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Review
. 2006 May;106(5):1836-61.
doi: 10.1021/cr040430y.

Early events in protein folding explored by rapid mixing methods

Heinrich Roder  1 , Kosuke Maki, Hong Cheng
Affiliations
Review

Early events in protein folding explored by rapid mixing methods

Heinrich Roder et al. Chem Rev. 2006 May.
No abstract available

PubMed Disclaimer

Figures

Figure 1
Figure 1
Continuous-flow capillary mixing apparatus in fluorescence mode. a Schematic of the solution delivery system, mixer, observation cell and optical arrangement. b Expanded view of the mixer. c Diagram illustrating continuous-flow measurement.
Figure 2
Figure 2
Continuous-flow measurements of the quenching of NATA fluorescence by NBS used to determine the experimental dead-time. a Plot of NATA fluorescence (>324 nm) vs. time at several NBS concentrations. b NATA-NBS reaction rates from exponential fitting of the data in panel a vs. NBS concentration. Linear regression (line) yields a second-order rate constant of 7.9 105 M−1 s−1.
Figure 3
Figure 3
Schematic of a microfluidic mixer designed by Bilsel et al.. The 127-μm-thick mixer is sandwiched between quartz windows and is sealed in a stainless-steel holder using Teflon gaskets (1.6 mm thick). Solutions are delivered to and from the mixing region through holes in the upper layers (arrows). Reprinted with permission from ref . Copyright 2005 American Institute of Physics.
Figure 4
Figure 4
Folding mechanism of SNase probed by tryptophan fluorescence. a Fluorescence emission spectra of the Trp76 variant of SNase under native and denaturing conditions (solid) and a folding intermediate populated at equilibrium (dashed). The spectrum of the intermediate was determined by global analysis of the fluorescence spectra as a function of urea concentration (pH 5.2, 15 °C). b Time-course of folding (triggered by a pH jump from 2 to 5.2) for wild-type SNase (Trp140) and a single-tryptophan variant (Trp76) measured by continuous-flow (< 10−3 s) and stopped-flow (> 103 s) fluorescence. c ANS fluorescence changes during ANS binding/folding of Trp76 SNase measured by continuous-flow experiments at 15°C in the presence of 160 μM ANS. U → A: Salt concentration jump from 0 M to 1 M KCl at pH2.0; U → N: refolding induced by a pH-jump from 2.0 to 5.2; A+ANS → A•ANS: ANS binding kinetics in the presence of 1 M KCl at pH 2.0; native control: ANS binding kinetics under the native condition (pH 5.2). Adapted from ref (Figures 3 and 5).
Figure 5
Figure 5
FRET-detection of an early folding intermediate in a helix-bundle protein, ACBP. a Ribbon diagram of ACBP, based on an NMR structure. The two tryptophan residues and the mutated C-terminal isoleucine are shown in ball and stick. The two lower panels show refolding kinetics of unmodified ACBP (b) and AEDANS-labeled ACBP,I86C (c) in pH 5.3 buffer containing 0.34 M GuHCl at 26 °C. In both panels data from continuous-flow (○) and stopped-flow (▽) experiments were matched and combined. Reprinted with permission from ref . Copyright 2002 National Academy of Sciences of the USA.
Figure 6
Figure 6
Initial stages of refolding of acid-denatured oxidized cyt c at pH 5 monitored by continuous-flow absorbance measurements at different wavelengths spanning the Soret heme absorbance band. The lines represent a global fit of a four-state folding mechanism to the family of kinetic traces. Reprinted with permission from ref . Copyright 2004 Elsevier Inc.
Figure 7
Figure 7
Characterization of an intermediate populated during folding of cytochrome c (pH 6, 10 °C). a Fractional degree of labeling vs. pulse pH for a representative set of NH groups measured by 2D NMR analysis of refolding cytochrome c samples that were exposed to a 50 ms labeling pulse of increasing pH at a folding time of 100 ms. Solid lines represent a fit of the data yielding the rates of formation and unfolding of the intermediate state (see text). Dashed lines indicate the labeling profiles expected in the absence of structure. b Equilibrium constant for formation of the intermediate, KUI = kUI/kIU, based on the fits of the labeling results in a. Values of KUI ≥ 1 indicative of persistent hydrogen bonded structure are mainly found for residues in the N- and C-terminal helices. Cys 14, Ala15 and His 18 (gray bars) are protected even in the unfolded state. Adapted from ref (Figures 3 and 4), with permission by the authors.
Figure 8
Figure 8
Stopped-flow fluorescence evidence for an unresolved rapid process (burst phase) during folding of cyt c (pH 5, 10 °C). a Tryptophan fluorescence changes during refolding of acid-unfolded cytochrome c (pH 2, ~15 mM HCl) at a final GuHCl concentration of 0.7 M. The initial signal S(0) at t=0 (determined on the basis of a separate dead-time measurement) falls short of the signal for the unfolded state under refolding conditions, Spred(U), obtained by linear extrapolation of the unfolded-state baseline (see dashed line in b). b Effect of the denaturant concentration on the initial (squares) and final (circles) fluorescence signal, S(0) and S(∞), measured in a series of stopped-flow refolding experiments at different final GuHCl concentration. Reprinted with permission from ref . Copyright 2004 Elsevier Inc.
Figure 9
Figure 9
Schematic log(rate) vs. [denaturant] plots (chevrons) for a two-state (a) and a three-state (b) folding/unfolding mechanism. The lower panels show the predicted amplitude for the main folding phases (a1), the burst phase predicted for a three-state process (a0 in panel d) and the equilibrium unfolding transition (aeq).
Figure 10
Figure 10
Folding kinetics of GB1 at pH 5.0, 20 °C, in the presence of 0.4 M sodium sulfate. Panel a shows a representative kinetic trace at 1.12 M GHCl monitored by continuous-flow (circles) and stopped-flow (diamonds) fluorescence along with controls for fully unfolded (U) and folded (N) solutions. Single- and double-exponential fits and residuals are shown with solid and dashed lines, respectively. Reprinted with permission from ref . Copyright 1999 Nature Publishing Group. Panel b shows a family of refolding traces at the final GuHCl concentrations indicated. Solid lines show the time course predicted on the basis of a three-state model (Scheme 3), using the following parameters: kUI° = 2300 s−1, mUI‡ = −0.6 kcal mol−1M−1, kIU° = 70 s−1, mIU‡ = 1.15 kcal mol−1M−1, kIN° = 600 s−1, mIN‡ = 0, kNI° = 0.14, mNI‡ = 0.3 kcal mol−1M−1 (see Figure 11a, ref 78). The relative signals for the N-, I- and U-states were sN = 0.29 ± 0.01, sI = sU = 0.98 ± 0.02. Dashed lines indicate a best “fit” of a two-state mechanism, using rate constants falling on a linear extrapolation of the chevron plot between 1.5 and 3 M GuHCl to lower denaturant concentrations (cf. ref 213).
Figure 11
Figure 11
GuHCl-dependence of the rate constants (a) and kinetic amplitudes (b) of the fast (squares) and slow (circles) kinetic phases observed during folding of GB1. c Free energy diagrams for folding of GB1 under conditions where the intermediate, I is well populated (0 M) and unstable (2.5 M GuHCl). α represents the change in solvent-accessible surface area relative to the unfolded state U. Reprinted with permission from ref . Copyright 1999 Nature Publishing Group.
Figure 12
Figure 12
Comparison of quadruple (a) and triple (b) exponential fitting of the kinetics of refolding of F45W ubiquitin at final GuHCl concentrations of 0.5 and 1.0 M (pH 5, 25 °C). Fluorescence traces measured in continuous- and stopped-flow experiments were normalized with respect to the unfolded protein in 6 M GuHCl. The residuals (top two traces in each panel) indicate that four exponentials are required to obtain a satisfactory fit of the data over the time window shown. Reprinted with permission from ref . Copyright 2005 Wiley-VCH.
Figure 13
Figure 13
Expanded region of the rate profile (log rate vs. [GuHCl[) for the two main phases (circles and squares) and a minor slower phase (dtriangles) observed during folding of F45W ubiquitin (pH 5, 25 °C). The solid lines show the rates predicted by a three-state model, and the dashed lines indicate the elementary rate constants used. The X-symbols show the apparent rates obtained by triple-exponential fitting (Figure 10b). Reprinted with permission from ref . Copyright 2005 Wiley-VCH.
Figure 14
Figure 14
a Ribbon diagram of horse cytochrome c, based on the crystal structure. b Refolding kinetics of acid-unfolded cyt c at pH 4.5, 22 °C measured by continuous-flow mixing, indicating heme-induced quenching of Trp59 fluorescence associated with chain collapse. Inset: Arrhenius plot for the rates of the major (circles) and the minor (squares) submillisecond phases. Adapted from ref (Figure 2).
Figure 15
Figure 15
Sequential four-state mechanism involving two partially structured ensembles, I1 and I2, in addition to the acid-unfolded (U) and native (N) states. Cartoons of various states apomyoglobin consistent with the fraction of helical residues (fH) and radius of gyration (Rg) reported in ref . Cylinders indicate the approximate position of α-helices in the native state and possible arrangement of core helices in the intermediates consistent with amide protection data.,
Figure 16
Figure 16
Kinetic mechanism of Im7 folding. a Ribbon diagram of Im7. b Representative kinetic trace measured by continuous-flow (●) and stopped-flow (○) fluorescence. The kinetics at this and all other urea concentrations measured is accurately predicted by on on-pathway mechanism (solid line) while schemes with off-pathway intermediates fail to reproduce the data (dashed line). c Observed (symbols) and predicted (solid lines) rates of folding and unfolding, based on mechanisms with on-pathway (left) and off-pathway (right) intermediates. Dashed lines indicate the corresponding elementary rate constants. Adapted from ref (Figures 2 and 3).
Scheme 1
Scheme 1
Scheme 2
Scheme 2
Scheme 3
Scheme 3
Scheme 4
Scheme 4
Scheme 5
Scheme 5
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