Transcript protein folding, madison 2009

Designing this teaching tidbit has
gotten completely out of hand
Group 1: Chemistry/Biology Interface
Protein Folding
Xinnian Chen, U Conn.
Alison Hill, Duke
Ron Grunwald, Duke
Dan Kiehart, Duke
Dan Mulkey, U Conn.
Carolyn Norris, Johns Hopkins
Joel Schildbach, Johns Hopkins
Thanks to Amy Prunuske and Brad Hyman
Course: Introductory level course in which
chemical foundations of biological function are
described.
Students have been exposed to:
•Basic chemistry of covalent and noncovalent
bonds
•DNA structure and sequence
•Protein primary sequence is (almost always)
specified by sequences in DNA.
•mRNA is translated on ribosomes
•Linear polypeptide chain, comes off the ribosome
and begins to fold.
Thus they have been exposed to:
Text
UNIT: INTRODUCTION TO PROTEINS
Goal 1: Protein Primary Structure = Amino acid sequence.
Goal 2: Protein Secondary, Tertiary and Quaternary
Structure
Goal 3, TIDBIT: Explore HOW proteins
fold
3. TIDBIT Goal: Students will begin to understand
the mechanism by which proteins fold into higher
order structures due to noncovalent interactions.
a. Outcome: Students will be able to evaluate
experimental data to decide whether or not a protein
can fold spontaneously.
b. Outcome: Students will be capable of predicting
and describing the mechanism of disruption of protein
structure by a chemical perturbant.
c. Outcome: Students will recognize that protein 3D
structure is required for function.
(CN)
(JS)
Proteins with different
functions have
different structures
•Proteins with different
shapes and sizes
•Proteins reproducibly
fold from their primary
structures
•Shape determines
function
(JS)
Even small proteins have very complex 3D structures:
Ribonuclease: (RNase) is a digestive enzyme
cuts RNA polymers into monomers
124 amino acids
>900 atoms (not counting hydrogens!)
Three different 3D representations of ribonuclease:
Ribbon Diagram
Highlights
Secondary
Structure
Stick Diagram
Space Filling
Shows the Position Model Features
of Heavy Atoms Surface Topology
(not H)
Hydrogen bonds stabilize protein structure, e.g., alpha
helices
H-bond
(JS)
(DM)
Urea offers an opportunity to investigate the bonds
that stabilize protein structure: How?
(DM)
How does urea interact with proteins?
1) 1 minute drawing: show the
interactions of 6 molecules of Urea with
the polypeptide backbone on the
handout
(DM)
2) Think/Pair/Share: What happens when
you add 8M Urea (a REALLY HIGH
CONCENTRATION) to 1 µM alpha helix?
Collectively choose a hypothesis for what 8M
Urea does to a protein...
(DM)
(DPK)
Next, we’ll use Urea as a probe of protein structure in an
experiment
(DPK)
Experimental Data
Ribonuclease
in
salt solution
?
+/- Urea
(DPK)
Clicker Question:
What do you think is the most likely effect of Urea on the
STRUCTURE of the RNase?
(DPK)
0%
activity
100%
100%
activity
Ribonuclease
in
dilute salt water
activity
A
B
C
+ 8M Urea
D
Ribonuclease,
dilute salt water,
Urea removed
Clicker Vote
Table discussion: rationale behind your choice
(Revote if wide distribution)
Class summarize why?
A
cleas
salt
er(DPK)
B
C
+ 8M
Urea
D
(RG)
Cut into Pieces? Aggregate? Specific Inhibitor?
You’ll learn later that good inhibitors function at nM
concentrations:
Remember that Urea works at M concentrations!
(DPK)
(RG)
Christian Anfinsen:
Nobel Prize in Chemistry - 1972
One minute essay:
Based on what you’ve learned, write a that relates
structure, function and reversible folding/unfolding
(RG)
For next class: Develop a hypothesis of
protein folding that relates primary
sequence to native structure and
function
(XC)
Adios!
?
Ribonuclease
in
dilute salt water
Ribonuclease,
dilute salt water,
+ “Stuff”
Ribonuclease,
dilute salt water,
“Stuff” removed
the “Stuff” is Urea (cute huh?):
Clicker Question: What kind of bond/force is Urea most
likely to disrupt?
A. covalent B. ionic C. van der Waals D. hydrogen
E. traxoline
Adios!
Catalog and know NC/sidechain interaction
Assessment LOCS
Manipulate strings, evaluate
Specific inteactions
Unique Structures
Next anfinsen with beta mercaptoethanol?
what kind of interactions mediate these different levels
of structure?
Focus on noncovalent bonds and the SS bonds that
stabilize it
Secondary Structure: H-Bonds
between atoms in the Polypeptide
Backbone
Structure of alpha helix and urea
what happens and how?
8
1
8
7
2
7
6
3
6
5
7 possible
4 partners
8
1
7
1
5
4
2
5
4
8
2
6
1
3
5 possible
partners
1
7
2
6
3
just one
4 option left
3
3 possible
partners
5