1. Leaning Goals for Protein Folding
Topic
Learning Goals
Learning Outcomes
Protein Folding
Understand how amino acids assemble into polypeptides
Understand how higher order structure arises in polypeptides and proteins
Understand how a protein’s location and function within the cell are dictated by higher order structure
Recognize sites of potential interaction between amino acid side chains in a given string of amino acids
Predict the location of an amino acid (or stretch of amino acids) in a protein’s tertiary/quaternary structure
Analyze effects of mutations on protein folding or protein-protein interactions
Predict the cellular location of a protein based on the properties of its amino acids

2. Meet the 20 Amino Acids! Backbone in yellow
“R groups” in blue – unique character of each amino acid
Note: Cysteine is weird – some books say it’s polar, some nonpolar (acts NP)
You WILL!!! need to know characteristics and unique features of each amino acid
You WILL NOT need to know how to draw each amino acid or recognize its specific structure (just its characteristics)
Helix breaker
Disulfide bonds
Can be phosphorylated
Ask students if they noticed any unique chemical groups while classifying the amino acids – organic students will know these as FUNCTIONAL GROUPS.
Positively charged
Negatively charged

3. Today in 41C… We will answer the questions:
How do proteins assemble from amino acids?
How do proteins fold accurately & reproducibly every time? (A protein can’t function without its proper structure, after all!)
Cdk
Cyclin
Cdk
Cyclin

4. Amino Acids Combine by Dehydration/Condensation Reactions

5. Model 2: Protein Structure & Folding
Primary structure: amino acid sequence
Secondary structure
Tertiary structure
Quaternary structure: more than one polypeptide in a complex
Which BONDS promote each level of structure? Between which parts of the amino acids?

7. Secondary Structures: The a-helix and b-sheet
H-bonds between backbone of every 4th amino acid
R-groups stick out of helix!
b-sheet
H-bonds between backbones of adjacent strands
R-groups stick out in alternating fashion (up, down, up, down)

8. If a high concentration of urea were added to a solution containing a protein with an a-helix, how would protein structure be affected?
Urea
Alpha helix

9. Model 2: Protein Structure & Folding
Primary structure: amino acid sequence
Secondary structure
Tertiary structure
Quaternary structure: more than one polypeptide in a complex
Which BONDS promote each level of structure? Between which parts of the amino acids?

10. Tertiary Structure and the “Hydrophobic Effect”
What would this protein look like when properly folded?
Note: tertiary structure always depends upon the surrounding environmental conditions
Figure 4-5 Hydrophobic forces help proteins fold into compact conformations.
The polar amino acid side chains tend to fall on the outside of the folded protein, where they can interact with water; the nonpolar amino acid side chains are buried on the inside to form a highly packed hydrophobic core of atoms that are hidden from water. In this very schematic drawing, the protein contains only about 30 amino acids.
remember that tertiary structure is strongly dependent upon CURRENT conditions, and can be changed by alterations in pH, salt, etc.
Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008)

11. Formation of Binding Pockets in 3° and 4° Structure

12. Clicker Question 1
In an aqueous environment, which of the following amino acids is most likely to be found in the protein’s interior? A B C D E
Correct answer is E, methionine. Because these amino acids are within the context of a protein, the amino and carboxyl group will be used to generate a peptide bond and will not be charged. Also, it is the R group that determines whether the amino acid is hydrophobic (and will be found in the interior of the protein in an aqueous solution) or hydrophilic (and will be found in the exterior of the protein in an aqueous solution)
To answer this question, look for hydrophobic side chains. The answer is methionine, whose R group is nonpolar (note that sulfur and carbon have the same electronegativity, so the covalent bond linking them is nonpolar)

13. Where in the cell would you find a protein with:
Mostly polar (hydrophilic) amino acids on its surface?
Distinct regions of polar & nonpolar amino acids?
cyclin
CDK
succinate dehydrogenase
Cyclin PDB =
CDK2 PDB = 3LFQ
Polar amino acids
Nonpolar amino acids

14. Clicker Question 2
You are conducting experiments on the human ribonuclease enzyme and want to denature (unfold) the protein. Which condition(s) could disrupt the majority of the folding patterns?
Heat the protein to 37oC
Add 6 molar urea
Decrease the pH to 6.5
Two of the above
All of the above
Room temperature: ~22oC
Urea:
Answer is (b) – urea. Urea is a polar molecule that will compete for the H-bonds between amino acids that hold the secondary structure together.
Thermal energy disrupts H-bonds, but 37C is body temperature. The proteins in our cells are doing just fine.
pH 6.0 is not that far off from physiological pH (7.0). The H-bonds that hold DNA strands together don’t start to denature until more alkaline conditions, around pH10
Tertiary structure is more easily disrupted by changes in heat, pH, polar molecules, etc. because of ionic bonds being disrupted

15. How do proteins achieve their 3D structure?
Each team gets a “protein” with a secondary structure.
Brown/black: nonpolar amino acids
Blue: polar uncharged amino acids
Orange: cysteine
Red: positively charged
Green: negatively charged
Possibly useful additional materials in the front of the room
Thin black pipe cleaners: covalent bonds
Black streamers: ionic bonds (“salt bridges”)
Fold your protein into a stable tertiary structure (in water)!
Each team has an identical string of “peptides” folded into a secondary structure, which are made out of fluffy pipe cleaners (Hobby Lobby sells them cheaply).
Give them 3-5 minutes to fold their proteins into tertiary structure, and have one representative come of the front of the room to model their group’s folded protein.
It is virtually guaranteed that there will be several different structures on display, which leads to the questions…
If each team had the same primary and secondary structure, why did each team come up with a different tertiary structure?
Wouldn’t this cause HUGE problems for the ability of this protein to carry out its normal function in the cell, since a protein or enzyme isn’t active unless it has achieved its proper conformation?
Therefore, there must be some mechanism in place that allows a given stretch of amino acids to fold into their proper shape every time a new protein is made via translation. 15

16. Brainstorm with your Team
Develop two or more distinct (different) hypotheses to explain how proteins might fold into specific, reproducible 3D structures.

17. What are the Instructions for Protein Folding
What are the Instructions for Protein Folding? Classic Experiments, Volume 1
Ribonuclease A
14 kDa
RuBisCo
560 kDa
Hypothesis 1:
Instructions for folding are inherent in the amino acid sequence
Hypothesis 2:
A separate enzyme or protein
is required for proper folding
Conditions: Protein activity:
Purified protein (from cells) 100%
Denature w/ 8M urea %
Renature (remove urea) %
_____________________
100% 0% 2%
Add cell extract (lacking enzyme) 100% %
Modified from Christian Anfinsen, John Ellis

18. What are the Instructions for Protein Folding
What are the Instructions for Protein Folding? Classic Experiments, Volume 1
Ribonuclease A
14 kDa
RuBisCo
560 kDa
Hypothesis 1:
Instructions for folding are inherent in the amino acid sequence
Hypothesis 2:
A separate enzyme or protein
is required for proper folding
Conditions: Protein activity:
Purified protein (from cells) 100%
Denature w/ 8M urea %
Renature (remove urea) %
_____________________
100% 0% 2%
Add cell extract (lacking enzyme) 100% %
Extract contained “chaperone” (helper) proteins that can help complex structures to fold, but the information is inherently embedded in the amino acid sequence
Modified from Christian Anfinsen, John Ellis

19. Protein Folding is Assisted by Chaperones
Prion disease
Cancer
Alzheimer’s

20. Protein Folding is Assisted by Chaperones & Chaperonins
Chaperones Prevent undesired interactions
Chaperone
Chaperones prevent undesired interactions.
Chaperonins promote proper folding.
Chaperonins Promote proper interactions
Chaperonin
Chaperonin

21. Bonds are important not only for folding,
but also for protein-protein interactions
Cdk
Cyclin
Cdk
Cyclin
Jeffrey et al., Nature (1995)

22. Let’s Design an Experiment…
The G1/S cyclin & Cdk interact at a defined interface, and you hypothesize that lysine (Lys, K) at position 178 of the Cdk protein is essential for this interaction.
What kind of mutations could you introduce at K178 that would allow you to test your hypothesis?
Cdk
Cyclin
Study below found that lysine (K) 178 of Cdk was essential for generating the cyclin-Cdk interface.
A novel binding pocket of cyclin-dependent kinase 2
Hao Chen1, Rachel Van Duyne2, Naigong Zhang1,†, Fatah Kashanchi2,3,4,*, Chen Zeng1. Proteins: Structure, Function, and Bioinformatics Volume 74, Issue 1, pages 122–132, January 2009
Discuss with your team for ~2 min.
Be prepared to describe to the class which mutations you would make and why you chose them (a rationale).
K178
Chen et al. (2009)

23. Protein Folding & Interactions in Disease
Hemoglobin & Sickle Cell Anemia

24. Hemoglobin H2S mutation  Sickle Cell Anemia
Hemoglobin b subunit
H2S mutation:
Glu6  Val6
Hydrophobic amino acids
Source:
In this depiction of the E6V mutation responsible for the HbS phenotype we can see Chain B (yellow) and Chain H (red). The hydrophobic residues are colored light blue and the Val6 side chain is also depicted. This solvent exposed surface of the Chain B contains a cluster of hydrophobic residues, and the replacement of the acidic Glu6 residue in the normal sequence with valine in this HbS mutant creates a hydrophobic patch on Chain H as well. These two surfaces are held closely together (hydrophobic effect) and are responsible for the linking of hemoglobin molecules and the subsequent changes in red cell morphology. 
Hemoglobin b subunit on a neighboring hemoglobin molecule

25. How Do We Determine Primary Structure? Genomics Mass Spectrometry

26. How Do We Determine Higher Order Structures?
Protein crystals
X-ray Crystallography
Crystal structure
Cdk
Cyclin
Rene Bodenstaff (2002)
X-ray diffraction pattern
X-rays
The crystal structure of Cdk vs. Cdk/cyclin revealed a flexible loop that prevents or allows ATP binding to the Cdk, respectively.
Jeffrey et al., Nature (1995)
Computation

27. How Do We Predict Secondary and Tertiary Structure?
LOTS of “prediction programs” on the internet that compare your protein sequence with similar motifs
PsiPred:
Phyre:
Jpred:
Protein Data Bank:
Searchable repository of all known protein structures
Based on a protein’s “crystal structure”
Example: Succinate Dehydrogenase (SDH)

28. Succinate Dehydrogenase (SDH)
Secondary Structure

29. Folding  3D Structure  Function
Cdk
Cyclin
Cdk
Cyclin
View 3D structures & lots of other info at:
Protein Data Bank
Jeffrey et al., Nature (1995)

30. Coming up in 41C… We will answer the question:
What the heck does Cdk actually do?!? (What are enzymes & how do they work?)

32. Polypeptide Structure & Folding
Primary
Secondary
Which bonds promote each level of structure?
b-sheet
a-helix
Tertiary
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Quaternary

33. pH of cell 
pKa of R-group 

34. Polypeptide Structure & Folding
Primary
Secondary
b-sheet
a-helix
Tertiary
Next year: Add data from classic ribonuclease folding experiment. And then the RUBISCO chaperone discovery if can find it. Teach about technique of reconstitution & in vitro experiments.
Which bonds promote each level of structure?
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Quaternary