An analysis of alterations in ribosomal conformation using reductive methylation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Ribosomes are essential for protein synthesis.
• Understanding ribosomal protein accessibility is key to deciphering ribosome function and regulation.
• Conformational changes in ribosomes are linked to cellular processes and disease states.

Purpose of the Study:

• To optimize reductive alkylation conditions for ribosomal proteins.
• To compare the accessibility of ribosomal proteins in native, denatured, and conformationally altered states.
• To identify specific ribosomal proteins involved in structural differences and drug-induced conformational changes.

Main Methods:

• Reductive alkylation of rat liver ribosomes using [14C]formaldehyde and NaBH4.
• Separation of ribosomal proteins from RNA and denaturation in 6 M guanidine.
• Differential labeling with formaldehyde and NaB3H4 to determine protein accessibility via 3H/14C ratios.
• Two-dimensional electrophoresis for protein separation and analysis.

Main Results:

• Identified proteins with differential accessibility in native vs. denatured states (e.g., S6, S11, S26, L3, L35 less accessible; S1, S15, L11, L12, L16, L24 more accessible).
• Observed distinct protein accessibility changes in normal polysomes versus monomers (involving S3, L7, L36).
• Detected ribosomal conformation changes induced by ethionine intoxication, implicating S26, L35, S3, S20, L7, L8, L24, L27, L28, and L34.

Conclusions:

• Established a method to probe ribosomal protein accessibility in various states.
• Provided a detailed map of ribosomal protein accessibility, highlighting structural differences.
• Demonstrated the utility of reductive alkylation in studying ribosome dynamics and drug-induced conformational alterations.