Ribosomes
Magnesium ion binds ribosomes and maintains native structure of 70S ribosomes [11-15]. To investigate the structural change in the absence of magnesium, freshly purified ribosomes were analyzed by HPLC at various concentrations of magnesium ions (Figure 9A). We did not observe apparent dissociation of 70S into smaller 50S and 30S subunits. Instead, the 70S peak gradually disappeared and re-appeared as a broader peak eluted at earlier time. The apparent molecular weights for the non-native ribosomes implied that higher order of reorganization might have occurred in the magnesium-free environment. To completely remove magnesium ions, increasing concentration of EDTA was supplemented into the running buffer. In the presence of 5 mM EDTA two peaks appeared at 6.2 and 6.8 min (Figure 9B). To explore the possible conformational reorganization of ribosomes, these two peaks were collected and examined under Cryo-EM (Figure 2B, 2C). In the presence of magnesium, 70 S ribosomes acquired a native conformation; in the absence of magnesium ribosomes appeared aggregates under EM. Judging by the size revealed by EM image, the 6.2 min peak consisted aggregate of 50S subunit while the 6.8 min peak appeared to be aggregate of 30S subunit. The appearance of aggregates revealed molecular sizes and weights much larger than the pore size of exclusion resin. It is likely that the collected peaks contain 50S and 30S subunits respectively. The subunits interacted with each other and presented molecular size larger than 70S but still smaller than the pore size of exclusion resin thus were eluted earlier than 70S ribosome but later than the exclusion peak. During the sample preparation for Cryo-EM, the collected fraction lost water and generated large aggregates. Structural characterization must be performed to verify this
statement.
HPLC has been applied to the analysis and purification for the protein components of ribosomes (ref). Hydrophobic interaction is applied as a final touch in the preparation of ribosomes for crystallography [21]. Affinity HPLC is so far the fastest purification tool [22]. However, both methods lack the ability of analyzing structural variation for the purified ribosomes. Gel-filtration HPLC provides an economic and fast tool to purify ribosomes from crude extract. The ability to resolve structural interaction of ribosomes indicates additional dimension for conformational study.
Fig 9:HPLC analysis of purified ribosomes at various concentrations of magnesium ions.
(A), and in the presence of EDTA (B). The concentrations of magnesium ions and EDTA are marked on the left of each chromatogram.
Chapter 5 : Conclusion
We applied HPLC-gel filtration system for the analysis and purification of prokaryotic ribosomes. The ribosomes thus purified were optically purer and more stable in solution for storage. The current study provided a fast and convenient procedure alternative for the purification of ribosome. In addition, HPLC was demonstrated as a handy tool to access structural information for ribosomes. There are some problems with our method. Although the ribosome become more purer and more stable, we can not purify great quantity of ribosomes with HPLC-gel-filtration. It is due to the injection volume of gel-filtration column.
Chapter 6 : References
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