PROTEIN STRUCTURE,

SORTING AND TARGETING

Mrs. Ofelia Solano

Saludar
Department of Natural Sciences
University of St. La Salle
 Overview of protein structure
and function.
(a) The linear sequence of amino
acids (10 structure) folds into
helices or sheets (20 structure)
which pack into a globular or
fibrous domain (30 structure).
Some individual
proteins self-associate into
complexes (40 structure).
(b) Proteins display functions
that arise from specific binding
interactions and conformational
changes in the structure of a
properly folded protein.
 Proteins have to be folded into the proper three dimensional
conformation to work properly.
 Sometimes the primary sequence of amino acids is sufficient to
spontaneously direct the folding of proteins into their proper shape.
 However, often newly-made proteins require the help of molecular
chaperones to attain their final shape. Members of the heatshock protein
family (Hsp70 and Hsp60) briefly bind to and stabilize hydrophobic regions
of proteins (especially rich in Trp, Phe, Leu) allowing proper folding instead
of aggregation with other immature proteins.
 Heat-denatured proteins can be renatured through the activity of molecular
chaperones and heatshock proteins are made during times of stress.
 A number of diseases, including Alzheimer's disease, may be considered to
be protein-folding diseases.
 Prion diseases, such as "mad cow" disease, may "self-propagate" based
upon a misfolded protein that can, in turn, misfold other versions of the same
protein.
 Chaperone- and chaperonin-mediated protein folding. (a) Many proteins fold into their proper 3-D
structures with the assistance of Hsp70-like proteins (top). These chaperones transiently bind to a nascent
polypeptide as it emerges from a ribosome. Proper folding of other proteins (bottom) depends on chaperonins
such as the prokaryotic GroEL, a hollow, barrel-shaped complex of 14 identical 60,000-MW subunits
arranged in two stacked rings. One end of GroEL is transiently blocked by the co-chaperonin GroES, an
assembly of 10,000-MW subunits. (b) In the absence of ATP or presence of ADP, GroEL exists in a “tight”
conformational state that binds partly folded or misfolded proteins. Binding of ATP shifts GroEL to a more
open, “relaxed” state, which releases the folded protein.
ER protein folding
The ER membrane-bound chaperone protein calnexin,
or a resident chaperone calreticulin binds to incompletely folded proteins,
trapping the protein in the ER. Glucosyl transferase determines whether the
protein is folded properly or not: if the protein is still incompletely folded, the
enzyme renews the protein's affinity for calnexin & retains it in the ER. The
cycle repeats until the protein has folded completely.
The export and degradation of misfolded ER proteins.
Misfolded soluble proteins in the ER lumen or membrane proteins are
translocated back into the cytosol, where they are deglycosylated,
ubiquitylated, and degraded in proteasomes. Misfolded proteins are
exported through the same type of translocator that mediated their import;
accessory proteins allow it to operate in the export direction.
 Ubiquitin proteolytic pathway
(a) Enzyme E1 is activated by
attachment of an ubiquitin (Ub)
molecule (1) and then transfers this Ub
molecule to E2 (2). Ubiquitin ligase
(E3) transfers the bound Ub molecule
on E2 to the side-chain-NH2 of a lysine
residue in a target protein (3).Ub
molecules are added to the target
protein by repeating steps 1–3 , forming
a polyubiquitin chain that directs the
tagged protein to a proteasome (4).
Within this complex, the protein is
cleaved into small peptide fragments
(5).
(b) Computer-generated image reveals
that a proteasome has a cylindrical
structure with a cap at each end of a
core region. Proteolysis of ubiquitin-
tagged proteins occurs along the inner
wall of the core.
After the amino chain is made, many
proteins undergo posttranslational
processing (including removal of stretches
of amino acids).
1. In prokaryotes, the N-formyl group is
always removed in the mature protein
and often the methionine and,
sometimes, a number of N-terminal
amino acids are cleaved away from the
final protein product.
 Example: Proinsulin is converted to the
active hormone by the enzymatic
removal of a long internal section of
polypeptide. The two remaining chains
continue to be covalently connected by
disulfide bonds connecting cysteine
residues in insulin.
2. Recently discovered, the process of
protein splicing (analagous to RNA
splicing) removes inteins and splices the
exteins together to make a mature
protein.
 PROTEIN TARGETING AND SORTING
 Free and bound populations of ribosomes are active participants
in protein synthesis.
 Free ribosomes are suspended in the cytosol and synthesize
proteins that reside in the cytosol.
 Bound ribosomes are attached to the cytosolic side of the
endoplasmic reticulum.
 They synthesize proteins of the endomembrane system as well
as proteins secreted from the cell.
 Secretory proteins are released entirely into the cisternal space,
but membrane proteins remain partially embedded in the ER
membrane.
 While bound and free ribosomes are identical in structure, their
location depends on the signal peptidase of proteins that they
are synthesizing.
Overview of major protein-sorting
pathways in eukaryotic cells.
 In cotranslational import, proteins to be targeted to the endoplasmic reticulum initially have an N-
terminal peptide, the ER signal sequence, translated by a cytosolic ribosome.
 The ER signal sequence is bound by a signal-recognition particle (SRP), a ribonucleoprotein complex
composed of 6 peptides and a 300 nucleotide RNA molecule.
 The SRP binds to the SRP receptor to dock the ribosome on the ER membrane.
 When the SRP receptor binds GTP, the nascent polypeptide enters the pore.
 The SRP is released with hydrolysis of the GTP.
 The growing polypeptide translocates through a hydrophilic pore created by one or more membrane
proteins called the translocon.
 The ribosome fits tightly across the cytoplasmic side of the pore and the ER-lumen side is somehow closed
off until the polypeptide is about 70 amino acids long.
 When the polypepide is complete, the signal peptidase cleave the signal to release the protein into the ER
lumen while retaining the signal peptide, for a time, in the membrane.
 Afterwards the ribosome is released and the pore closes completely.
 Other kinds of signal peptides are used to target polypeptides to mitochondria,
chloroplasts, the nucleus, and other organelles that are not part of the
endomembrane system.
 In these cases, translation is completed in the cytosol before the polypeptide is
imported into the organelle.
 Each of these polypeptides has a “postal” code that ensures its delivery to the
correct cellular location.
 In principle, a signal could be required for either retention in, or exit from a
compartment.
Major topological classes of integral membrane proteins synthesized on the rough
ER. The hydrophobic segments of the protein chain form helices embedded in the
membrane bilayer; the regions outside the membrane are hydrophilic and fold into various
conformations. All type IV proteins have multiple transmembrane helices. The type IV
topology depicted here corresponds to that of G protein–coupled receptors: seven helices,
the N-terminus on the exoplasmic side of the membrane, and the C-terminus on the cytosolic
side. Other type IV proteins may have a different number of helices and various orientations
of the N-terminus and C-terminus.
Integral membrane proteins are inserted into the ER membrane as
they are made, rather than into the lumen.
Posttranslational import allows some polypeptides to enter organelles after
protein synthesis. Like cotranslational import into the ER, posttranslational
import into a mitochondrion (and chloroplast) involves a signal sequence
(called a transit sequence), a membrane receptor, pore-forming membrane
proteins, and a peptidase.
 In the mitochondrion, the membrane receptor recognizes
the signal sequence directly without the intervention of a
cytosolic SRP.
 Furthermore, chaperone proteins play several crucial roles
in the mitochondrial process:
o Chaperones keep the polypeptide partially unfolded
after synthesis in the cytosol so that binding of the
transit sequence and translocation can occur.
o Chaperones drive the translocation itself by binding to
and releasing from the polypeptide within the matrix, an
ATP-requiring process
o Chaperones often help the polypeptide fold into its final
conformation.
 Protein import into the mitochondrial
matrix. Precursor proteins synthesized on
cytosolic ribosomes are maintained in an
unfolded or partially folded state by bound
chaperones, such as Hsc70 (1). After a
precursor protein binds to an import receptor
near a site of contact with the inner membrane
(2), it is transferred into the general import
pore (3). The translocating protein then moves
through this channel and an adjacent channel
in the inner membrane (4-5). Note that
translocation occurs at rare “contact sites” at
which the inner and outer membranes appear to
touch. Binding of the translocating protein by
the matrix chaperone Hsc70 and subsequent
ATP hydrolysis by Hsc70 helps drive import
into the matrix. Once the uptake-targeting
sequence is removed by a matrix protease and
Hsc70 is released from the newly imported
protein (6), it folds into the mature, active
conformation within the matrix (7). Folding of
some proteins depends on matrix chaperonins.
Pathways for transporting proteins from the cytosol to the inner
mitochondrial membrane.
 In all three pathways, proteins cross the outer membrane via the Tom40
general import pore.
 Proteins delivered by pathways A and B contain an N-terminal matrix-
targeting sequence that is recognized by the Tom20/22 import receptor in
the outer membrane.
 Although both these pathways use the Tim23/17 inner-membrane
channel, they differ in that the entire precursor protein enters the matrix
and then is redirected to the inner membrane in pathway B. Matrix Hsc70
plays a role similar its role in the import of soluble matrix proteins.
 Proteins delivered by pathway C contain internal sequences that are
recognized by the Tom70 import receptor.
 A different inner-membrane translocation channel (Tim22/54) is used in
this pathway.
 Two intermembrane proteins (Tim9 and Tim10) facilitate transfer
between the outer and inner channels.
The major difference is that the internal Two pathways for transporting
targeting sequence in proteins such as proteins from the cytosol to the
cytochrome b2 destined for the intermembrane mitochondrial intermembrane space.
space is recognized by an innermembrane Pathway A, the major one for delivery to
protease, which cleaves the protein on the the inter-membrane space, is similar to
inter-membrane-space pathway A for delivery to the inner
side of the membrane. The membrane.
released protein then folds
and binds to its heme
cofactor within the
intermembrane
space. Pathway B
involves direct
delivery to the

intermembrane
space through the
Tom40 general
import pore in the
outer membrane.
 Two of the four pathways for transporting
proteins from the cytosol to the thylakoid
lumen. In these pathways, unfolded precursors are
delivered to the stroma via the same outer-membrane
proteins that import stromal-localized proteins.
Cleavage of the N-terminal stromal-import sequence by
a stromal protease then reveals the thylakoid-targeting
sequence. At this point the two pathways diverge. In the
SRP dependent pathway (left), plastocyanin and similar
proteins are kept unfolded in the stromal space by a set
of chaperones and, directed by the thylakoid targeting
sequence, bind to proteins that are closely related to the
bacterial SRP, SRP receptor, and SecY translocon,
which mediate movement into the lumen. After the
thylakoid-targeting sequence is removed in the
thylakoid lumen by a
separate endoprotease, the protein folds
into its mature conformation. In the pH dependent
pathway (right), metal-binding
proteins fold in the stroma, and complex
redox cofactors are added. Two arginine
residues (RR) at the N-terminus of the
thylakoid-targeting sequence and a pH
gradient across the inner membrane are
required for transport of the folded protein
into the thylakoid lumen. The translocon in
the thylakoid membrane is composed of
at least four proteins related to proteins in
the bacterial inner membrane.
 (1) Catalase and most other peroxisomal matrix
proteins contain a C-terminal PTS1 uptake-
Import of targeting sequence (red) that binds to the
cytosolic receptor Pex5. (2) Pex5 with the bound
peroxisomal
matrix protein interacts with the Pex14 receptor
matrix located on the peroxisome membrane. (3) The
proteins matrix protein–Pex5 complex is then transferred
directed by to a set of membrane proteins (Pex10, Pex12, and
PTS1 Pex2) that are necessary for translocation into the
targeting peroxisomal matrix by an unknown mechanism.
sequence. (4) At some point, either
during translocation or in the lumen,
Pex5 dissociates from the matrix
protein and returns to the
cytosol, a process that involves
the Pex2/10/12 complex and
additional membrane and
cytosolic proteins. Note that
folded proteins can be imported
into peroxisomes and that the
targeting sequence is not
removed in the matrix.
 Mutations can affect protein
structure and function
 Mutations are changes in the genetic material of a cell
or virus. MUTATION AND DNA REPAIR MECHANISMS.pptx
 These include large-scale mutations in which long
segments of DNA are affected (for example,
translocations, duplications, and inversions).
 A chemical change in just one base pair of a gene
causes a spontaneous or point mutation.
 If these occur in gametes or cells producing gametes,
they may be transmitted to future generations.
 For example, sickle-cell disease is caused by a mutation of a
single base pair in the gene that codes for one of the
polypeptides of hemoglobin.
 A change in a single nucleotide from T to A in the DNA
template leads to an abnormal protein.
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anslation/translation.htm