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. 2008 Sep 16;105(37):13859-64.
doi: 10.1073/pnas.0804033105. Epub 2008 Sep 8.

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

Takanori Uzawa  1 , Chiaki Nishimura, Shuji Akiyama, Koichiro Ishimori, Satoshi Takahashi, H Jane Dyson, Peter E Wright
Affiliations

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

Takanori Uzawa et al. Proc Natl Acad Sci U S A. .

Abstract

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 time-resolve 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Representative plots of amide proton occupancy in partially refolded apomyoglobin as the 3.6-ms labeling pulse pH is varied from 7.0 to 10.7. (A) V10 (A helix), representative of the highly protected residues (class S). (B) A143 (H helix), representative of moderately protected residues (class M). (C) V66 (E helix), representative of weakly protected residues (class W). Red circles and blue triangles represent the data obtained at tf of 0.4 and 6 ms, respectively. The red lines and blue lines represent the fits to the 0.4- and 6-ms data, respectively, obtained by using Eqs. 2–5. The red and blue lines are coincident in A and B. The dotted lines show the expected curves for unprotected amides at tp of 3.6 ms.
Fig. 2.
Fig. 2.
Plots of stability and [NHopen] as a function of residue number. (A) The estimated stability (kcl/kop) for each amide. For highly protected amides with pH-independent proton occupancies, kcl/kop values are assigned a lower limit of 100. (B) [NHopen] values, estimated from kcl, kop, and tf. The red circles and blue triangles represent [NHopen] at tf of 0.4 and 6 ms, respectively. [NHopen] for highly protected amides is assigned a limiting value of 0. The location of the helices in the holomyoglobin structure is indicated at the top.
Fig. 3.
Fig. 3.
Equilibration of amide proton exchange at 0.4 and 6 ms. (A) [NHopen] versus kcl/kop for a refolding time of 6 ms. Experimental data points are shown as blue triangles. The solid line depicts equilibrium values of [NHopen] at long refolding time, calculated by using the equation [NHopen] = kop/(kop + kcl) derived from Eq. 4. (B) Plot of [NHopen] versus kcl/kop for a refolding time of 0.4 ms. Data points are colored according to their location in the apomyoglobin helices, as depicted in Fig. 2. The highly protected amides are assigned values of kcl/kop = 100 and [NHopen] = 0. The solid line depicts the equilibrium values of [NHopen], and the broken line was calculated by using Eq. 4 with kop = 200 s−1 and tf = 0.4 ms.
Fig. 4.
Fig. 4.
Location of the most rapidly protected amides. (A) Distribution of fully and nonequilibrated amides in the 0.4-ms intermediate ensemble. The structure of holomyoglobin (38) is represented as a tube of varying radius. Residues whose amides are fully equilibrated after 0.4 ms of refolding, i.e., for which [NHopen] has reached the equilibrium value, are depicted in magenta with increased tube radius. Residues for which [NHopen] has not reached the equilibrium values and which are fluctuating between folded and unfolded states are depicted by a thinner blue tube. Very thin tubes represent regions that do not exhibit exchange protection and probably remain unstructured in the folding intermediate. (B) Mapping of kcl values onto the holomyoglobin structure. Closing rates are depicted by variations in color and tube radius. Red, kcl > 9,500 s−1; yellow, 5,000 < kcl < 9,500 s−1; green, 2,500 < kcl < 5,000 s−1; blue, kcl < 2,500 s−1. This figure was prepared by using MolMol (39).
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