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

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.

An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

“…The particularly large nonpolar solvent-exposed surface of L23 (Fig. 4f ), the known unfolded or partially folded nature of native chains 21 , 22 , 36 , 72 , and the data of Fig. 7b suggest that both hydrophobic effects and Mg +2 play a role in the detected interactions.…”

Nascent-Chain Interaction Networks and Their Effect on the Bacterial Ribosome

“…Next, we performed fluorescence depolarization decay experiments in the frequency domain (69–71) to probe the rotational dynamics of nascent chains encoding foldable sequences. This technique has been previously employed to assess the rotational correlation time (ρ c ) and amplitude of rotational motions of RNCs (17, 18, 34, 49, 72). The goal of this experiment was to probe whether RNCs harboring long nascent chains display any degree of compaction.…”

“…80-100 Å long, 10-35 Å wide (7–10) and is able to fit 30 to 40 nascent residues. (11–16) Within the ribosomal exit tunnel and its nearby regions across the highly negatively charged outer surface of the ribosome, (3) nascent chains encoding single-domain proteins become compact (17, 18) and acquire some secondary (19–23) and tertiary structure (5, 24–28). This set of observations proves the importance of the ribosome in nascent-protein structure formation.…”

Critical Beginnings: Selective Tuning of Solubility and Structural Accuracy of Newly Synthesized Proteins by the Hsp70 Chaperone System

“…coli . Third, apoMb is a well-biochemically characterized protein ,, that has been an important model system for detailed experimental investigations on protein folding paths upon refolding into buffered solution ,,, and within the cellular environment. ,,,,,, Fourth, apoMb is a quintessential example of the many proteins that are unable to reach their native state at 100% level upon refolding from denaturant . Indeed, purified chemically denatured wild-type apoMb forms some soluble aggregates upon refolding into buffer .…”

“…The above geometrical arguments are supported by our knowledge of the ribosome structure, including the conformational features of the ribosomal exit tunnel and by the known number (30–35) of nascent-chain residues buried inside the tunnel within known proteomes. , In addition, given the size of the known compact N-terminal domain of ribosome-bound nascent apoMb (ca. 60–80 N-terminal residues , ), the negatively charged aspartic acid of the I28D variant must reside within the compact N-terminal region outside of the ribosomal tunnel core. , Therefore, the I28D apoMb variant may perturb conformational sampling either co- or post-translationally, given that the perturbed compact domain can exert its effect during biosynthesis or after nascent-protein release from the ribosome.…”