Download I. Characteristics of amino acids and folding of nascent polypeptides

I. Characteristics of amino acids and folding of nascent polypeptides
You will be responsible for knowing the 3 letter code for the different amino acid
residues (e.g., Trp for tryptophan), and their general characteristics, i.e, hydropholic, polar, and
charged. Please see pp 59-64 for an overview of proteins.
Secondary structural elements (alpha helices and beta strands) will often form in a newly
translated protein, largely determined by the polypeptide’s primary sequence (amino acid
sequence). Higher order folding (tertiary and quaternary structures) will ofter require the
assistance of other proteins called molecular chaperones (Fig. 7.32). A chaperone interacts with
a newly synthesized (nascent) polypeptide before it folds or with an improperly folded or
unfolded protein (often caused by environmental stress such as elevated temperature). The
chaperone does not become part of the final structure of the protein, but prevents aggregation of
proteins and enables efficient acquisition of proper folded conformation. Chaperones that work
in the cytoplasm of Bacteria often rely on ATP hydrolysis for their function.
Related proteins are also important for targeting nascent polypeptides to different cellular
compartments. A large percentage of proteins in cells are translocated outside of the cytoplasm
(where translation occurs) to the cytoplasmic membrane, to the extracellular environment, and
(in Gram negative bacteria) to the periplasm/outer membrane. This targeting is often dependent
on the proteins interacting with chaperones before or during the translocation process.
II. Localization of unfolded secreted proteins (Experimental model:
periplasm/outer membrane of E. coli)
A. Signal sequences of exported proteins (Text pp 203-204).
(also called leader or signal peptides--SS's).
Proteins destined for export beyond the cytoplasm of the cell are often synthesized with an
amino terminal extension--a signal sequence--that is proteolytically removed during protein
export from the cytoplasm. There is not a defined sequence that acts as a SS, but rather a
characteristic arrangement of charged and hydrophobic amino acids. A polypeptide with an
attached SS is often described as the “precursor” form of the protein (precursor protein). The SS
is recognized by components of the bacterial secretion or Sec apparatus that mediates precursor
export.
B. Secretory (Sec) apparatus (Text pp 77; 202-204).
The Bacterial Sec apparatus (i.e., theSecE & SecY proteins) is highly related to the eukaryotic
SEC translocation channel in the ER membrane of cells .
i.
Essential components: SecA, SecE, SecY, Leader peptidase (Lep); SRP
The secretion process relies on both ATP and Proton Motive Force; The core secretion
channel in the cytoplasmic membrane is a complex of SecE, SecY and SecG (= SecYEG or
SecEYG….all the same)
ii.
Other components: SecB, SecD, SecF (unknown what SecDF do)
iii.
SS-containing polypeptides are targeted to a cytoplasmic membrane-bound
machinery that uses energy to export a polypeptide across the cytoplasmic membrane.
For a protein to be a suitable substrate for export via the secretion (Sec) machinery,
the polypeptide must be relatively unfolded (no tertiary or quaternary structure).
Post-translational process: After translation of the SS-containing precursor polypeptide, SecA
binds to it (at the SS) while in the cytoplasm; an additional chaperone like SecB may also bind to
other regions of the polypeptide to keep it from folding. The SecA-precursor protein complex
then binds to the SecYEG secretion apparatus in the cytoplasmic membrane. SecA inserts part of
itself and of the precursor protein into the SecYEG channel (leaving the N-terminus of the
precursor on the cytoplasmic face of the channel). SecA hydrolyzes ATP, comes back out of the
channel (leaving the precursor associated with SecYEG). and binds another segment of the
precursor; the process repeats, resulting in sequential threading of the unfolded precursor protein
in 20-30 amino acid segments through the Sec YEG channel. This sequential process relies on
conformational changes in SecYEG, energized by PMF. Early in the secretion process, Leader
Peptidase (= Lep) cuts the SS from the precursor protein.
Co-translational process: Proteins that are localized to the cytoplasmic membrane in Bacteria
have segments approximately 20 aa’s in length, which are highly enriched for hydrophobic
amino acids. These are “transmembrane domains,” which will span the cytoplasmic membrane
of the cell. The first transmembrane domain of a cytoplasmic membrane protein will act like a
SS in some ways (see above): during the translation of a cytoplasmic membrane protein, the
Signal recognition particle (SRP: a complex of a small RNA and a few proteins) will bind to the
first hydrophobic transmembrane domain of the protein and bring the translating ribosome with
the nascent polypeptide to the membrane. There, it will bind to its receptor (FtsY in the handout
figure), and then the ribosome will continue to translate the nascent cytoplasmic membrane
protein into the SecYEG channel. From there, the protein will be incorporated into the
membrane. The process is similar to post-translational export (see above), expect that no SecBlike chaperone is required and no SS-cleavage occurs. The bacterial SRP is related to the SRP
complex that acts during eukaryotic protein secretion.
III. Tat apparatus: for the export of folded proteins
Bacteria also rely on a different export apparatus to localize particular proteins that fold in the
cytoplasm before localization.
Core apparatus: TatABC proteins in the cytoplasmic membrane of the cell
Tat exported proteins are targeted to the TatABC apparatus via an N-terminal signal sequence,
which contains a Twin Arg Tag (hence the Tat). The Tat SS is about 15 aa’s longer than a Secrecognized SS and contains a motif composed of the residues Ser or Thr-Arg-Arg, followed by
any aa, then two hydrophobic aa’s.
The Tat localization process has not been as well studied as the Sec process/apparatus.
Nevertheless, it appears that the process is:
i.
A folded Tat-exported protein associates with TatBC, via its TAT sequence.
ii.
TatBC-bound to its protein then associates with the TatA channel.
iii.
The folded protein translocates through the membrane in a PMF-dependent manner.
The TAT sequence is removed from the protein during this process.
While the Tat machinery is commonly found in Bacteria and Archaea, it is only found in
chloroplasts of eukaryotic cells (no Tat export in the ER).