Structural Biochemistry/Proteins/Protein Folding on the Ribosome

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Although much work has been done on protein folding "in vitro", few research has significantly advanced the work contributing to "in vivo" protein folding. The importance of the latter comes as a consequence that protein folding is presumably guided by a molecular mechanism instead of a protein independently folding according to the lowest energy conformation. Although it has proven that proteins are highly successful at reaching their native state only by chaperone proteins, it seems that at the creation of a new protein, something must assist the development of the secondary and tertiary structure. The authors of a current opinion article in Structural Biology, Lisa D. Cabrita, Christopher M. Dobson, and John Christodoulou have published an update on the recent discoveries of how the nascent chains of a newly synthesized protein emerges in the article entitled, "Protein Folding on the Ribosome."

Folding on Ribosome[edit]

The place where the protein chain begins to fold is a topic that is greatly studied. As the nascent chain goes through the “exit tunnel” of the ribosome and into the cellular environment, when does the chain begin to fold? The idea of cotranslational folding in the ribosomal tunnel will be discussed. The nascent chain of the protein is bound to the peptidyl transferase centre (PTC) at its C terminus and will emerge in a vectorial manner. The tunnel is very narrow and enforces a certain rigidity on the nascent chain, with the addition of each amino acid the conformational space of the protein increases. Co translational folding can be a big help in reducing the possible conformational space by helping the protein to acquire a significant level of native state while still in the ribosomal tunnel. The length of the protein can also give a good estimate of its three dimensional structure. Smaller chains tend to favor beta sheets while longer chains (like those reaching 119 out of 153 residues) tend to favor the alpha helix.

The ribosomal tunnel is more than 80 ampere in length and its width is around 10-20 ampere. Inside the tunnel are auxiliary molecules like the L23, L22, and L4 proteins that interact with the nascent chain help with the folding. The tunnel also has hydrophilic character and helps the nascent chain to travel through it without being hindered. Although rigid, the tunnel is not passive conduit but whether or not it has the ability to promote protein folding is unknown. A recent experiment involving cryoEM has shown that there are folding zones in the tunnel. At the exit port (some 80 ampere from the PTC), the nascent chain has assumed a preferred low order conformation. This enforces the suggestion that the chain can have degrees of folding at certain regions. Although some low order folding can occur, the adoption of the native state occurs outside the tunnel, but not necessarily when the nascent chain has been released. The bound nascent chain (RNC) adopts partially folded structure and in a crowded cellular environment, this can cause the chain to self-associate. This self-association, however, is relieved with the staggered ribosomes lined along the exit tunnel that maximizes the distances between the RNC.

The current understanding of protein folding has come from in vitro studies of renaturation of proteins through a variety of different environments as well as in silico computer simulations. These studies can only help to extrapolate fractions of the in vivo process of protein formation. Protein folding is initiated following the synthesis of the nascent polypeptide chain as it is synthesized by the ribosome. The start of protein folding is therefore coupled with the continuing synthesis of the polypeptide chain.

Currently, protein folding is view as a process that takes place as a consequence of interactions been the amino acid of that protein which can take certain paths to achieve a lowest energy state, the native state. However, there are certain paths a protein may start to fold by and lead to a conformation that is of low energy but not the native state. The protein has not way of coming of this conformation without a significant amount of energy input. This non-native state is a way a protein can be misfolded and lead to aggregation. Another factor that can influence the likelihood of obtaining the native state is the fact that larger proteins have more possibilities of folding, this decreases the likelihood of forming the most energetically favorable state. Proteins us the "co-translational folding' to reduce the extent of conformational space available to the protein. Adding to this, molecular chaperones help to further assist proteins in achieving their native conformational state.

Generation of RNC for studies[edit]

One technique of generating RNC and taking snapshots as it emerges from the tunnel is to arrest translation. A truncated DNA without a termination sequence is used. This allows for the nascent chain to remain bound until desired. To determining the residues of the chain, they can be labeled by carbon-13 or nitrogen-15 and later detected by NMR spectroscopy. Another technique is the PURE method and it contains the minimal components required for translation. This method has been used to study the interaction of the chains and auxiliary molecules like the TF chaperone. This method is coupled with quartz-crystal microbalance technique to analyze the synthesis by mass. An in vivo technique in generating RNC chain can be done by stimulating it in a high cell density. This is initially done in an unlabeled environment, the cells are then transferred to a labeled medium. The RNC is generated by SecM. The RNC is purified by affinity chromatography and detected by SDS-PAGE or immunoblotting.

By generating the RNCs, many experiments can be done to study more about the emerging nascent chain. As mentioned above, the chain emerges from the exit tunnel in a vectorial manner. This enables the chain to sample the native folding and increases the probability of folding to the native state. Along with this vectorial folding, chaperones also help in favorable folding rates and correct folding.

Ribosome Structure and Co-translational Protein Folding[edit]

In E. coli the 70S ribosomal particle is composed of 50 proteins and three RNA molecules. The most interesting structural feature in the 70S ribosomal particle in regards to protein folding is the ribosomal exit tunnel. This is a channel that links PTC(peptidyl transferase centre) with the cellular environment. The dimensions include a length of 80 angstoms, width between 10-20 angstroms. 70S is lined with a large RNA molecule and L4 and L22 ribosomal proteins. Also L23 serves as a docking point for other molecules to assist in the folding process. L4 and L22 proteins in the ribosomal exit tunnel have been shown by recent cryoEM studies that they can interfere with proteins synthesis along with other interactions with the nascent chain. In addition, arginine residues have been observes to stop the translation process by changing electrostatic potentials. Although ribosomal exit tunnel is presumably to have a more or less rigid structure, it seems that it does partake to a degree support nascent chain folding. This is evidence by the fact that on average the tunnel is able to accommodate about 30-40 residues, which is considerably more than a polypeptide chain sequence that is fully extended. The degree to which a nascent chain folds seems to vary depending on the kind of protein being synthesized. Certain nascent chains transmembrane protein sequences appear to possibly already construct an alpha-helical structure inside the tunnel. Studying nascent chains emerging from the ribosomal exit tunnel has proven to be a significant challenge for any of the current methods of structural and cellular biology. One idea presented in this paper is to take be able to have "snapshot" of the elongation process. In order to due this, translation must be arrested artificially which would involve engineering DNA strands that lacks a stop codon. Another issue is also in focusing on the particular residues of interest on the nascent chain within the sea of other residues form the ribosome.

Understanding Co-translational Folding by Biochemical and Biophysical Studies[edit]

Once examples illuminated in the article is using SDS-Page on the risbosomal bound nascent chains(RNCs) of influenza haemagglutinin which showed they can form disulfide bonds and undergo glycosylation. Also, using monoclonal antibodies, it has been discovered that there is variability in the emergence of the nascent chain from the tunnel. These examples among others demonstrate that not only can nascent chains acquire structure but also activity while still being attached to the ribosome. The speed of folding for nascent chains seems to be related to the number of stop and rare codons present. The reasoning is that a discontinuous translation rate will slow down the folding process. However, slower rates seem to produce more efficient folding since the nascent chain has more time to develop its native structure. Most of the biochemical and physical methods illuminating the understanding of co-translational folding has been eluded by x-ray crystallography because of the dynamic nature of the folding process which in crystallography is very difficult to obtain.

Auxiliary Factors in Co-translational folding[edit]

As the nascent chain starts emerging from the tunnel, it has to opportunity to interact with molecules that will assist int eh folding process. These include molecular chaperones, peptide deformylase, and the signal recognition particle. The first molecule in assisting the nascent chain in folding is the 48kDa TF which docks on L23. This protein in the absence of a nascent chain will dock on and off however iwth the presence of the nascent chain its affinity to bind to L23 increases. TF undergoes a conformational change in a where a protective cavity is formed for the nascent chain. TF enables enough of the polypeptide chain to emerge such that a significant degree of folding can be achieved. It does this by binding to hydrophobic segments of the chain even after is has released from L23. Once hydrophobic regions of chain are no longer exposed, TF seem to unbind and allow further helper molecules to assist in protein folding. TF seems to increase folding efficiency but at the expense of being slower to fold. Protein translocation is then done by SRT which shuttles the TF to a heterotrimeric integral membrane protein. This then allow further processing and folding.

Ribosome subunit in prokaryote cells and eukaryote cells[edit]

The ribosomes catalyze peptide bond formation, in a process called peptidyl transfer catalysis, and synthesize polypeptides by reading the genetic code of the mRNA. The ribosome is composed of a large and a small subunit both in prokaryote and eukaryote cells. Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit. Due to the differences in their structures, the bacterial 70S ribosomes are vulnerable to these antibiotics while the eukaryotic 80S ribosomes are not. Within the cellular structure, mitochondria have ribosomes similar to the bacterial ones; however, mitochondria within eukaryote cells are not affected by these antibiotics because they are surrounded by membrane around its organelle. The initiation of the translation process in bacteria was found to locate on 30s subunit. This process requires the increase of both the incubation temperature and ionic strength in order to assemble into the correct tertiary structure contained with its amino acid sequence. The research experiments done by Dr. Masayasu’s research on the synthesis of ribosomes and ribosomal components in E-coli, also found that the correct assembly of the ribosomal particles is locating in the structures of their own molecular component and not by other nonribosomal factors.

A ribosome is the essential contributing factor in protein synthesis where it is assembled on the translation initiation region (TIR) of the mRNA during the initiation phase of translation. The mRNA is decoded as it slides through the large ribosomal subunit and places the a polypeptide chain in the other subunit of the ribosome. Newly synthesized protein will then dissociate once the stop codon is reached in the ribosome. In the final ribosome recycling phase, the ribosomal subunits dissociate and the mRNA is released. The main events of the translation process are relatively similar in both prokaryotic and eukaryotic cells. Major differences in the detailed mechanism of each phase exist. Bacterial translation involves relatively few factors, in contrast to the more complex process in eukaryotes.

Peptidyl Transfer Catalysis By Ribosome[edit]

During protein elongation, the ribosome PTC acts as a catalyst to cleave the

See also[edit]

Structural Biochemistry/Proteins/Protein Folding

Reference[edit]

Ki Yun Leung, Edward, et al. (2011). [1] The Mechanism of Peptidyl Transfer Catalysis by the Ribosome, 80(1):527-555.