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Folding of proteins
•Proteins are synthesized on ribosomes as linear chains of amino acids.
•In order to be biologically active, they must fold into a unique threedimensional structure.
•The tunnel that leads from the peptidyl transferase site to the exterior of the
ribosome is ~100A long, enough to shelter 30 residues. (1A=10 -9 nm)
•Once the polypep emerges it has to fold properly and reach its destination
•The tunnel is too narrow for formation of secondary structure other than
helices. Folding can start before translation is complete
Molecular chaparones are unrelated protein families whose role is to
stabilize unfolded proteins, unfold them for translocation across
membranes or for degradation,
• Chaparones bind to the end terminus of polypeptides to prevent their
aggregation, to facilitate their folding and to promote association with other
subunits
•Ribosomal proteins seem to play a role in recruiting chaperones e.g. in E coli
the trigger protein associates with a ribosomal protein located at the outlet of
the peptide exit tunnel
•Trigger recognizes relatively short hydrophobic protein segments.
• Members of the Heat shock group of proteins are also
involved early on , they bind to and protect longer
polypeptides. GroEL and GroES facilitate protein folding
after translation termination
• Chaperones include the heat shock proteins such as
HSP-60 and HSP-70
– They stabilize non-native conformation and facilitate correct
folding of protein subunits.
• They do not interact with native proteins, nor do they
form part of the final folded structures.
• Some chaperones are non-specific, and interact with a
wide variety of polypeptide chains, but others are
restricted to specific targets.
• They often couple ATP binding/hydrolysis to the folding
process.
• Essential for viability, their expression is often
increased by cellular stress.
GroEL
• GroEl is a large, multi-subunit protein that can
accommodate small- and medium-sized proteins
• It consists of a central container formed from a
stack of rings, and a 'cap' that seals off the
folding protein from the external environment
• GroEl also has ATP-binding sites that use
energy to promote the proper folding and
release of the protein
• If a protein is trapped in a particularly deep local
minimum, we call it ‘misfolded’ as thermal energy may
not be enough to kick it out of the state.
• There has been tremendous biomedical interest in
misfolded proteins as serious human diseases such as
Alzheimers’s, Parkinson’s and mad cow disease
coincide with the accumulation of misfolded proteins in
the form of amyloids.
• no consensus on how the amyloids and the disease itself
is linked but it is becoming clear that a misfolded protein
can be used as a template to cause more misfolded
proteins etc, to aggravate the situation.
• Theory is that cellular chaperone machineries are
overwhelmed by the presence of amyloids so that they
do not have enough time to help fold other essential
proteins.
There are 3 major classes of chaperones:
The Hsp70 family
DnaK is the best known member of this family in E. coli. It is a very
highly conserved protein.
The Hsp 60 family
The E. coli member of this family, GroEL forms a double-layered
cylindrical structure which provides a protected environment in
which proteins can fold. The structure is capped at one end by
another heat-shock protein: GroES, a member of the Hsp 10 family.
The Hsp 90 family
• All are named because these proteins were first identified as Heat
Shock Proteins - their synthesis increased in response to a sudden
rise in temperature.
• In this role, chaperones protect important cell proteins from
denaturation as a result of a sudden rise in temperature.
Targeting in Bacteria
• As a protein is being synthesized, decisions must be
taken about sending it to the correct location in the cell
where it will be required.
• The information for doing this resides in the nascent
protein sequence itself.
• Once the protein has reached its final destination, this
information may be removed by proteolytic processing.
• In bacterial cells, the targeting decision is relatively
straightforward: is the protein destined to be an
intracellular protein or an extracellular one?
• Some more details required in a gram-negative
bacterium, such as E. coli, there must be some way of
knowing whether a protein is destined to go to the cell
membrane, the periplasmic space, or the outer
membrane.
Secreted proteins contain a signal sequence.
This is a short (6 - 30) stretch of hydrophobic amino acids, flanked on the Nterminal side by one or more positively charged amino acids and containing
neutral amino acids with short side-chains at the cleavage site
E. coli Signal Sequences from the SwissProt Database
Entry
Description
P31550
THIAMIN-BINDING PERIPLASMIC
PROTEIN PRECURSOR
P29679
ACYL-COA THIOESTERASE I
PRECURSOR (PROTEASE I)
P19935 TOLB PROTEIN PRECURSOR
Sequence
MSAPAVAVTAPVFA
MMNFNNVFRWHLPFLFLVLLTFRAAA
MKQALRVAFGFLILWASVLHA
GLYCEROL-3-PHOSPHATEP10904
BINDING PERIPLASMIC
PROTEIN PRECURSOR
MKPLHYTASALALGLALMGNAQA
D-XYLOSE-BINDING
P37387
PERIPLASMIC PROTEIN
PRECURSOR
MKIKNILLTLCTSLLLTNVAAHA
As proteins with signal sequences are
synthesized, they are bound by the SecB
protein.
This prevents the protein from folding.
SecB delivers the protein to the cell membrane
where it is secreted through a pore formed by
the SecE and SecY proteins.
Secretion is driven by the SecA ATPase.
After the protein has been secreted, the signal
sequence is removed by a membrane bound
leader peptidase.