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. 2021 Jun 24;125(24):6543-6558.
doi: 10.1021/acs.jpcb.1c04473. Epub 2021 Jun 10.

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins

Rachel B Hutchinson  1 , Xi Chen  1 , Ningkun Zhou  1 , Silvia Cavagnero  1
Affiliations

Fluorescence Anisotropy Decays and Microscale-Volume Viscometry Reveal the Compaction of Ribosome-Bound Nascent Proteins

Rachel B Hutchinson et al. J Phys Chem B. .

Abstract

This work introduces a technology that combines fluorescence anisotropy decay with microscale-volume viscometry to investigate the compaction and dynamics of ribosome-bound nascent proteins. Protein folding in the cell, especially when nascent chains emerge from the ribosomal tunnel, is poorly understood. Previous investigations based on fluorescence anisotropy decay determined that a portion of the ribosome-bound nascent protein apomyoglobin (apoMb) forms a compact structure. This work, however, could not assess the size of the compact region. The combination of fluorescence anisotropy with microscale-volume viscometry, presented here, enables identifying the size of compact nascent-chain subdomains using a single fluorophore label. Our results demonstrate that the compact region of nascent apoMb contains 57-83 amino acids and lacks residues corresponding to the two native C-terminal helices. These amino acids are necessary for fully burying the nonpolar residues in the native structure, yet they are not available for folding before ribosome release. Therefore, apoMb requires a significant degree of post-translational folding for the generation of its native structure. In summary, the combination of fluorescence anisotropy decay and microscale-volume viscometry is a powerful approach to determine the size of independently tumbling compact regions of biomolecules. This technology is of general applicability to compact macromolecules linked to larger frameworks.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Hypothetical limiting models describing the ribosome-bound apoMb nascent chain (RNC) compaction. Models a and b can be eliminated due to the known highly spatially confined environment of the nascent chain (cone semiangle = 20 ± 1°). Model c can be eliminated because the compact region explored in this work independently tumbles on the low-ns timescale (see fluorescence anisotropy decay analysis). This timescale is incompatible with the much slower tumbling of the ribosome. Models d, e, and f are consistent with previously published work but could not be discriminated from each other in the past.
Figure 2.
Figure 2.
Summary of workflow design to determine degree of compaction of RNCs. The rotational correlation time (τc) describing nascent-chain compact-subdomain motions and RNC macroscopic viscosity (ηmacro) were determined via fluorescence anisotropy decay in the frequency domain and microscale-volume viscometry, respectively. Experimentally determined values were employed to determine the size of the compact subdomain. Spherical, prolate ellipsoidal, and oblate ellipsoidal models were used to model the compact region of the nascent chain.
Figure 3.
Figure 3.
Effect of molecular crowding and excluded volume on microscale-volume viscosity. (a) The excluded volume comprises the space occupied by molecules (green) and the depletion layer surrounding them (gray). (b) Within a crowded solution, two molecules cannot occupy the same space, but their depletion layers can overlap. (c) The viscosity within the depletion layer (ηdl) surrounding a particle is between the pure-buffer (solvent system) viscosity (ηs) and the experimentally measured macroscopic viscosity (ηmacro).
Figure 4.
Figure 4.
Experimentally measured viscosity of RNC solutions (blue) and buffer (yellow).
Figure 5.
Figure 5.
Fluorescence anisotropy decay in the frequency domain enables resolving multiple rotational modes, including one corresponding to an apoMb RNC compact subdomain (rotational correlation time τc = 5–9 ns). (a) Simulations of 2- and 3-component frequency-domain anisotropy decay data. (b) Representative physical models for samples with one global tumbling motion one or two local tumbling motions. (c) Prior anisotropy decay data revealed that ApoMb nascent chains become compact and tumble independently starting from chain lengths of ca. 57 residues.
Figure 6.
Figure 6.
Order parameters and cone semiangles define the spatial confinement of nascent chain dynamics. (a) Dependence of order parameter on cone semiangle, assuming a square-well potential. (b) General features of cone-semi angle models. The vector μ⇀ defines the fluorophore’s symmetry axis, whose orientation can fluctuate so that its angle with the z-axis (θ) is less than or equal to the cone semiangle θ0. The motions are randomly distributed within the XY plane and can assume any value of the azimuthal angle ϕ) The z-axis is defined to be perpendicular to the macromolecular surface the tumbling species is attached to. (c) Cartoon description of macromolecules bearing either one or two local motions that are spatially confined within a cone.
Figure 7.
Figure 7.
Representative frequency-domain fluorescence anisotropy-decay data for full-length apoMb RNCs.
Figure 8.
Figure 8.
The size of the RNC compact subdomain can be deduced from the experimental rotational correlation time (for the low-ns motion) and from the experimentally determined viscosity. Three limiting molecular shapes were considered: a. spherical, prolate ellipsoid (axial ratio = 3.5) and oblate ellipsoid (axial ratio = 0.5). b. Plot illustrating the experimentally determined rotational correlation time for the intermediate-timescale motions (τc,I) as a function of number of amino acids in the compact subdomain. Domain sizes compatible with experimentally determined rotational correlation times are shaded in color (blue, orange, green).
Figure 9.
Figure 9.
RNC models compatible with experimentally determined rotational correlation times and viscosity values.
Figure 10.
Figure 10.
RNC models compatible with spatially-confined dynamics described by experimental cone-semi angles. The spatial confinement of the nascent chain dynamics suggests that the compact domain lies within the ribosomal vestibule or is somehow spatially confined within the outer surface of the ribosome.
Figure 11.
Figure 11.
Compact nascent-apoMb subdomain does not include residues corresponding to the native C-terminal helices. a. Size of independently tumbling compact subdomain mapped onto the structure of native apoMb. b. RNC structures compatible with our experimental data.
Figure 12.
Figure 12.
Computational data rationalizing why the nascent-apoMb compact subdomain lacks residues corresponding to the native G helix. (a) Native apoMb structure of with G and H helices colored in blue and green, respectively. (b) Elongation net-charge nonpolar (NECNOP) plot for apoMb generated according to Yaeger-Weiss et al. The black discriminant line separates regions corresponding to folded (right of line) and disordered proteins (left of line). (c) Fraction of nonpolar solvent-accessible surface area (NSASA) at different apoMb chain lengths. Adapted with permission from Kurt, N.; Cavagnero, S. J. Am. Chem. Soc. 2005, 127 (45), 15690–15691. Copyright (2005) American Chemical Society. (d) Predicted free energy of folding for different apoMb chain lengths. See Figure S6 for predicted folding free energies of additional chain lengths. All free energy calculations were carried out with FoldX version 5. PDB files for apoMb (RCSB PDB: 1mbc) were generated with Pymol version 2.0.0.
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