Structural characterization of partially folded intermediates of apomyoglobin H64F

Schwarzinger, Stephan; Mohana‐Borges, Ronaldo; Kroon, Gerard; Dyson, H. Jane; Wright, Peter E.

doi:10.1110/ps.073187208

Cited by 17 publications

(16 citation statements)

References 35 publications

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“…2) in zero denaturant. In comparison, the native-like pH 4 molten-globule state of apomyoglobin retains between 50 and 80% native structure for its four most structured helices (68), and 10 -20% native structure in its pH 2.3 acid-denatured state (69).…”

Section: Resultsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

Journal of Biological Chemistry

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Prion propagation involves a conformational transition of the cellular form of prion protein (PrP C ) to a disease-specific isomer (PrP Sc ), shifting from a predominantly ␣-helical conformation to one dominated by ␤-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP Sc , is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant ␤-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a ␤-rich conformation. This precursor state is almost as compact as the folded PrP C structure and, as it assembles, only residues 126 -227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

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Section: Resultsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

Journal of Biological Chemistry

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“…A further site of frustration of E helix folding is the distal histidine (H64), a residue that is of functional importance in holomyoglobin to enhance O 2 binding and to discriminate against competing ligands such as CO. Substitution of H64 with a non-polar phenylalanine greatly stabilizes helical structure in the entire E helix region in both the burst phase conformational ensemble and in the equilibrium molten globule intermediate and results in a 2-fold increase in the apoMb folding rate 17,18. The frustration caused by the distal histidine has been attributed to the energetic cost of desolvation and deprotonation of the imidazole group required for packing in the hydrophobic core 18.…”

Section: Discussionmentioning

confidence: 99%

“…Substitution of H64 with a non-polar phenylalanine greatly stabilizes helical structure in the entire E helix region in both the burst phase conformational ensemble and in the equilibrium molten globule intermediate and results in a 2-fold increase in the apoMb folding rate 17,18. The frustration caused by the distal histidine has been attributed to the energetic cost of desolvation and deprotonation of the imidazole group required for packing in the hydrophobic core 18. It will be of interest in future work to determine whether the effects of B helix stabilization and mutation of H64 to Phe are additive or synergistic.…”

Section: Discussionmentioning

confidence: 99%

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

Nishimura

Dyson

Wright

2010

Journal of Molecular Biology

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Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B-helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the G-H turn region in the G23A/G25A mutant, suggesting that stabilization of the B-helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix.

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“…These results demonstrate that, although the folding intermediate is locked into the native-like conformation, this is not sufficient to promote correct packing of the E helix, and shows that there must be additional sites of energetic frustration that impede native docking of the E helix and progression to the transition state. In addition to the non-native G-H helix packing, instability of the N-terminal region of helix B 37 and burial of the distal His64 38 also frustrate the folding process by impeding the native docking interactions of helix E. Translocation of the H helix appears to promote dynamic disorder in the E and F helix regions, preventing excessive stabilization of compact non-native structures and allowing efficient search of conformational space until additional sources of frustration are relieved and folding can progress. 36 …”

Section: Why Does the Kinetic Folding Intermediate Occur?mentioning

confidence: 99%

How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway

Dyson

Wright

2016

Acc. Chem. Res.

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Conspectus Although each type of protein fold and in some cases individual proteins within a fold classification can have very different mechanisms of folding, the underlying biophysical and biochemical principles that operate to cause a linear polypeptide chain to fold into a globular structure must be the same. In an aqueous solution, the protein takes up the thermodynamically most stable structure, but the pathway along which the polypeptide proceeds in order to reach that structure is a function of the amino acid sequence, which must be the final determining factor, not only in shaping the final folded structure, but in dictating the folding pathway. A number of groups have focused on a single protein or group of proteins, to determine in detail the factors that influence the rate and mechanism of folding in a defined system, with the hope that hypothesis-driven experiments can elucidate the underlying principles governing the folding process. Our research group has focused on the folding of the globin family of proteins, and in particular on the monomeric protein apomyoglobin. Apomyoglobin (apoMb) folds relatively slowly (~2 seconds) via an ensemble of obligatory intermediates that form rapidly after the initiation of folding. The folding pathway can be dissected using rapid-mixing techniques, which can probe processes in the millisecond time range. Stopped-flow measurements detected by circular dichroism (CD) or fluorescence spectroscopy give information on the rates of folding events. Quench-flow experiments utilize the differential rates of hydrogen-deuterium exchange of amide protons protected in parts of the structure that are folded early; protection of amides can be detected by mass spectrometry or proton nuclear magnetic resonance spectroscopy (NMR). In addition, apoMb forms an intermediate at equilibrium at pH ~ 4, which is sufficiently stable for it to be structurally characterized by solution methods such as CD, fluorescence and NMR spectroscopies, and the conformational ensembles formed in the presence of denaturing agents and low pH can be characterized as models for the unfolded states of the protein. Newer NMR techniques such as measurement of residual dipolar couplings in the various partly folded states, and relaxation dispersion measurements to probe invisible states present at low concentrations, have contributed to providing a detailed picture of the apomyoglobin folding pathway. The research summarized in this review was aimed at characterizing and comparing the equilibrium and kinetic intermediates both structurally and dynamically, as well as delineating the complete folding pathway at a residue-specific level, in order to answer the question “What is it about the amino acid sequence that causes each molecule in the unfolded protein ensemble to start folding, and, once started, to proceed towards the formation of the correctly folded three-dimensional structure?”

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Structural characterization of partially folded intermediates of apomyoglobin H64F

Cited by 17 publications

References 35 publications

Conformational Properties of β-PrP

Conformational Properties of β-PrP

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway

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