1. Chapter 5
CHM 341
Fall 2016

2. I. Amino Acids Most proteins are composed of 20 “standard” amino acids
 - amino acids with exception of proline because all have primary amino group and carboxylic acid group on SAME carbon

3. I. Amino Acids
Hydrophobic
Have non-polar side chains
Charged
Polar
Side chains interact with water because have H-bonding ability

4. I. Amino Acids
D. Peptide Bonds
 - amino acids polymerize though elimination of water
Can make dipeptide, tripeptides, polypeptides
Polypeptides are linear polymers

5. I. Amino Acids
E. pka values are determined by induction and resonance

6. I. Amino Acids
F. Ionization properties of amino acids
Estimate the net charge of the polypeptide chain at pH 7.0 Asp – Tyr – Tyr – Glu – Cys Estimate the net charge of the polypeptide chain at pH 7.4 Ala – Asp – His – Lys – Glu – Arg – Gln

7. II. Protein Structure
Proteins are at center of action in biological process
Enzymes
Regulators
Messengers
Transport/store molecules like metals, O2
Proteins are best understood in terms of its structure (3-D form)

8. 3-D Structure of Proteins
Primary (1o)
Secondary (2o) structure:
Tertiary (3o)
Quaternary (4o) structure:

9. A. Primary (1o) Structure)
First protein structure determined was insulin in 1953
Since then 10,000s of proteins sequenced

10. Linked several amino acids in a polypeptide chain
B. 2o Characteristics
Linked several amino acids in a polypeptide chain
Electrons are delocalized so peptide bond has 2 resonance forms

11. 2. Cis conformation Less stable Likely with Pro 1. Trans Conformation
Consequence of resonance interactions that give a peptide bond ~40% double bond character which gives peptide group a rigid planar structure
2. Cis conformation
Less stable
Likely with Pro

12. Rotation around C-N bond () Rotation around C-C bond ()
3. Torsion Angles
Conformation of the backbone can be described by torsion angles (dihedral angles)
Rotation around C-N bond ()
Rotation around C-C bond ()
and are defined at 180o when chain is in its fully extended conformation and increases clockwise when viewed along C
Some conformations are going to be more likely than others

13. 3. Torsion Angles and Ramachandran Diagrams
Sterically allowed values of  and  can be calculated
Information summarized in Ramachandran diagram
Most areas are forbidden because of steric hindrance

14. Most recognizable structure Characterized by
4. a-Helical Structure
Most recognizable structure
Characterized by
number (n) of peptide units per helical turn
pitch (p) distance the helix rises along it axis per turn
Has both favorable H-bonding and  and  fall in favorable Ramachandran diagram
Right handed
3.6 residues per turn, pitch of 5.4 A
Amino acid side chains avoid steric hindrance

15. 5. Beta Structure
Repeating  and  angles that fall in allowed Ramachandran diagram
Utilize full H-bonding, BUT H-bond between chains (not within a chain)
Anti-parallel:
Parallel:

16. Contain 2-22 polypeptide strands containing up to 15 residues
5.  Structures
Often will be pleated
Contain 2-22 polypeptide strands containing up to 15 residues
When are requirement for a b-sheet to form?

17. 6. Irregular Secondary Structures
Coils and loops (~50% of protein structure)
NOT random structures

18. C. 3o Structure and Protein Structure: Globular Proteins
Exist as a compact spheroidal molecule

19. Connected by 1 – 2 polypeptide segments
a. Domains
Connected by 1 – 2 polypeptide segments
Neighboring domains are structurally independent units that have the characteristics of small globular proteins
Domains have specific functions – binding of small molecules
Two – domain protein
glyceraldehyde-3-phosphate
dehydrogenase

20. b. Stabilizing Globular Proteins
Folded only marginally more stable than unfolded

21. Protein will fold as soon as it emerges from ribosome
c. Protein Folding
Protein will fold as soon as it emerges from ribosome
Protein folding is not random
Requires assistance and other proteins called molecular chaperones assist in folding

22. D. Quaternary Structure
Proteins with molecular mass > 100 kD generally have more than one polypeptide chain.
Individual chains are called sub-units
Subunits will arrange themselves with specific geometry = quaternary structure

23. Small intercellular protein
III. Myoglobin
Small intercellular protein

24. Heme contains 4 pyrrole groups
A. Heme group
Heme contains 4 pyrrole groups
Fe(II) atom at the center is coordinated by the 4 porphyrin N atoms and one N from a His side chain

25. B. Equilibrium of O2 binding
Myoglobin binding of O2 is simple equilibrium

26. C. Binding Curve
Steepness of hyperbola increases as K decreases

27. IV. Hemoglobin Structure & Mechanism
4 polypeptide chains
2  subunits
2  subunits

28. III. Hemoglobin Structure & Mechanism
Oxygenation causes extensive quaternary structural changes
Oxy- and Deoxy- Hb have different forms

29. A. Binding of O2
T-state (deoxy)
R-state (oxy)
In T state (blue) Fe(II) located 0.6 Å out of heme plane
When O2 binds Fe-N porphyrin bonds contract and Fe(II) moves in plane (red)

30. Difference between T and R occur at 1-2 and 2-1 interface
B. 2 Stable Positions
Difference between T and R occur at 1-2 and 2-1 interface

31. C. Role of Globin in Binding of O2
Protect Fe(II)
His attached to backside of porphyrin

32. D. Relative Stability of T and R
With no O2 present: T more stable
With O2 present: R more stable

33. V. Hemoglobin binding and pH
Effect of pH on Hb transport
Lung pH = 7.6
Blood pH = 7.2
pO2 in tissues = 30 torr
pO2 in lungs = 95 torr

34. Bohr Effect

35. VI. 2 – 3 Bis-phosphoglycerate
Red blood cells use BPG to fine tune hemoglobin function

36. VII. Abnormal Hemoglobins
Sickle Cell Anemia
Deoxyhemoglobin S forms insoluble filaments that deform red blood cells
Rigid sickle shaped cells cannot pass through the capillaries
Results in tissue death: lack of oxygen
Mutant hemoglobin where hemoglobin S contains Val instead of Glu at the 6th position of the  chain
Causes polymerization of hemoglobins

37. VII. Structural Proteins
Typical eukaryotic cells have 3 types of cytoskeletal proteins that form fibers

38. A. Microfilaments
Made of actin
Network of microfilaments support plasma membrane

39. B. Microfilaments extend/retract
Polymerization of actin monomers is reversible process so the polymer undergoes constant shrinking and growing as subunits add to and dissociate from one or both ends of the microfilaments

40. C. Microtubules
Microtubules are cytoskeletal fibers built from globular protein subunits
Microtubules can assemble and disassemble on a time scale that allow the cell to rapidly change shape in response to external or internal stimuli

41. D. -Keratin
Intermediate filaments are structural proteins
Chemically un-reactive
Component of hair, horns, nails and feathers
-helix shape, but exhibits smaller than expected spacing - due to coiled coil structure

42. E. Collagen
Most abundant animal protein
Major stress-bearing components of connective tissues (bone, teeth, tendons)
Has distinct amino acid composition
Every 3rd amino acid = glycine

43. E. Collagen
Cross-linking between fibrils also increases insolubility
Can’t be S-S bonds
Cross-link between Lys and His chains using Lysyl oxidase
Tends to occur near termini

44. Analyzing Protein Structure
Chromatography
Amino acid sequence of protein determine its overall chemical characteristics
1. Size Exclusion
2. Ion Exchange

45. B. Edman Degradation