Structural studies of MS2 bacteriophage virus particle disassembly by nuclear magnetic resonance relaxation measurements

Abstract

In this article we studied, by nuclear magnetic resonance relaxation measurements, the disassembly of a virus particle-the MS2 bacteriophage. MS2 is one of the single-stranded RNA bacteriophages that infect Escherichia coli. At pH 4.5, the phage turns to a metastable state, as is indicated by an increase in the observed nuclear magnetic resonance signal intensity upon decreasing the pH from 7.0 to 4.5. Steady-state fluorescence and circular dichroism spectra at pH 4.5 show that the difference in conformation and secondary structure is not pronounced if compared with the phage at pH 7.0. At pH 4.5, two-dimensional (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum shows approximately 40 crosspeaks, corresponding to the most mobile residues of MS2 coat protein at pH 4.5. The (15)N linewidth is approximately 30 Hz, which is consistent with an intermediate with a rotational relaxation time of 100 ns. The average spin lattice relaxation time (T(1)) of the mobile residues was measured at different temperatures, clearly distinguishing between the dimer and the equilibrium intermediate. The results show, for the first time, the presence of intermediates in the process of dissociation of the MS2 bacteriophage.

FIGURE 1

¹⁵N-edited proton spectra (HMQC) of MS2 coat protein at pH 4.5 (A) and 7.0 (B) at 40°C. The spectra were obtained with 1024 scans and a recycle delay of 3 s at pH 4.5, and 8 s for pH 7.0.

FIGURE 2

¹⁵N-¹H HMQC spectra of MS2 (A, B) and VLP (C) at pH 7.0 (A) and 4.5 (B, C). All spectra were acquired with 1024 × 128 points. Spectrum A was obtained with 400 scans and a recycle delay of 8 s. Spectrum B was recorded with 160 scans and a recycle delay of 3 s. The processing was performed with zero filling and a square sine multiplication shifted by 90°, in the indirect dimension and exponential multiplication with 10 Hz of line broadening in the direct dimension. States-time proportional phase incrementation method was used for quadrature detection in the indirect dimension. All spectra were done at 40°C.

FIGURE 3

Tryptophan steady-state fluorescence and circular dichroism spectra at pH 7.0 (gray) and 4.5 (black) at room temperature. The fluorescence spectra were obtained with excitation wavelength of 280 nm at room temperature.

FIGURE 4

Theoretical value of linewidth of the ¹⁵N line as a function of τ_m (overall correlation time) in several values of S² (order parameter), as indicated. The simulations were done using the Lipari-Szabo model free formalism with a fixed value of τ_e (internal correlation time) of 100 ps.

FIGURE 5

Theoretical value of T₁ as a function of τ_e (internal correlation time) in several values of τ_m (overall correlation time): (a) τ_m, 2 ns; (b) τ_m, 8 ns; (c) τ_m, 50 ns; (d) τ_m, 100 ns; and (e) τ_m, 500 ns. The simulations were done using the Lipari-Szabo model free formalism with a fixed value of order parameter (S² = 0.7). Theoretical values of T₂ are between 240 and 350 ms for a, 90 and 130 ms for b, 15 and 20 ms for c, 7.7 and 10.4 ms for d, and 2.7 and 3.5 ms for e. The experimental ¹⁵N linewidth of ∼30 Hz (obtained from Fig. 2) corresponds to τ_m = 100 ns.

FIGURE 6

Theoretical value of T₁ as a function of τ_e (internal correlation time) in several values of order parameters (S²): (a) S², 0; (b) S², 0.6; (c) S², 0.7; (d) S², 0.8; and (e) S², 0.9. The simulations were done using the Lipari-Szabo model free formalism with a fixed value of overall correlation time (τ_m = 100 ns). Theoretical values of T₂ are between 7.4 and 7.7 ms for a, 7.8 and 12.2 ms for b, 7.7 and 10.4 ms for c, 7.5 and 9.11 ms for d, and 7.4 to 8.1 ms for e. Theoretical values of T₂ in all order parameters are in agreement with the experimental ¹⁵N linewidth of ∼30 Hz (obtained from Fig. 3) when τ_m = 100 ns.

FIGURE 7

The same as Fig. 6 plus regions representing all the possible values of T₁av at each temperature. (a) S², 0; (b) S², 0.6; (c) S², 0.7; (d) S², 0.8; and (e) S², 0.9. The criteria used to find the regions are the following. At 30°C, τ_e is 2.5 ns, and the maximum and minimum S² are the T₁ corresponding to T₁av within an experimental error of 15%. For temperatures >30°C, the maximum order parameter is the one obtained at 30°C. The minimum order parameter is ‘0’, maximum disorder. The internal correlation is necessarily lower than the one at 30°C, and the minimum and maximum T₁ will be T₁av within the range of the experimental error of 15%. For temperatures <30°C, the minimum order parameter is the minimum obtained at 30°C. The maximum order parameter will be ‘1’, maximum order. The internal correlation is necessarily higher then the one at 30°C, and the minimum and maximum T₁ will be T₁av within the range of the experimental error of 15%.

FIGURE 8

Schematic diagram showing two mechanisms for MS2 disassembly. (A) Here, there are intermediates in the disassembly: a hexamer of trimers, the trimer that corresponds to the asymmetric unit, and the dimeric form that is the conformation where the capsid protein works as a repressor. (B) The two-states dissociation is shown, where the final conformation is the dimeric form.