Consequences of Stabilizing the Natively Disordered F Helix for the Folding Pathway of Apomyoglobin

Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

doi:10.1016/j.jmb.2011.05.028

Cited by 16 publications

(34 citation statements)

References 39 publications

(63 reference statements)

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“…This effect perhaps allows a protein to assume its kinetic intermediate state, even for a protein that is predicted to be a two‐state folder by purely native centric models. Actually, there are some reports that non‐native interactions contribute to the accumulation of the kinetic intermediate states . Inhibiting the non‐local contacts, especially the N‐ and C‐terminal interactions, may also destabilize the overstabilized intermediate state of iFABP.…”

Section: Resultsmentioning

confidence: 99%

Topological and sequence information predict that foldons organize a partially overlapped and hierarchical structure

2015

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It has been suggested that proteins have substructures, called foldons, which can cooperatively fold into the native structure. However, several prior investigations define foldons in various ways, citing different foldon characteristics, thereby making the concept of a foldon ambiguous. In this study, we perform a Gō model simulation and analyze the characteristics of substructures that cooperatively fold into the native-like structure. Although some results do not agree well with the experimental evidence due to the simplicity of our coarse-grained model, our results strongly suggest that cooperatively folding units sometimes organize a partially overlapped and hierarchical structure. This view makes us easy to interpret some different proposal about the foldon as a difference of the hierarchical structure. On the basis of this finding, we present a new method to assign foldons and their hierarchy, using structural and sequence information. The results show that the foldons assigned by our method correspond to the intermediate structures identified by some experimental techniques. The new method makes it easy to predict whether a protein folds sequentially into the native structure or whether some foldons fold into the native structure in parallel.

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Section: Resultsmentioning

confidence: 99%

Topological and sequence information predict that foldons organize a partially overlapped and hierarchical structure

2015

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“…Mutants with increased helical structure in the F helix (P88K/S92K (F2) and P88K/A90L/S92K/A94L (F4)) were designed to alter the AABUF of the F helix to increase the likelihood that this region of the protein would participate in the earliest steps of folding. 41 NH resonances were visible in the HSQC spectrum for the F helix residues of both mutant proteins at neutral pH, indicating that the F helix has been stabilized, but only the F4 mutant protein had a high enough AABUF to stabilize the F helix in the burst phase intermediate. Interestingly, the overall rate of folding is slowed in the F4 mutant, because the folded F helix stabilizes the intermediate and inhibits translocation of the H helix to its native position.…”

Section: Apomyoglobin Folding Efficiency Is Subordinated To Functionmentioning

confidence: 99%

“…Interestingly, the overall rate of folding is slowed in the F4 mutant, because the folded F helix stabilizes the intermediate and inhibits translocation of the H helix to its native position. 41 The structural heterogeneity observed for the F helix in the native wild-type protein at pH 6 may be related to the in vivo insertion of the heme after the protein is folded. Flexibility in the F helix would allow insertion of the bulky heme, after which the heme pocket closes and the F helix becomes stably folded in the holoprotein.…”

Section: Apomyoglobin Folding Efficiency Is Subordinated To Functionmentioning

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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“…24 The folding/unfolding kinetics of this portion of the protein has evolved to be carefully optimized. 25,26 Globins from all organisms are characterized by a small number (ca. 6) of highly conserved core residues that do not play any functional role, 27 and may therefore be important for folding 28,29 and (or) stability.…”

Section: ■ Introductionmentioning

confidence: 99%

Sub-millisecond Chain Collapse of the Escherichia coli Globin ApoHmpH

et al. 2013

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Myoglobins are ubiquitous proteins that play a seminal role in oxygen storage, transport, and NO metabolism. The folding mechanism of apomyoglobins from different species has been studied to a fair extent over the last two decades. However, integrated investigations of the entire process, including both the early (sub-ms) and late (ms-s) folding stages, have been missing. Here, we study the folding kinetics of the single-Trp Escherichia coli globin apoHmpH via a combination of continuous-flow microfluidic and stopped-flow approaches. A rich series of molecular events emerges, spanning a very wide temporal range covering more than 7 orders of magnitude, from sub-microseconds to tens of seconds. Variations in fluorescence intensity and spectral shifts reveal that the protein region around Trp120 undergoes a fast collapse within the 8 μs mixing time and gradually reaches a native-like conformation with a half-life of 144 μs from refolding initiation. There are no further fluorescence changes beyond ca. 800 μs, and folding proceeds much more slowly, up to 20 s, with acquisition of the missing helicity (ca. 30%), long after consolidation of core compaction. The picture that emerges is a gradual acquisition of native structure on a free-energy landscape with few large barriers. Interestingly, the single tryptophan, which lies within the main folding core of globins, senses some local structural consolidation events after establishment of native-like core polarity (i.e., likely after core dedydration). In all, this work highlights how the main core of the globin fold is capable of becoming fully native efficiently, on the sub-millisecond time scale.

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Consequences of Stabilizing the Natively Disordered F Helix for the Folding Pathway of Apomyoglobin

Cited by 16 publications

References 39 publications

Topological and sequence information predict that foldons organize a partially overlapped and hierarchical structure

Topological and sequence information predict that foldons organize a partially overlapped and hierarchical structure

How Does Your Protein Fold? Elucidating the Apomyoglobin Folding Pathway

Sub-millisecond Chain Collapse of the Escherichia coli Globin ApoHmpH

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