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Comparative Study
. 2003 Oct;85(4):2606-18.
doi: 10.1016/s0006-3495(03)74683-9.

Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4

Said A Ali  1 , Noriyuki Iwabuchi, Takuro Matsui, Ken Hirota, Shun-Ichi Kidokoro, Munehito Arai, Kunihiro Kuwajima, Peter Schuck, Fumio Arisaka
Affiliations
Comparative Study

Reversible and fast association equilibria of a molecular chaperone, gp57A, of bacteriophage T4

Said A Ali et al. Biophys J. 2003 Oct.

Abstract

The association of a molecular chaperone, gp57A, of bacteriophage T4, which facilitates formation of the long and short tail fibers, was investigated by analytical ultracentrifugation, differential scanning microcalorimetry, and stopped-flow circular dichroism (CD) to establish the association scheme of the protein. Gp57A is an oligomeric alpha-helix protein with 79 amino acids. Analysis of the sedimentation velocity data by direct boundary modeling with Lamm equation solutions together with a more detailed boundary analysis incorporating association schemes led us to conclude that at least three oligomeric species of gp57A are in reversible and fast association equilibria and that a 3(mer)-6(mer)-12(mer) model described the data best. On the other hand, differential scanning microcalorimetry revealed a highly reversible two-step transition of dissociation/denaturation, both of which accompanied decrease in CD at 222 nm. The melting curve analysis revealed that it is consistent with a 6(mer)-3(mer)-1(mer) model. The refolding/association kinetics of gp57A measured by stopped-flow CD was consistent with the interpretation that the bimolecular reaction from trimer to hexamer was preceded by a fast alpha-helix formation in the dead-time. Trimer or hexamer is likely the functional oligomeric state of gp57A.

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Figures

FIGURE 1
FIGURE 1
Dependence of the weight average buoyant molar mass (Mb) upon the weight concentration of gp57A, determined from sedimentation equilibrium experiments using a range of loading concentrations from 0.02 to 7.0 mg/ml, at 20°C at rotor speeds of 12,000, 15,000, and 18,000 rpm. Absorbance and Rayleigh optics as well as different optical pathlength centerpieces are used to cover the whole range of concentration as indicated by the different symbols, and at each concentration global modeling was used to calculate the best-fit buoyant molar mass. The whole data sets are globally fitted to the trimer-hexamer-dodecamer model (solid line). The binding constants, K3–6 and K6–12, are (2.17 ± 0.10) × 104 M−1 and (2.63 ± 0.10) × 104 M−1, respectively.
FIGURE 2
FIGURE 2
Sedimentation coefficient distribution c(s) derived from sedimentation velocity data at 50,000 rpm and 20°C. Loading concentrations were 0.1, 0.31, 0.52, 0.84, 1.05, and 2.1 mg/ml. The c(s) distribution was derived with the frictional ratio (f/f0) and the base line offset as a floating parameters, and with maximum entropy regularization with a confidence limit of p = 0.95.
FIGURE 3
FIGURE 3
Raw sedimentation velocity data and global fit incorporating an explicit trimer-hexamer-dodecamer model. Shown are only every 10th experimental absorbance profile (circles) obtained at a rotor speed of 50,000 rpm at 20°C at pH 8.0, at concentrations of 0.1 mg/ml (a), 0.52 mg/ml (c), and 2.1 mg/ml (e). The scans shown are taken from equivalent timepoints for each loading concentration. A global fit with a trimer-hexamer-dodecamer model was achieved with a larger set of sedimentation data at loading concentrations of 0.1, 0.31, 0.52, 1.05, and 2.1 mg/ml which resulted in binding constants of K3–6 = (3.4 ± 0.6) × 104 M−1 and K6–12 = (7.62 ± 1.3) × 103 M−1, with sedimentation coefficients s3 = 2.16 ± 0.09 S, s6 = 3.25 ± 0.04 S, and s12 = 4.59 ± 0.08 S. The solid lines show the best-fit sedimentation distributions (including time-invariant noise components). The residuals of the fits corresponding to the data in a, c, and e are shown in b, d, and f, respectively.
FIGURE 4
FIGURE 4
The pH dependence of the self-association of gp57A. The protein was dialyzed against buffers at different pH and sedimented at loading concentrations of 0.1, 0.36, 0.63, 1.05, 1.57, and 2.1 mg/ml at a rotor speed of 50,000 rpm and a temperature of 20°C. Shown are the sedimentation coefficient distributions for 0.63 mg/ml at pH values of 2.8, 5.2, 6.2, 7.3, 8.0, and 9.0, respectively. The buffers used are 5 mM NH4-acetate, 100 mM NaCl at pH 2.8; 100 mM Na-acetate, 50 mM NaCl at pH 5.2; 100 mM MES buffer, 50 mM NaCl at pH 6.2; 100 mM Tris-HCl, 50 mM NaCl at pH 7.3 and pH 8.0; and 100 mM glycine, 50 mM NaCl at pH 9.0, respectively. (Inset: sw vs. pH.)
FIGURE 5
FIGURE 5
(a) Temperature dependence of the self-association of gp57A. Sedimentation velocity experiments were conducted at temperatures of 4, 12, 20, 28, and 37°C, at a rotor speed of 50,000 rpm and at pH 8.0. Shown are the sedimentation coefficient distributions c(s) derived from the data at a loading concentration of 0.63 mg/ml. A global analysis using a trimer-hexamer-dodecamer model was globally fitted to the sedimentation data from loading concentrations of 0.42, 0.52, and 0.63 mg/ml, using the predetermined sedimentation coefficients for each oligomer. (b) Shows a van't Hoff plot of the temperature dependence of the trimer-hexamer and the hexamer-dodecamer association step.
FIGURE 5
FIGURE 5
(a) Temperature dependence of the self-association of gp57A. Sedimentation velocity experiments were conducted at temperatures of 4, 12, 20, 28, and 37°C, at a rotor speed of 50,000 rpm and at pH 8.0. Shown are the sedimentation coefficient distributions c(s) derived from the data at a loading concentration of 0.63 mg/ml. A global analysis using a trimer-hexamer-dodecamer model was globally fitted to the sedimentation data from loading concentrations of 0.42, 0.52, and 0.63 mg/ml, using the predetermined sedimentation coefficients for each oligomer. (b) Shows a van't Hoff plot of the temperature dependence of the trimer-hexamer and the hexamer-dodecamer association step.
FIGURE 6
FIGURE 6
Observed heat capacity of gp57A in pH 8.0, 50 mM sodium phosphate buffer with various protein concentration and theoretical functions. Open circles are the observed and the solid lines are the best-fitted functions by global analysis, with the same thermodynamic parameters for all the concentrations, represented by thick lines (trimer intermediate model) and thin lines (dimer intermediate model), respectively. The protein concentrations are indicated in the figure.
FIGURE 7
FIGURE 7
Observed heat capacity of gp57A in pH 2.8, 20 mM glycine buffer with different protein concentration and theoretical functions. (Meaning of symbols and lines are the same as in Fig. 6.)
FIGURE 8
FIGURE 8
(a) Temperature dependence of the residue ellipticity, [θ222], of gp57A at 222 nm in 50 mM sodium phosphate buffer, pH 8.0. (b) Derivative of the [θ222] in a, with respective to temperature reveals two peaks corresponding approximately to the two Tm values in DSC, indicating that both transitions accompany a decrease in α-helicity.
FIGURE 8
FIGURE 8
(a) Temperature dependence of the residue ellipticity, [θ222], of gp57A at 222 nm in 50 mM sodium phosphate buffer, pH 8.0. (b) Derivative of the [θ222] in a, with respective to temperature reveals two peaks corresponding approximately to the two Tm values in DSC, indicating that both transitions accompany a decrease in α-helicity.
FIGURE 9
FIGURE 9
(a) A representative kinetic refolding curve of gp57A monitored by stopped-flow CD at 222 nm (black line). Inset shows the curve within 0.4 s of the refolding reaction. Protein concentration was 9.87 μM. The fitting curves assuming the bimolecular, trimolecular, and tetramolecular reactions (see Materials and Methods) are also shown by red, blue, and green lines, respectively. (b) Dependence of the apparent rate constant (kapp) on the protein concentration. The straight line is the best fit using the equation kapp = 2[gp57A]kf with kf = (1.45 ± 0.04) × 106 M−1 s−1. (c) The ellipticity value at zero time of the refolding reaction plotted against the protein concentration. The straight line shows the average value of the data (−7560 ± 240° cm2 dmol−1).
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