Download Lecture on PROTEIN FOLDING

Protein Folding
Cell/mol bio lab
Proteins are like a long spaghetti
noodle, folded back upon itself over
and over
Why study the 3-D shape of a
protein?
(go to Cell Biology web site from my home page)
First though, what is Protein Folding?
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Proteins are very rickety; their shape is easily
distorted. Mother Nature uses this to control
enzymes (bind something to an enzyme, and
distort the enzyme, turn it off or on)
Proteins are rickety because their 3-D shape is
largely due to weak bonds (not strong covalent
bonds)
Biomolecules/drugs bind to proteins through
weak bonds (only rare poisons bind through
covalent bonds) to distort enzymes
Pages 30-33; 44-53 130, 134, 696, 706-707
(6th ed of World of Cell)
Protein Folding
„ Some day (as in Star Trek), we
hope to design new proteins to cure
disease
„ But bad protein folding can
CAUSE disease
Protein Folding Diseases page 697 in
text (6th ed)
„ Alzheimer patients have extracellular
amyloid plaques made up of amyloid-β (Aβ)
„ Because protein A-β is not folded properly
„ ApoE is a protein that may cause normal Aβ to misfold
„ APP (a membrane protein) is cut (cleaved)
to form A-β- so many study APP cleavage;
prevent the cleavage and prevent amyloid
plaques (?)
Prions induce scrapie, & mad cow
disease and, in humans, CreutzfeldtJakob disease (vCJD)
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Prions do not contain DNA or RNA
Prions (PrPSC) are believed to be misfolded
versions of normal proteins (PrPC)
When Prions (PrPSC) bind to folding protein,
they cause the protein to fold abnormally and
then clump (aggrate) –why? What weak bond is
involved?
This is how the Prions (PrPSC) reproducescauses more misfolded proteins without DNA
Protein Folding Diseases page 697 in text (6th ed)
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Fig. 22A-1
http://www.faseb.org/opar/protfold/protein.html
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View the member societies www.faseb.org/faseb/societies.html
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“Alzheimer's disease. Cystic fibrosis. Mad
Cow disease. An inherited form of
emphysema. Even many cancers. Recent
discoveries show that all these apparently
unrelated diseases result from protein
folding gone wrong.”
“As though that weren't enough, many of
the unexpected difficulties biotechnology
companies encounter when trying to
produce human proteins in bacteria also
result from something amiss when proteins
fold.”
Figure 2-18 The Spontaneity of Polypeptide
Folding
rare
Proteins loose their 3-D shape (denature) when the
weak bonds that hold together the structure are
broken.
Weak bonds are broken when the temperature is
raised from body temp (37 C) to about 60 C
Or by changing the pH
Or by using chemical agents
Why is ribonuclease weird?
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It can renature (most proteins unravel, and
aggregate and precipitate because they reveal their
nonpolar amino acids)- most proteins cannot
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renature
It can fold without chaperones (proteins that
help other proteins fold)
Chaperones do not provide info, they merely
bind to the folding protein to make sure that
the wrong weak bonds are not formed
Heat up a cell, you could denature proteins,
so more Chaperones are made to protect
proteins (Chaperones were originally called heat shock proteins)
Figure 3-2 The Structures of the 20 Amino
Acids Found in Proteins
Don’t memorize
The structures
Just know that there are
3 types of
Amino acids
Polar (polar cov bond,
no full charge but
partial charge)
Charged Amino acid
Nonpolar amino acid
(hidden inside spherical
protein)
Oil in
Swimming
pool
Figure 3-5
Bonds and
Interactions
Involved in
Protein
Folding and
Stabilitywhich are
weak bonds
and which
strong?
Strong covalent bonds are about 50 times stronger than weak bonds
Figure 3-6 The Four Levels of Organization of
Protein Structure- memorize this crucial slide
Disulfide bridge, all
Weak bonds stabilize
3 and 4th deg structure
List of
Amino acids
From N terminus
To C terminus
Covalent bond
Connects amino
acids
subunits
Small loops: Helix or
B pleated Sheet
(only H bonding)
Big loops
Figure 3-7 The Primary Structure of Insulin
Figure 3-8 The α Helix and β Sheet
H bond only stabilizes
This level of structure
Figure 3-9 Common Structural Motifs
Combinations
Of helix and
Beta pleated
Sheets (purple arrows
Point to the end or C
Terminus)
Helix turn helix
Found in
Transcriptions
Factors (they bind
DNA and turn off/on
Genes)
So motifs often do
Same thing but
are found in
Different proteins
Figure 3-4 Hemoglobin: what levels are present?
Figure 3-10 The Structure of Hair
KERATIN
What levels of protein
Are in the hair protein
KERATIN?
Proteins are spherical
(globular) or
Long and thin
(called fibrous)
Figure 3-11 The Three-Dimensional Structure
of Ribonuclease- A GLOBULAR protein
Ball and stick model
Spiral and Ribbon
model--Where are
B pleated sheets?
Helices?
Figure 3-12 Structures of Several Globular
Proteins- “Domains” make up proteins
Domains are about 50-350, have similar function in different proteins, a unit of
Tertiary structure (made up with secondary structures)
Figure 3-13 An
Example of a
Protein
Containing Two
Functional
Domains- each
domain binds
something
different
Figure 6-2 Molecular Structures of Lysozyme
and Carboxypeptidase A
We will be looking at this movement in the enzyme
Hexokinase with a program called MAGE. You click
and animate the binding of substrate and
the movement of the two domains (along the hinge;
where is the hinge and
2 domains?)
Figure 6-6 The
Conformational
Change in
Enzyme Structure
Induced by
Substrate Binding
(rickety protein
distorts due to
the new weak
bonds forming
between substrate
and protein)
Calmodulin binds 4 Calcium ions,
causing the calmodulin to bend and
wrap around other proteins – this turns
on other proteins
We will
View this
In MAGE
Fig. 14-13
Page 403
2 globular
Ends
Joined by
A helix
Calcium makes
new weak ionic
bonds to the R groups of
amino acids of the protein
calmodulin
this really
changes the protein;
calmodulin breaks
in the middle and
hinges – to
bind another
protein (and turn
this other protein on)
With 4 Ca bound to
Calmodulin
(from Introduction
to Protein Structure,
also in lab exercise file)
Maybe most impt protein in cell: cdk
-an enzyme that turns on cell division (or G1
to S phase). Another protein called cyclin
builds up in the cell, and then binds to and
activates cdk. Cdk then causes progression
to cell division
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We will be studying cdk’s later (another kinase
called map kinase can turn it on)
We will be using MAGE to view the binding of
cyclin and cdk
HOW DOES THE CYCLIN BINDING TO CDK
TURN ON THE CDK KINASE ACTIVITY?
CYCLIN BINDING FORMS NEW WEAK BONDS THAT CAUSE
A BIT OF PEPTIDE CALLED THE T LOOP TO MOVE OUT
OF THE ACTIVE SITE OF Cdk; THIS UNBLOCKS
THE ACTIVE SITE.
(from Mol Bio of the Cell; Alberts et al.)
CDK2 is off,
no cell division
PSTAIRE is
not in the
active site, and
T Loop Blocks
the binding of
substrate
CYCLIN (GREEN) BINDS
AND MOVES PSTAIRE AND T LOOP
TO CORRECT LOCATIONS
CELL DIVISION STARTED
—OR CANCER?
Space filling model of CDK2
No Cyclin binding
Cyclin binding
Membrane proteins are different from soluble
globular proteins Fig 2-13
Globular arrangement
of amino acids at the
ends in the cytoplasm
and outside the cell
but…..
Nonpolar amino acids
found in the center (the
Alpha helix that goes
through the
hydrophobic section of
the membrane
Porins are membrane proteins that
travese the membrane not with alpha
helix but with Beta pleated sheets
Chaperones Page 696, 706-707
„ Chaperones are of two general types
(with different mechanism, but do the
same thing):
Hsp70 and Hsp 60 (heat shock
protein)
„ Chaperones bind to a folding protein to
help the protein fold properly
„ As protein is made, it begins to fold
properly and bind other subunits of the
protein (if the protein has 4o structure)
„ Chaperones bind to a folding protein at
the folding protein’s hydrophobic
region (nonpolar amino acids),
prevents the folding protein from
aggregating/precipitating
„ Releases protein by use of ATP
breakdown (nonspontaneous reaction
without ATP)
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Remember: in a typical soluble protein, the
nonpolar amino acids are hidden on the
inside of the protein so the protein will not
precipitate