Protein secondary structure elements such as α helices and β

sheets constitute the major regular folding patterns in

proteins. With regard to these elements, …

A. hydrogen-bonding between the amino acid side chains

defines the type of secondary structure.

B. a certain short amino acid sequence always adopts the

same secondary structure.

C. only a few specific amino acid sequences can adopt these

repetitive structures.

D. the folding patterns result from hydrogen-bonding

between the N–H and C=O groups in the polypeptide
backbone.

E. All of the above.

For each of the following cartoon representations from
left to right, indicate whether the protein structure is
composed of only
(1) α helices,
(2) only parallel β sheets ,
(3) only antiparallel β sheets,
(4) α helices plus parallel β sheets,
(5) or α helices plus antiparallel β sheets

as repetitive secondary structure elements. Your answer

would be a four-digit number composed of digits 1 to 5
only, e.g. 5513.
For each of the following cartoon representations from
left to right, indicate whether the protein structure is
composed of only
(1) α helices,
(2) only parallel β sheets ,
(3) only antiparallel β sheets,
(4) α helices plus parallel β sheets,
(5) or α helices plus antiparallel β sheets
as repetitive secondary structure elements. Your answer
would be a four-digit number composed of digits 1 to 5
only, e.g. 5513.
5 1 3 4
 Question 1.1
You run a mixture of proteins on an SDS-PAGE and identify two
proteins: Cyclin-B and Ribonucleotide reductase. From the position
on the gel you estimate that these proteins are about 57 kDa and 44
kDa respectively.
• The predicted molecular masses based on the sequences
of the genes for these two proteins:
46 kDa = cyclin B
44 kDa = ribonucleotide reductase

Why is the SDS-PAGE estimate for the molecular mass of

cyclin B so different from its predicted value?

A) Because it contains a high fraction of hydrophobic amino

acids
B) Because it contains a high fraction of positive amino acids
C) Because it contains a high fraction of negative amino
acids
D) Because it contains more amino acids than predicted by
the gene
Alberts, 8-17
 How SDS Works

sodium dodecyl sulfate) is a detergent (soap) that can dissolve

hydrophobic molecules but also has a negative charge (sulfate) attached
to it. Therefore, if a cell is incubated with SDS, the membranes will be
dissolved, all the proteins will be soluablized by the detergent, plus all
the proteins will be covered with many negative charges.
Alberts, 8-18
Alberts, 8-17
 How SDS Works

sodium dodecyl sulfate) is a detergent (soap) that can dissolve

hydrophobic molecules but also has a negative charge (sulfate) attached
to it. Therefore, if a cell is incubated with SDS, the membranes will be
dissolved, all the proteins will be soluablized by the detergent, plus all
the proteins will be covered with many negative charges.
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

Alberts, 8-18
 Coomassie
Brilliant Blue

Western blot using

antibody
 What is antibody
Y-shaped protein produced mainly by plasma cells that is used
by the immune system to identify and neutralize pathogens

Monoclonal

Polyclonal
Monoclonal antibodies are monospecific antibodies that are made by
identical immune cells that are all clones of a unique parent cell
Polyclonal antibody:
Antibodies that are secreted by different B cell lineages
with the body. They are a collection of immunoglobulin
molecules that react against a specific antigen, each
identifying a different epitope
The difference between monoclonal
and polyclonal antibodies
 Question 1.2
You are studying a protein of 100 kilodaltons (kd). You found a way to
cleave this protein at a single site ¼ of the length from the N-terminus.
You have raised a monoclonal antibody that binds to the N-terminus
of protein X.
You run on an SDS-PAGE a sample of intact protein X (on lane 1) and
a sample of protein X that has been cut at that single site (lane 2). If
you do a Western Blot analysis with the monoclonal antibody what
will be the size of protein band(s) observed in lanes 1 and 2?

A) Lane 1 = 100 kd Lane 2 = 25 + 75 kd

B) Lane 1 = 25 + 100 kd Lane 2 = 25 kd
C) Lane 1 = 75 kd Lane 2 = 75 kd
D) Lane 1 = 100 kd Lane 2 = 25 kd
E) Lane 1 = 25 kd Lane 2 = 25 kd
Lecture 2 Protein Folding by Chaperones

Protein Degradation by the Ubiquitin

/ Proteasome Pathway
 Protein degradation is
the final system to
control protein
concentration in the cell

Most cytosolic proteins are

degraded in the Proteasome

Alberts, 6-97
Protein structure
Few proteins can refold after complete
denaturation.

Alberts, 3-6
Most proteins begin to fold as they are synthesized

Alberts, 6-84
 Two Generic Types of Chaperones

Monomeric Multimeric
HSP-70 HSP-60
Heat Shock Proteins = proteins involved in the folding of new proteins or refolding
of proteins that started to unfold after heat treatment.
The Hsp70 family of molecular chaperones
 Folding of Proteins by HSP-70

• HSP-70 uses ATP to fold proteins

• Many monomers bind along the length of the protein
• Can bind as soon as protein is out of the ribosome
• Can also Refold a partially misfolded protein
• Useful to maintain a protein in an extended conformation
Alberts, 6-86
The folding of a protein is controlled by its
amino acid sequence

•Some proteins fold on their own

•Most proteins need help to fold
Alberts, 3-5
 Folding of Proteins by HSP-60

• Hsp60 forms a Large Complex (14 x HSP-60 + accessory prot.)

• Has 2 Folding Chambers
• Folds using ATP
• Works in partnership with HSP-70 which delivers unfolded
protein
Alberts, 6-87
The structure and function of the Hsp60 family
molecular chaperons

http://tools.medicine.yale.edu/horwich/www/
 Folding pathways for a protein

Proteins that fail to fold need to be degraded

otherwise they will aggregate
Alberts, 6-88
 Protein aggregation can be harmful to cells

b Amyloid

Normal AD

Alzheimer’s Disease is Thought

To Result From The Aggregation
of Specific Neuronal Proteins
 The Proteasome: The Chamber of Doom!

cap
The 19s cap
core
core
The 20S
core protein

The proteasome degrades proteins

that are tagged by ubiquitin chains
Alberts, 6-89
The Proteasome Cap contains
an “Unfoldase” that denatures
proteins

The Unfoldase uses the

energy of ATP to unfold
proteins and feed them into
the Proteasome core Alberts, 6-90
 Degradation of tagged proteins by the Proteasome

The proteasome cap removes ubiquitin

subunits for recycling and ‘feeds’ the
targeted protein to the protease core
Alberts, 6-90
 Ubiquitin Structure

Ubiquitous = Universal,
Found Everywhere
(“pup” in prokaryotes)

•Only 76 amino acids long

•Made as a polyubiquitin chain
then cut into single ubiquitins

Alberts, 6-92
 First two steps in ubiquitylation

C-terminal of ubiquitin link to E1

E1: ubiquitin activating enzyme

E2: ubiquitin conjugating enzyme
E3: ubiquitin ligase
Alberts, 6-92
 Third step in ubiquitylation

E1: ubiquitin activating enzyme

E2: ubiquitin conjugating enzyme
E3: ubiquitin ligase
Alberts, 6-92
 Degradation of tagged proteins by the Proteasome

The proteasome cap removes ubiquitin

subunits for recycling and ‘feeds’ the
targeted protein to the protease core
Alberts, 6-90
Different forms of Ubiquitylation have different outcomes

Alberts, 6-93
Ubiquitylation can be controlled in two ways

A) by activation of a ubiquitin ligase

Alberts, 6-94
B) by activation of a Degradation Signal on
the target protein

Alberts, 6-94
 Properties of Chaperones:

Which statement about Chaperones is true?

A) Most proteins need the assistance of Chaperones to

fold inside the cell.
B) Some proteins are folded only by HSP-60 and do not
need HSP-70
C) HSP-70 binds to proteins as they are being
synthesized by the ribosome
D) HSP-60 binds to proteins after they have been
completely synthesized
 Properties of Chaperones:

How do Chaperones recognize proteins that are NOT

completely folded? (so they can bind to those proteins
to help them fold)
A) Chaperones can recognize the shape of unfolded
proteins.
B) Chaperones can recognize the shape of folded
proteins.
C) Chaperones bind to patches of hydrophobic amino
acids on other proteins.
D) Chaperones bind to patches of hydrophilic amino
acids on other proteins.
E) Chaperones bind equally well to a folded or unfolded
protein.
 Ubiquitylation

Which of the following proteins determines which protein

lives or dies by ubiquitin-mediated degradation?

A) HSP-70
B) Ubiquitin Activating Enzyme
C) E2
D) E3
E) Proteasome core
 Experimental Question:
You have a drug that blocks the activity of one of the
enzymes involved in ubiquitylation of proteins. You treat cells
with this drug and you determine the effect of treatment on
the abundance of different proteins by Western blot analysis.
Cdc2 is a control of a protein that is not ubiquitylated. From
the literature you know that the other 4 proteins are
ubiquitylated by different E2+E3 ligase proteins.

Based on this information you can conclude that:

A = The drug blocks the activity of Ubiquitin Activating Enzyme (E1)

B = The drug blocks the activity of a Ubiquitin E2 protein
C = The drug blocks the activity of a Ubiquitin E3 protein
D = Protein synthesis is increased by the drug treatment
 Experimental Question:
You have a drug that blocks the activity of the Proteasome. You treat cells
with this drug and you determine the effect of treatment on the abundance of
your favorite protein by Western blot analysis.

The arrow points to the size of your favorite protein (YFP).

Blot probed with anti-YFP

antibodies

YFP

0 6 12 hours
Based on this Western blot you can conclude that:
A = proteins larger than YFP are synthesized
B = drug treatment does not affect YFP in any way
C = drug treatment causes YFP to be destroyed in the proteasome
D = drug treatment causes YFP to become ubiquitylated
E = drug treatment caused the accumulation of ubiquitylated-YFP
 Experimental Question:
How can you show that the bands larger than YFP represent
polyubiquitylated forms of YFP?

YFP

0 6 12 hours

Blot probed with anti-YFP

antibodies