Mechanical and chemical unfolding of a single protein: a comparison - PubMed (original) (raw)

Comparative Study

Mechanical and chemical unfolding of a single protein: a comparison

M Carrion-Vazquez et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.

PubMed Disclaimer

Figures

Figure 1

Construction of poly(I27) proteins. (A) Agarose gel stained with ethidium bromide showing the size of the I27–RS multiples (right lane). (B) Coomassie blue staining of the purified I27RS8 protein (≈90 kDa) separated by using SDS/PAGE. (C and D) Summary of the sequence of the I27RS8 and I27GLG12 constructs.

Figure 2

Force–extension relationships for recombinant poly(I27) measured with AFM techniques. (A and B) Stretching of single I27GLG12 (A) or I27RS8 polyproteins (B) give force–extension curves with a sawtooth pattern having equally spaced force peaks. The sawtooth pattern is well described by the WLC equation (continuous lines). (C) Unfolding force frequency histogram for I27RS8. The lines correspond to Monte Carlo simulations of the mean unfolding forces (n = 10,000) of eight domains placed in series by using three different unfolding rate constants, _k_u0, an unfolding distance, Δxu, of 0.25 nm, and a pulling rate of 0.6 nm/ms.

Figure 3

Stretching single I27RS8 proteins at different pulling speeds. Each symbol (■) is the average of (from left to right) 16, 19, 16, 266, 21, 21, and 9 data points. The solid lines correspond to Monte Carlo simulations obtained for three different unfolding rate constants, similar to those shown in Fig. 2. The best fit was obtained with _k_u0 = 3.3 × 10−4 s−1 and a Δxu = 0.25 nm. The dashed line corresponds to a Monte Carlo simulation using _k_u0 = 3.3 × 10−4 s−1 and a Δxu = 0.35 nm.

Figure 4

(A) Unfolding and refolding cycles of an I27RS8 protein probed with a double-pulse protocol. The protein is first stretched to count the number of domains that unfold, Ntotal, (a_–_c, upper traces), and then it is relaxed to its initial length. A second extension after a delay, Δ_t_, measures the number of refolded domains, Nrefolded (a_–_c, lower traces). (B) Plot of the refolded fraction, Nrefolded/Ntotal versus Δ_t_. Each symbol is the average of 53, 17, 20, 8, and 5 data points obtained from six separate experiments. The solid line is a fit of the data to the function Pƒ(t) = 1 − _e_−_tk_f0, where _k_f0 = 1.2 _s_−1. ●, data from a Monte Carlo simulation of a two-state folding/unfolding kinetic model using a folding rate constant, _k_f0 = 1.2s−1.

Figure 5

Refolding depends on the degree of relaxation of the I27RS8 protein. (A) A three-step pulse protocol (Inset in A) allows a first extension to completely unfold the I27RS8 protein (upper traces), then the protein is rapidly relaxed to a length L0 for a fixed period of time (5 s). A second extension then allows us to count the number of domains that refold during the relaxation period at that particular length, L0 (bottom traces). (B) Plot of Nrefolded/Ntotal vs. L0/L for 5, 6, 10, 5, 5, 4, 8, 2, 6, and 5 data points obtained from three separate experiments. The black solid line corresponds to the prediction of Eq. 2, using _k_f0 = 1.2 s−1 and a Δxf = 2.3 nm. ● corresponds to a Monte Carlo simulation of a two-state kinetic model with a folding distance Δxf = 2.3 nm.

Figure 6

(A) Chevron plot of the folding kinetics of isolated I27s. Natural logarithm of unfolding (⋄) and refolding (○) rate constants vs. denaturant concentration. The extrapolation of ln_k_u to 0M denaturant is shown (dashed line). A model of the expected kinetics for a two-state kinetic system is shown (solid line). The refolding and unfolding rate constants from AFM are shown (● and ■, respectively). (B) Plot of pulling rate ÷ 28.5 vs. unfolding force, redrawn from the data shown in Fig. 3. Extrapolation of the AFM data (■) to zero force predicts the spontaneous rate of unfolding of the protein obtained by chemical denaturation (⋄).

Figure 7

Comparison of the folding pathway of an Ig domain denatured by an applied force or chemical denaturants. (A) Model of the stretching of a single Ig domain. Under an applied force, an Ig domain unravels, causing an increase in the end-to-end length, Δx. (B) Diagram of the folding pathway for an Ig domain as determined by using AFM. The changes in free energy (Δ_G_) are plotted vs. the reaction coordinate (end-to-end extension; Δx). Three distinct states are identified: native (N, Δx = 0), condensed denatured (CD, Δx = 25.5Å), and extended denatured (ED, 25.5 < Δx < 284 Å). The transition state, ‡, is located 2.5 Å away from the native state and 23 Å away from the condensed denatured state. (C) The folding pathway determined by using chemical denaturants. The changes in free energy (Δ_G_) between the native, N, and the denatured, D, state are shown vs. the reaction coordinate characterized by a fractional distance δ, where 0 < δ < 1 and δ‡ = _m_‡−N/_m_D−N = 0.1. The putative intermediate is not shown, as its position in the folding coordinate has not been determined.

References

1. Bork P, Downing A K, Kieffer B, Campbell I D. Q Rev Biophys. 1996;29:119–167. - PubMed
1. Bork P, Helm L, Sander C. J Mol Biol. 1994;242:309–320. - PubMed
1. Fong S, Hamill S J, Proctor M, Freund S M V, Benian G M, Clothia C, Bycroft M, Clarke J. J Mol Biol. 1996;264:624–639. - PubMed
1. Plaxco K W, Spitzfaden C, Campbell I D, Dobson C M. Proc Natl Acad Sci USA. 1996;93:10703–10706. - PMC - PubMed
1. Clarke J, Hamill S J, Johnson C M. J Mol Biol. 1997;270:771–778. - PubMed

Mechanical and chemical unfolding of a single protein: a comparison - PubMed (original) (raw)