1. The Three-Dimensional
Chapter 4
The Three-Dimensional
Structure of Proteins
Part 2

2. Chapter 4, Part 2: Learning Goals
Know the structures and functions of collagens, role of ascorbic acid (vitamin C) in collagen structure.
Know globular protein structure and families.
Know how de-naturation and re-naturation works or sometimes doesn’t.

3. Collagen Triple Helix Left Handed, 3 aa/turn
Collagen triple helix is a helix that exists nowhere else and makes up the several types of collagens of our connective tissues and bone.

4. The triple helix has an obvious structure as seen by electron microscopy. What is not shown here is that the interconnections involve hydroxy-proline and hydroxy-lysine. These are prolines and lysines that are modified after translation.

5. Prolyl-4-hyroxylase Hydroxylates Protein as Procollagen
Hyrdoxproline is necessary to keep some prolines in the “exo” form to allow the collagen triple helix to form.

6. Prolyl-4-hydroxylase is a Di-oxygenase can Catalyze Two Reactions
Scurvy is characterized by connective tissue disorders: recycling of connective tissue does not form solid collagen without Hyp. Symptoms include fragile blood vessels, skin lesions, poor wound healing and eventually death. Vitamin C is common in fruits and vegetables which on long sailing voyages are not available. The British were often call limeys because they sailed with a good supply of limes to be consumed during the voyage (this insight is credited to James Cook one of the early British explorers).
Without Vitamin C, the iron of the first enzyme becomes oxidized and Inactive. Ascorbate actually keeps the enzyme iron reduced although this diagram does not show it.

7. Hydroxylysine Cross Links Collagen Triple Helix Strands

8. Iriquois showing Jacques Cartier how to make Cedar Tea - a source of Vitamin C
Scurvy was the bain of long sailing voyages due to lack of vitamin C (which hadn’t been discovered then). What country is Jacques Cartier “discovering”?

9. James Lind’s experiment could not be done today. Why?
Did he lack a control group?
Was there something else?

10. Newly Discovered Bond in Collagen IV
The Sulfilimine Bond
Between a hydroxylysine and methionine
Vanacore, R, et al A sulfilimine bond identified in collagen IV. Science. 325: Sept 4, 2009

11. Sulfilimine Bond – Evolutionary Conservation
This newly discovered bond is widely distributed in other animal collagens.

12. Human Serum Albumin (Mr = 64,500) if it was:
Overall shape of a protein if it was….
This figure has a flaw. Horizontal dimensions are OK, Verticals are off in two ways: different scale and globular form is way too small.

13. Structures of Myoglobin
Note the prophyrin ring which has a Fe in the center. These are all different ways to represent the same protein.

14. What about “random coil” or “random structure”?
Where is it in myoglobin? - go back to previous slide, it represents 22% of the amino acids in myoglobin!
Is it random? Yes and No!! Both are correct why?
Is it coil? Yes and No!! Both are correct why?
Random structure is usually called random coil even though it may not be coiled. It is random because no other protein has exactly the same structure, but it is not unstructured or totally random. There are proteins that have random structures that change their conformation.

15. Heme in Myoglobin
The iron in heme has two available orbitals for coordination complexes.

16. Structures of some Small Proteins
It is easy to see the mix of alpha helix, beta structure and random coil in the “ribbon” structures.

17. How much random coils is in cytochrome-c?

18. Each Domain has a Distinct Function: Binding Ca++
Troponin has 2 Domains
Domains are defined as protein part having a common 3-D structure and distinct function.
Each Domain has a Distinct Function: Binding Ca++

19. Two Small Motifs Here alpha helix connects Alpha turn alpha are
two beta-structures common on some DNA
binding proteins

20. FIGURE 4–20 Stable folding patterns in proteins
FIGURE 4–20 Stable folding patterns in proteins. (a) Connections between β strands in layered β sheets. The strands here are viewed from one end, with no twisting. Thick lines represent connections at the ends nearest the viewer; thin lines are connections at the far ends of the
β strands. The connections at a given end (e.g., near the viewer) rarely cross one another. An example of such a rare crossover is illustrated by
the yellow strand in the structure on the right. (b) Because of the righthanded twist in β strands, connections between strands are generally
right-handed. Left-handed connections must traverse sharper angles and are harder to form. (c) This twisted β sheet is from a domain of
photolyase (a protein that repairs certain types of DNA damage) from E. coli (derived from PDB ID 1DNP). Connecting loops have been
removed so as to focus on the folding of the β sheet.

21. Smaller Motifs into Large Motifs

22. Protein Families – Classes and Folds
There are hundreds of protein families. The protein databases are outstandingly FULL and Growing.

23. All Beta Protein Families

24. Alpha/Beta Protein Families
Alpha/Beta Families use alpha helix to link up parallel beta structrue.

25. Alpha + Beta Protein Families

26. Max Perutz and John Kendrew
Max is holding the first electron density structure of myoglobin, John is holding the high resolution structure determined some years later. Both developed x-ray crystallography.

27. Quaternary Structure of Hemoglobin
2 α and 2 β

28. Quaternary Structure: Symmetry

30. This is the structure of some viral capsid proteins that make up the bacteriophage head.

31. Polio Virus and Tobacco Mosaic Virus

32. Protein Stability and Folding
A protein’s function depends on its 3D-structure
Loss of structural integrity with accompanying loss of activity is called denaturation
Proteins can be denatured by:
heat or cold
pH extremes
organic solvents
chaotropic agents: urea and guanidinium hydrochloride

33. Thermal and Chemical Protein Denaturation
Irreversible
Reversible
Protein folding can get wacked, or gently unfolded. It just depends how the unfolding is done.
or Urea

34. Ribonuclease Refolding Experiment
Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds
Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease
When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed
The sequence alone determines the native conformation
Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize

35. Reversible Unfolding with Mercaptoethanol
CH3-CH2-SH
This can only be done by reversible unfolding methods.
This step must be done very slowly

36. Simulated Folding

38. FIGURE 4-29a,b The thermodynamics of protein folding depicted as a free-energy funnel. At the top, the number of conformations, and hence the conformational entropy, is large. Only a small fraction of the intramolecular interactions that will exist in the native conformation are present. As folding progresses, the thermodynamic path down the funnel reduces the number of states present (decreases entropy), increases the amount of protein in the native conformation, and decreases the free energy. Depressions on the sides of the funnel represent semistable folding intermediates, which in some cases may slow the folding process.

39. Proteins folding follow a distinct path
FIGURE 4–28 A protein-folding pathway as defined for a small protein. A hierarchical pathway is shown, based on computer modeling. Small regions of secondary structure are assembled first and then gradually incorporated into larger structures. The program used for this model has been highly successful in predicting the three-dimensional structure of small proteins from their amino acid sequence. The numbers indicate the amino acid residues in this 56 residue peptide that have acquired their final structure in each of the steps shown.

40. Creutzfledt-Jakob Disease: Human Spongiform Encephalopathy
This is the same as Mad Cow Disease, and is a protein miss-folding disease brought about by a prion (infectious protein).
Vacuoles Contain a Missfolded Protein – in Brain Tissue

41. Prions Infectious proteins
Inherited and transmissible by ingestion, transplant, & surgical instruments
PrPC, normal cellular prion protein, on nerve cell surface
PrPSc, scrapie protein, accumulate in brain cells forming plaques
Scrapie is the prion disease in sheep: the miss-folded protein in the brain producing the same spongiform encephalopathy causes the sheep to scrape themselves against bushes, trees, fences, etc to their death.

42. Prion Miss-folding
PrPSc
PrPc 1 2 3 4
Lysosome
Endosome 5 6 7 8

43. PrP Folding
The form with the pronounced beta structure is the pathology inducing form.

44. Chaperones prevent misfolding
FIGURE 4-30 Chaperones in protein folding. The cyclic pathway by which chaperones bind and release polypeptides is illustrated for the E. coli chaperone proteins DnaK and DnaJ, homologs of the eukaryotic chaperones Hsp70 and Hsp40. The chaperones do not actively promote the folding of the substrate protein, but instead prevent aggregation of unfolded peptides. For a population of polypeptide molecules, some fraction of the molecules released at the end of the cycle are in the native conformation. The remainder are rebound by DnaK or diverted to the chaperonin system (GroEL; see Figure 4-30). In bacteria, a protein called GrpE interacts transiently with DnaK late in the cycle (step 3), promoting dissociation of ADP and possibly DnaJ. No eukaryotic analog of GrpE is known.

45. Chaperonins facilitate folding
FIGURE 4-31a Chaperonins in protein folding. (a) A proposed pathway for the action of the E. coli chaperonins GroEL (a member of the Hsp60 protein family) and GroES. Each GroEL complex consists of two large chambers formed by two heptameric rings (each subunit Mr 57,000). GroES, also a heptamer (subunit Mr 10,000), blocks one of the GroEL chambers after an unfolded protein is bound inside. The chamber with the unfolded protein is referred to as cis; the opposite one is trans. Folding occurs within the cis chamber, during the time it takes to hydrolyze the 7 ATP bound to the subunits in the heptameric ring. The GroES and the ADP molecules then dissociate, and the protein is released. The two chambers of the GroEL/Hsp60 systems alternate in the binding and facilitated folding of client proteins.

46. GroEL and GroES
See the similarity to the Proteasome ! Both are very large barrels.

47. Protein Folding  Alzheimer’s Disease, Type 2 Diabetes and Parkinson’s Disease

48. Amyloid Fibers Stabilized by F
Different Amyloid diseases depend on organ the fibers occur A A

49. Summary of Forces Driving Protein Structure
1.  hydrophobic interactions contribute strongly to protein folding and stabilization  ultimately burring hydrophobic R groups with at least two layers of secondary structure covering them up to exclude water.  
2.  alpha and beta structures are usually in different layers. Their R-groups generally do not allow mixing.
3.  Secondary structure near each other (in primary sequence) are usually stacked (except in quaternary structure).
4.  beta structure is most stable when slightly twisted. The great example being the beta-barrel (Fig 4-20) of many membrane proteins.
5. Beta bends can not form knots.

50. Things to Know and Do Before Class
Know collagen structure and the role of vitamin C.
Structure of globular proteins, circular dichroism, and the main idea of protein families (there are over 800).
Denaturation and Renaturation (or not) of proteins
4. One of the largest unsolved puzzles in modern biochemistry: the details of how proteins fold.
Roles of Chaparones.
Be able to do EOC Problems Problem 12 makes you calculate the molecular weight of the DNP-aa in the diagram.