Proteins

1
Proteins
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 solns
• 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 solns
• Scleroproteins Insoluble in water or salt solns

4
CompositionSimple vs. Conjugated

• Simple-
• Conjugated-
• Apoprotein-
• Holoprotein-
• Prosthetic group-

5
Shape Globular vs. Fibrous

• Rasmol Demo
• 101 ratio arbitrary division

6
Physical Properties

• Not discussed

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?

11
Protein Purification Salting-in/salting out

• Process
• Bigger salts work better (NH4)2SO4
• Bigger proteins ppt at lower salt
• Dialysis

12
Protein Purification Size exclusion
chromatography

• Separate by size (number of amino acids)

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

14
Protein Purification Affinity chromatography

• Takes advantage of a specific binding property of
  the protein.

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?
• Sangers reagent
• 3. What is carboxy terminus?
• Limited carboxypeptidase digestion
• 4. What is sequence?
• Edman degradation

18
Protein Sequencing Continued

• 5. Fragment protein
• cyanogen bromide
• proteases
• 6. Align fragments

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

21
Protein Sequencing Example

• 3. What is amino terminus? Serine
• Sangers reagent

22
Protein Sequencing Example

• 4. What is carboxy terminus? Valine
• Carboxypeptidase
• Limited digestion (short time, low temp) gives a
  single major a.a.

23
Protein Sequencing Example

• 5. What is sequence?
• Edman degradation

24
Protein Sequencing Example

• 6. Fragment protein
• cyanogen bromide 2 pieces
• Chymotrypsin 5 pieces
• Trypsin ? Pieces
• Data on board

25
Protein Sequencing Example

• 7. Align fragments
• Ser-...

26
Much Data, Much Work
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

28
Forces Involved in 1o Structure

• Strong
• peptide bond
• disulfide bond

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

39
Denaturation

• Denaturation- disruption of the normal 3D shape
• agents
• alcohol
• weak acid or base
• heat
• detergents
• reducing agents

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

42
4o Structure and Sickle-cell Anemia

• Glu ß6 Val