified trigger sequence. Furthermore, using amide-to-ester backbone mutations, we are able to demonstrate that helix formation is not necessary for acquiring the transition state in the folding of the helical subdomain of villin headpiece, discrediting the backbone-centered view of protein folding.
    Dissertations available from ProQuest. 01/2008;
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    ABSTRACT: 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 06/2004; 43(20):6230-40. · 3.38 Impact Factor
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    ABSTRACT: Molecular dynamics simulations of N-terminal peptides from apo-myoglobin with several lengths were executed to study their stability to testify the possibility of cotranslational folding. By analyzing 10 ns MD simulations in water, we found that the secondary and tertiary structures of a short N-terminal peptide (36 residues) are unstable whereas those of longer N-terminal peptides (more than 77 residues) are relatively stable. In addition, we confirmed that the structural changes are driven by the free energy. Our results suggest that a nascent apo-myoglobin chain starts to form α-helix structures after elongation of at least 77 amino acid residues.
    Chemical Physics Letters 01/2006; · 2.15 Impact Factor


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