Site-specific collapse dynamics guide the formation of the cytochrome c' four-helix bundle -- Kimura et al. 104 (1): 117 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Kimura et al. 10.1073/pnas.0609413103.

Supporting Information

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Supporting Table 1
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. Kinetics of quenching of N-acetyl-tryptophanamide (NATA) fluorescence by varying concentrations of N-bromosuccinimide (NBS) using the CTF mixer. The folding time was estimated from the position of the fiber based on the bimolecular rate constant (~4.2 ´ 10⁵ M^-1·s^-1), which was determined independently and is comparable to previous results (1, 2). The final concentrations of NBS are 5.0 mM (black), 10.0 mM (red), and 20.0 mM (blue). Solid lines are single exponential fits to the data with rate constants of 2.2 ´ 10³ s^-1 (black), 4.3 ´ 10³ s^-1 (red), and 8.0 ´ 10³ s^-1 (blue). The data were acquired by using a flow rate of 5.4 ml/min. The NATA concentration was 18 mM in 20 mM sodium phosphate buffer at pH 7.0 and 18°C.

1. Shastry MCR, Luck SD, Roder H (1998) Biophys J 74:2714-2721.

2. Peterman BF (1979) Anal Biochem 93:442-444

Supporting Figure 7

Fig. 7. Probability distributions of D-A distances [P(r)] for the unfolded states of cyt c' obtained by using linear least squares with a nonnegativity constraint (LSQNNONEG) (black bar), maximum entropy analysis (ME) (red bar), and freely jointed chain model (blue line) analyses. (A) P(r) extracted from the FET kinetics of W32 in 2.0 M GuHCl. (B) P(r) extracted from the FET kinetics of W72 in 3.5 M GuHCl.

Supporting Figure 8

Fig. 8. Moments of the P(r) distributions for refolding kinetics probed by W32 (Left) and W72 (Right) cyt c'. Folding of cyt c' was triggered by dilution of GuHCl in an ultrafast mixer (pH 7.0; ~18°C; W32 [GuHCl] = 2.0 - 0.33 M; W72 [GuHCl] = 3.5 - 0.58 M). (A and D) First moments (M₁). (B and E) Second moments (M₂). (C and F) Variance as a function of refolding time.

Table 1. Characteristics of the distributions of D-A distances for unfolded W-labeled variants of cyt c′ extracted from the fits of the FET kinetics according to freely jointed chain model (FJC; Eq. 2), linear least squares with a nonnegativity constraint (LSQNONNEG), and maximum entropy analysis (ME)

Variant	N *	FJC				LSQNONNEG		ME
Variant	N *	<r²>,Å^{2 †}	l _p, Å ^‡	r , Å^§	<r>_rms, Å^¶	r , Å^§	<r>_rms, Å^¶	r , Å^§	<r>_rms, Å^¶
W32	81	2.12 x 10³	5.1	42.4	46.0	37.3	38.3	35.6	36.5
W72	41	1.95 x 10³	6.9	40.6	44.1	35.9	37.1	34.8	35.9

*Number of residues separating tryptophan (W) and the heme (Cys-113).

^†Mean-squared end-to-end distance. ^‡Persistence length extracted from <r²> and N. ^§Mean value given by the first moment of the distribution. ^¶Root-mean-squared value derived from the second moment of the distribution.