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. 2014 Jan 15;136(2):733-40.
doi: 10.1021/ja410437d. Epub 2013 Dec 31.

Ultrafast Hydrogen Exchange Reveals Specific Structural Events During the Initial Stages of Folding of Cytochrome C

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Ultrafast Hydrogen Exchange Reveals Specific Structural Events During the Initial Stages of Folding of Cytochrome C

Hossein Fazelinia et al. J Am Chem Soc. .
Free PMC article

Abstract

Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two α-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of α-helical segments rather than a general hydrophobic collapse.

Figures

Figure 1
Figure 1
Microfluidic mixing device. A. Photograph of PEEK chip (25×25 mm2) showing laser microfabricated surface features, including mixing regions M1 through M5 and access channels. B. Schematic of an expanded region (circled in A) covering mixing regions M1 through M3. PB: high-pH pulse buffer in H2O; UP: unfolded protein in D2O; QB: low-pH quench buffer in H2O. C. Photograph of the steel holder containing the multi-mixer chip illustrated in A. D. Calibration of the mixing device using alkaline hydrolysis of phenol chloroacetate (PCA; inset). The absorbance of the product (phenol) was measured at 270 nm vs. aging time.
Figure 2
Figure 2
pH-dependent competition between D-H exchange and folding of cyt c at constant time (140 µs, 22 °C). Normalized proton occupancies for representative residues (symbols, solid lines) are compare with the pH profiles predicted for the corresponding residues in the unfolded state (dashed lines). Solid lines represent fits of Eq. 1 to the data.
Figure 3
Figure 3
Time-dependence of D-H exchange/folding competition at a urea concentration of 0.6 M (filled symbols) and intrinsic exchange kinetics in the urea-unfolded state (open symbols) for representative residues (pH 9.8, 22 °C). Solid lines represent fits of Eq. 2 to the competition data and dashed lines are single-exponential fits of the exchange data at 6 M urea.
Figure 4
Figure 4
Sequence dependence of time-dependent competition and exchange results. The bar graphs in panels A and B show effective protection rates (kf) and intrinsic exchange rates (kcU), respectively, at 0.6 M urea obtained from least-squares fitting of Eq. 2 to the competition data in Figure S3. Panel C shows direct exchange rates for unfolded cyt c at 6 M urea vs. residue number.
Figure 5
Figure 5
Summary of pH-dependent exchange/folding competition data for the compact intermediate, I, of cyt c populated at a folding time of 140 µs. Panels A and B show bar graphs of the local rate constants for formation (kUIloc) and unfolding (kIUloc) of the I-state vs. residue number obtained by fitting Eq. 1 to the data in Figure S2. The corresponding equilibrium constants, KUIloc = kUIloc/kIUloc, are plotted in panel C.
Figure 6
Figure 6
Ribbon diagrams of the X-ray structure of native horse cytochrome c (pdb 1hrc) color-coded to show amide protection patterns at folding times of 140 µs (A) and ~1 ms (B). In both panels, residues that are protected in the native state, but unprotected in the I-state (KUIloc < 2; kfloc <200) are shown in gray. The pH-dependent competition results (Figure 5C) are summarized in panel A, where residues with low (2 ≤ KUIloc < 3), intermediate (3 ≤ KUIloc < 4) and high (KUIloc ≥ 4) are colored blue, orange and red, respectively. The time-dependent competition data (Figure 4A) are summarized in panel B, where residues with low (200 ≤ kfloc < 300), intermediate (300 ≤ kfloc < 400) and high (kfloc ≥ 400) are colored blue, orange and red, respectively.
Scheme 1
Scheme 1
H/D exchange/folding competition

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