1. Protein folding
Primary structure itself results in some folding constraints:
See bottom of handout 3-3

3. These 4 red atoms are in one plane (C of C=O central)
And these 4 atoms are in
one plane (N central)
These 4 red atoms are in one plane
(C of C=O central)
 so 6 atoms in one plane

8. There’s still plenty of flexibility

9. H Secondary structure: the alpha helix Amino acids shown
simplified, without
side chains and H’s. H
Almost every N-H and C=O
group can participate

10. Side chains = -CH3 (lighter gray) H’s not shown
Alpha helix depictions
C = grays
N = blue
O = red
Poly alanine
Side chains = -CH3 (lighter gray)
H’s not shown

11. Linus Pauling and a model of the alpha helix.1963

12. beta pleated sheet
Secondary structure:
H-bond
AA residue

13. Beta sheet (i.e., beta pleated sheet)
antiparallel
antiparallel
parallel

14. Beta-sheets
Anti-parallel
Parallel

15. secondary structure (my definition):
structure produced by regular repeated interactions between atoms of the backbone.

16. Tertiary structure: The overall 3-D structure of a polypeptide.
Neither
This is a popular “ribbon” model
of protein structure. Get familiar
with it. The ribbons are stretches
of single polypeptide chains. A
single ribbon is NOT a sheet.
3 alpha helices
A beta sheet
These “ribbon” depictions do not show the side chains, only the backbone

17. Tertiary structure (overall 3-D)
ionic
hydrophobic
H-bond
cys
Ion - dipole interaction
covalent
Van der Waals
Examples of bonds
determining 3D structure
Exist in loop regions and in regions of secondary structure

18. Disulfide bond formation
(covalent, strong)
Sulfhydryl group
½ O2
R-CH2-SH +
HS-CH2-R
R-CH2-S-S-CH2-R
+ HOH
cysteine
cysteine
cystine
Two sulfhydryls have been oxidized (lost H’s)
Oxygen has been reduced (gained H’s).
Oxygen was the oxidizing agent (acceptor of the H’s).
An oxidation-reduction reaction: Cysteines are getting oxidized
(losing H atoms, with electron; NOT losing a proton, not like acids.)
Oxygen is getting reduced, gaining H-atoms and electrons
Actually it’s the loss and gain of the electrons that constitutes oxidation and reduction, respectively.
No catalyst is usually needed here.

19. Stays intact in the jacuzzi at 37 deg C
Overall 3-D structure of a polypeptide is tertiary structure
Stays intact in the jacuzzi at 37 deg C
Usually does not require the strong covalent disulfide bond to maintain its 3-D structure
[Tuber mode]l

20. Protein structures are depicted in a variety of ways
Backbone only
Ribbon
Small molecule
bound
Drawing attention
to a few side groups
Continuous lines, ribbons=
backbone (not sheets)
Space-filling
Space-filing,
with surface charge
blue = +
red = -

21. Most proteins are organized into

22. Handout 4-2

23. Two different
proteins with almost the same 3-D structure !
Handout 4-2

24. 4o, QUATERNARY STRUCTURE
Monomeric protein (no quaternary structure)
Dimeric protein (a homodimer)
The usual
weak
bonds
Dimeric protein (a heterodimer)
Also called:
multimeric proteins
A heterotetramer
A heteropolymeric protein (large one)

25. Hemoglobin  $ One protein  $ Four polypeptide chains,
2 identical alphas
and 2 identical betas
Four “subunits”
Molecular weight 
16,000
Subunit molecular weight $
16,000
Subunit molecular weight $ 
64,000
Protein molecular weight  $
64,000, even though the 4 chains are
not covalently bonded to each other

26. Interchain disulfide bonds
Tetramer
Two heavy chains (H),
Two light chains (L)
Interchain disulfide bonds
The 4 weak bond types

27. Sickle cell disease
Normal
Sickle cell
glu
glu
val

28. Some small molecules can be bound tightly to a protein.
Such associated small molecule are called “prosthetic groups”.
Some are even covalently bound to the protein.
Pyridoxal
phosphate
AA side chain
Enzyme
= Vitamin B6

29. Tetrahydrofolic acid Riboflavin ~ vitamin B2 Heme
Most prosthetic groups are bound tightly via weak bonds.
Tetrahydrofolic acid
~ vitamin B9
Riboflavin ~ vitamin B2
Heme

30. Membrane proteins
Hydrophobic side chains on the protein exterior for the portion in contact with the interior of the phospholipid bilayer.
Anions are negatively charged.
Cations are positively charged

31. Small molecules bind with great specificity to pockets on protein surfaces
Too far

32. Ligand
Protein

33. Testosterone Estrogen Ligand binding can be equisitely specific:
the estrogen reeptor binds estrogen but not testosterone.
Testosterone
Estrogen

34. Protein separation methods Ultracentrifugation
Mixture of proteins

35. Ultracentrifuge

36. Causing sedimentation:
centrifugal force = m(omega)2r
m = mass
omega = angular velocity
r = distance from the center of rotation
Opposing sedimentation = friction = foV.
fo = frictional coefficient (shape)
V = velocity
Constant velocity is soon reached; then, no tnet force
So: centrifugal force = frictional force (balanced each other out)
And so: m(omega)2r =  foV
And:  V = m(omega)2r/fo,
Or:  V = [(omega)2r] x [m / fo]
V proportional to mass (MW)
V inversely proportional to fo
V inversely proportional to “non-sphericity”
(spherical shape moves fastest)

37. Sample loaded here + + + +++ +++ +++ + + + +++ +++ +++ (“native”)
Glass
plates
Sample loaded here
Winner:
Small, +++
High positive charge + + +
Loser:
Large, +
low positive charge
+++
+++
+++
poly-
acrylamide
fibers +
Intermediate:
Large, +++
high positive charge + +
Intermediate:
Small, +
Low positive charge
+++
+++
+++
Molecules shown after several
hours of electrophoresis

38. Upper resevoir
Cut out for contact of buffer with gel

39. Cut out of glass plate for contact of buffer with gel
Clamped glass sandwich
Electrode connection (~ 150 V)
Reservoir for buffer

40. Power supply
Happy
post-doc
Tracking dyes

42. SDS PAGE = SDS polyacrylamide gel electrophoresis
sodium dodecyl sulfate, SDS (or SLS): CH3-(CH2)11- SO4--
CH3-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-SO4--
SDS
All the polypeptides are denatured and behave as random coils
All the polypeptides have the same charge per unit length
All are subject to the same electromotive force in the electric field
Separation based on the sieving effect of the polyacrylamide gel
Separation is by molecular weight only
SDS does not break covalent bonds (i.e., disulfides) (but can treat with mercaptoethanol for that) (and perhaps boil for a bit for good measure)

43. Disulfides between 2 cysteines can be cleaved in the laboratory by reduction, i.e., adding 2 Hs (with their electrons) back across the disulfide bond.
One adds a reducing agent:
mercaptoethanol (HO-CH2-CH2-SH).
In the presence of this reagent, one gets exchange among the disulfides and the sulfhydryls:
Protein-CH2-S-S-CH2-Protein  + 2 HO-CH2CH2-SH  --->
Protein-CH2-SH + HS-CH2-Protein   +  HO-CH2CH2-S-S-CH2CH2-OH
The protein's disulfide gets reduced (and the S-S bond cleaved), while the mercaptoethanol gets oxidized, losing electrons and protons and itself forming a disulfide bond. 

44. P.A.G.E. Molecular weight markers (proteins of known molecular weight)
e.g., “p53”
Molecular weight
markers
(proteins of known
molecular weight)

45. Molecular sieve chromatography
(= gel filtration, Sephadex chromatography)
Sephadex bead

46. Molecular sieve chromatography
Sephadex bead

47. Molecular sieve chromatography
Sephadex bead

48. Molecular sieve chromatography
Sephadex bead

49. Molecular sieve chromatography
Sephadex bead

50. Plain
Fancy
4oC (cold room)

51. Larger molecules get to the bottom faster, and ….
Non-spherical molecules get to the bottom faster
~infrequent
orientation
Non-spherical molecules get to the bottom faster

52. Handout 4-3: protein separations

53. Similar to handout 4-3, but Winners & native PAGE added
Largest and most spherical
Lowest MW
Winners:
Largest and least spherical
Similar to handout 4-3, but Winners & native PAGE added
Most charged and smallest
Winners:

54. Enzymes =
protein catalysts

55. Each arrow = an ENZYME

56. Chemical reaction between 2 reactants
H2 + I2
2 HI
H2 + I2
2 HI + energy
“Spontaneous” reaction:
Energy released
Goes to the right
H-I is more stable than H-H or I-I here
i.e., the H-I bond is stronger, takes more energy to break it
That’s why it “goes” to the right,
i.e., it will end up with more products than reactants
i.e., less tendency to go to the left, since the products are more stable

57. { 2H + 2I Change in Energy (Free Energy) H2 + I2 2 HI -3 kcal/mole
say, 100
kcal/mole
say, 103
Atom pulled completely apart
(a “thought” experiment)
Change in Energy (Free Energy)
H2 + I2 {
-3 kcal/mole
2 HI
Reaction goes spontaneously to the right
If energy change is negative: spontaneously to the right = exergonic: energy-releasing
If energy change is positive: spontaneously to the left = endergonic: energy-requiring

58. H2 + I2 2 HI H2 + I2 2 HI H2 + I2 2 HI H2 + I2 2 HI H2 + I2 2 HI
Different ways of writing chemical reactions
H2 + I2
2 HI
H2 + I2
2 HI
2 HI
H2 + I2
H2 + I2
2 HI
H2 + I2
2 HI

59. { 2H + 2I Change in Energy (Free Energy) H2 + I2 2 HI
But: it is not necessary to break
molecule down to its atoms in order
to rearrange them
say, 100
kcal/mole
say, 103
kcal/mole
Change in Energy (Free Energy)
H2 + I2 {
-3 kcal/mole
2 HI

60. + + + H I I I H I H H H I (H2 + I2) Transition state (TS) (2 HI)
Reactions proceed through a transition state H + I
(H2 + I2) I I H
Transition state
(TS) I + H H + H I
(2 HI)
Products

61. { 2H + 2I Change in Energy H2 + I2 2 HI ~100 kcal/mole H-H | | I-I
(TS)
Say,
~20 kcal/mole
H2 + I2
Activation
energy {
-3 kcal/mole
2 HI

62. { HHII (TS) Change in Energy (new scale) H2 + I2 2 HI
Allows it to happen
Energy needed
to bring molecules
together to form
a TS complex
Change in Energy (new scale)
determines speed =
VELOCITY =
rate of a reaction
Activation
energy
H2 + I2 {
3 kcal/mole
2 HI
Net energy change:
Which way it will end up.
the DIRECTION
of the reaction, independent of the rate
2 separate
concepts

63. Concerns about the cell’s chemical reactions
Direction
We need it to go in the direction we want
Speed
We need it to go fast enough to have the cell double in one generation

64. (to be discussed next time)
Example
Biosynthesis of a fatty acid
3 glucose’s
18-carbon fatty acid
Free energy change: ~ 300 kcal per mole of glucose used is REQUIRED
So: 3 glucose
18-carbon fatty acid
So getting a reaction to go in the direction you want is a major problem
(to be discussed next time)

65. Concerns about the cell’s chemical reactions
Direction
We need it to go in the direction we want
Speed
We need it to go fast enough to have the cell double in one generation
Catalysts deal with this second problem, which we will now consider

66. The catalyzed reaction
The velocity problem is solved by catalysts
The catalyzed reaction
The catalyst takes part in the reaction,
but it itself emerges unchanged

67. HHII (TS) Change in Energy H2 + I2 2 HI Activation energy TS without
catalyst TS
complex
with
catalyst
Change in Energy
Activation
energy
WITH the
catalyst
H2 + I2
2 HI

68. Reactants in an enzyme-catalyzed reaction = “substrates”

69. substrate binding site (not exactly synonymous,
Reactants (substrates)
Active site or
substrate binding site
(not exactly synonymous,
could be just part of the active site)
Not a substrate

70. Unlike inorganic catalysts, enzymes are specific
Substrate Binding

71. Small molecules bind with great specificity to pockets on ENZYME surfaces
Too far

72. Unlike inorganic catalysts, enzymes are specific
                                      succinic dehydrogenase
HOOC-HC=CH-COOH < > HOOC-CH2-CH2-COOH H
fumaric acid                                                     succinic acid
NOT a substrate for the enzyme:
1-hydroxy-butenoate:    HO-CH=CH-COOH
(simple OH instead of one of the carboxyl's)
Maleic acid
maleic acid
Platinum will work with all of these, indiscriminantly

73. Enzymes work as catalysts for two reasons: +
Enzymes work as catalysts for two reasons:
They bind the substrates putting them in close proximity.
They participate in the reaction, weakening the covalent bonds
of a substrate by its interaction with their amino acid residue side groups (e.g., by stretching).