2. Proteins
Macromolecules built of amino acids.
Huge number of possibilities
Classified in many ways:
• solubility
• composition
• shape
• physical properties
• function
• 3-D structure
3. Solubility
Albumins Soluble in water and salt soln’s
Globulins Sparingly soluble in water but
soluble in salt solutions
Prolamines Soluble in 70-80% EtOH but
insol in water and absolute EtOH
Histones Soluble in salt soln’s
Scleroproteins Insoluble in water or salt soln’s
7. Function
Enzymatic catalysts- next chapter
Transport and storage of molecules- Hb, ferritin
Mechanical functions- elastin
Movement- myosin
Protection- Ab
Information processing- rhodopsin
Regulatory- renin
Other
8. Structure
Primary (1o)- sequence of amino acids
Secondary (2o)- local 3-D shape
a-helix
ß-sheet
collagen triple helix
Tertiary (3o)- global 3-D shape
Quaternary (4o)- relation of polypeptides
9. 1o Structure
1o Structure- sequence of amino acids (disulfide
bond locations)
MUST have pure protein
10. Protein Purification
Starting Material
Start with a source very rich in protein:
Organism, tissue, cell type
Can you isolate a particular organelle as a starting
purification step?
13. Protein Purification
Ion-exchange chromatography
Separate on basis of protein charge
lys/arg vs asp/glu
net charge
Positively charged beads
Negatively charged beads
15. Protein Purification
Electrophoresis
Preparative electrophoresis can be used, but
analytical electrophoresis used more often to
see how pure the protein is.
16. Protein Amino Acid Composition
Acid hydrolysis- destroys Ser, others
Base hydrolysis- destroys Gln, others
17. Protein Sequencing
1. What is amino acid composition?
2. What is amino terminus?
Sanger’s reagent
3. What is carboxy terminus?
Limited carboxypeptidase digestion
4. What is sequence?
Edman degradation
19. Protein Sequencing
1. What is amino acid compostion?
2. What is amino terminus? -Sangers reagent
3. What is carboxy terminus? -Carboxypeptidase
4. What is sequence? -Edman degradation
5. Fragment protein
cyanogen bromide
proteases
6. Align fragments
20. Protein Sequencing Example
1. Isolated pure protein
2. What is amino acid composition?
Acid hydrolyze, 2D chromatography and detect
Gly- 2 Val- 2 Pro- 2 Ser- 2
His- 1 Phe- 1 Tyr- 1 Trp- 1
Met- 1 Arg- 3 Lys- 3 Glx- 1
22. Protein Sequencing Example
4. What is carboxy terminus?: Valine
Carboxypeptidase
Limited digestion (short time, low temp) gives a
single major a.a.
26. Much Data, Much Work
a c i d o r b a s e
h y d r o l y s i s
A m i n o A c id
C o m p o s i t io n
S a n g e r 's
T y p e t it l e h e r e
A m i n o
t e r m i n u s
C a r b o x y p e p t i d a s e
C a r b o x y
t e r m in u s
F r a g m e n t 1
E d m a n
f r a g m e n t
s e q u e n c e
F r a g m e n t 2
E d m a n
f r a g m e n t
s e q u e n c e
C N B r
F r a g m e n t 1
( S e q u e n c e )
E d m a n
f r a g m e n t
s e q u e n c e
F r a g m e n t 2
E d m a n
f r a g m e n t
s e q u e n c e
F r a g m e n t 3
E d m a n
f r a g m e n t
s e q u e n c e
F r a g m e n t 4
E d m a n
f r a g m e n t
s e q u e n c e
F r a g m e n t 5
E d m a n
f r a g m e n t
s e q u e n c e
T r y p s in , e t c .
P u r e P r o t e in
T y p e t i t l e h e r e
27. The Process Just Described is a
Huge Amount of Work
• Method of choice today is to sequence DNA:
drawback
• Only with prior knowledge do we move
forward
29. Primary Structure Determines
Secondary Structure
Dipeptide model- not all conformations are
possible
Ramachandran plot
Secondary (2o)- local 3-D shape
a-helix
ß-sheet
collagen triple helix
30. a-helix
Compact
pitch: rise/residue
know the dimensions
location of R groups
every fourth amino acid R group interacts
amino acids
NOT Pro
forces responsible
h-bonds parallel to axis
31. ß-sheet
More extended conformation
location of R groups
alternate
amino acids
“all”
forces involved
h bonds perpendicular
Parallel vs. Antiparallel
Usually short
32. collagen triple helix
Kinky
Pro-X-Gly or Hyp-X-Gly
Pro makes kinks
X varies
Why Gly?
33. Forces Involved in 2o Structure
Weak
hydrogen bonds
electrostatic interactions
metal ion coordination
hydrophobic effect
34. 3o Structure
3o Structure- Global 3-D shape
How are the 2o structures arranged in relation to
each other?
35. Predicting 3o Structure
Shape of 6,000 proteins determined by X-ray
crystallography
More than 500,000 sequenced
Computers allow prediction if sequence known
36. 3o Structure
Huge number of possible structures
Generalizations
interior hydrophobic, exterior hydrophillic
form follows function: motifs
EX: helix-loop-helix motif
EX: beta bend motif
EX: Greek Key motif
EX: ß-a-ß motif
37. Protein Folding
Not just any old way
For a given protein, all molecules have the same
shape.
folding occurs in stages
Domains-several motifs usually combine to form
compact globular structures
38. Forces Involved in 3o Structure
Weak
hydrogen bonds
electrostatic interactions
metal ion coordination
hydrophobic effect
40. 4o Structure
• 2 or more subunits arranged in relation to each
other
• held together by noncovalent interactions
• 2 or more subunits ---> dimer, trimer, etc.
• homodimer vs. heterotrimer, etc.
41. Forces Involved in 4o Structure
Weak
hydrogen bonds
electrostatic interactions
metal ion coordination
hydrophobic effect
?Can you give me some examples of what chemicals you think youve used, or how you think chemistry may have impacted your life?
Total lipid 570 mg/dL in average human (range 450-1000 mg/dL)
of that, 12 mg/dL is free fatty acids (FFA)
Salmonella, e.g. have unbranched and odd chain length fatty acids
Beef fat: 16:0, 29%; 18:0, 21%; 16:n and 18:n, 46%
