Chapter 5: Proteins: Primary Structure

1
Chapter 5Proteins Primary Structure
2
Proteins Primary Structure

• Purification
• Sequencing
• Evolution

3
Protein Purification

• Cloning has dramatically changed protein
  purification.
• Yields increase from 1-2 to 40

4
Protein Purification

• STEP 1 Breaking open the cell (mechanical
  disruption)
• Grinding
• Sonication
• Followed by filtration and/or centrifugation
• Where is your protein?

5
Protein Purification

• STEP 2 - Quantification
• Why do we measure our yield?
• How do we measure our yield?
• Mass
• Activity
• Enzymatic or Coupled Enzymatic Assays
• Immunoassays
• RIA radioactive product
• ELISA colored product

6
Protein Purification(Some Definitions)

• STEP 2 - Quantification
• Measuring our yield
• Specific Activity The number of units of enzyme
  per unit mass of protein, usually in milligrams
  of protein.
• International Unit (1 U) the amount of enzyme
  that catalyzes the formation of 1 micromole of
  product per minute under defined conditions
  (usually saturating substrate).

7
Protein Purification(Problem Solving)

• Step 1 of your purification yields 100 mL of a
  crude extract containing 10 mg/mL of protein and
  1000 U/mL of enzyme. Step 2 results in the
  collection of a fraction with a volume of 25 mL
  containing 10 mg/mL protein and 3000 U/mL enzyme.
  Was this a worthwhile purification step? What
  fold purification was achieved?

8
Protein Purification(Problem Solving)

• Step 1
• 100 mL x 10 mg/mL protein 1,000 mg protein
• 100 mL x 1,000 U/mL enzyme 100,000 U enzyme
• Specific Activity 100,000 U/1,000 mg 100 U/mg
• Step 2
• 25 mL x 10 mg/mL protein 250 mg protein
• 25 mL x 3000 U/mL enzyme 75,000 U enzyme.
• Specific Activity 75,000 U/250 mg 300 U/mg
• Net purification 3 fold Enzyme
  yield 75

9
Protein Purification(Problem Solving)

• What is the net charge of the following peptide
  at pH 7.0?
• A-D-L-A-P-M-I-F-W-Y-V
• (Written N to C terminus)
• Assuming that peptide bond formation has no
  effect on the pKs of other non-bonded ionizable
  groups, what is the isoelectric point on this
  peptide?

10
Protein Purification(Problem Solving)

• What is the net charge of the following peptide
  at pH 7.0?
• A-D--L-A-P-M-I-F-W-Y-V-
• (Written N to C terminus)
• What is the isoelectric point on this peptide?
• pK ?-COOH 2.29
• pK ?-COOH 3.90
• pK ?-NH3 9.87
• pI (2.29 3.90)/2 3.10

11
Separation TechniquesChromatography

• Hydrophobic Interaction Chromatography

Eluant ? ionic strength ? detergent ?pH
-Octyl or Phenyl groups on matrix
hydrophobicity
1
1
2
3
4
12
Separation TechniquesChromatography

• Gel Filtration / Size Exclusion / Molecular Sieve
  Chromatography

Eluant Constant buffer
-Gel Bead with pores Sephadex
Smallest particles come of last in a linear
relationship between eluant volume and log of MW
Decreasing size
2
1
3
4
13
Separation TechniquesChromatography

• Affinity Chromatography

Eluant 1 - Load and Wash with binding
buffer 2 - Elute ? pH or ionic strength (altered
binding)
-Matrix contains bound ligand for protein of
interest
2
1
3
4
14
Separation TechniquesPolyacrylamide Gel
Electrophoresis (PAGE)
-

• Separation by size and charge
• Charge is NOT uniform with size
• Run pH 9.0 net charge on proteins is (-)

15
Separation TechniquesPolyacrylamide Gel
Electrophoresis (PAGE)
-

• Band Detection
• Protein Stain
• Autoradiography
• Enzymatic Stain
• Immunoblotting (Western Blots)

16
Separation TechniquesSDS Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
-

• Proteins bind 1 SDS/2 a.a. giving strong negative
  charge THUS, Separation by size only
• Used to determine MW of subunits (relative
  mobility vs. log mass)

17
Separation TechniquesCapillary Electrophoresis
(CE)

• Uses microcapillaries (20 75 micrometers)
• Dissipate heat, allowing high electric fields
• Good for small samplesrapid and quantitative

18
Separation TechniquesUltracentrifugation -
Analytical

• Svedberg found that proteins CAN be precipitated
  at exceptionally high g forces (about 600,000g)
• Sedimentation rate is proportional to mass in set
  conditions (standard conditions 20º C in pure
  water) can be monitored optically
• Now used only for non-covalently associated
  molecules due to SDS-PAGE and gel filtration
  chromatography.
• The sedimentation coefficient Svedberg Unit
  (usually in 10-13 sec)

19
Separation TechniquesUltracentrifugation -
Preparative

• Zonal Ultracentrifugation Inert (sucrose)
  Gradient Separation monitored and stopped when
  separation is achieved (optical monitoring)
• Equilibrium Density Gradient Centrifugation CsCl
  forms a semi-stable gradient that holds bands
  of corresponding densities in place.
• Following either type of run, the bottom of the
  centrifuge tube is punctured and fractions are
  collected.

20
Protein Sequencing

• Sanger sequenced insulin in 1953.
• Took 10 years and 100 g protein
• Automation now allows average proteins to be
  completed in several days with micrograms of
  material
• Mass spectrometry can be used for small (25
  residue) peptides
• DNA sequencing can be used if corresponding DNA
  is known

21
Protein Sequencing

• IMPORTANCE
• Primary sequence predicts 3-D structure and
  mechanisms
• Sequence homologies are used to determine
  evolutionary relationships
• Point a.a. substitutions are often the cause of
  lack of function, causing disease

22
Protein Sequencing

• Preliminary Steps Terminal Group Analyses
• N-terminal analysis Dansyl Chloride reacts with
  primary amines
• Tells of peptides in a protein (how?)
• C-terminal analysis Carboxypeptidase
• May yield confusing results if penultimate a.a.
  has strong affinity for enzyme than the last a.a.

23
Protein Sequencing

• Preliminary Steps Disulfide Cleavages
• Separates polypeptide chains for separate
  sequencing
• May use performic acid (harsh-destroys Trp and
  oxidizes Met) or mercaptans

24
Protein Sequencing

• Total Amino Acid Analysis
• Total hydrolysis by acid, base or enzyme followed
  by HPLC separation
• Acid bad for Asn, Gln
• Base bad for Cys, Ser, Thr Arg
• Enzymatic incomplete and self-contaminating

25
Protein Sequencing

• Step 1
• Cleave proteins to 40-100 residue segments using
  endopeptidases (DNA Mapping)
• Trypsin (Arg, Lys)
• Chymotrypsin
• Thermolysin
• Pepsin
• Endopeptidase
• Cyanogen Bromide (Met)

26
Protein Sequencing

• Step 2
• Edman Degradation
• PITC (phenylisothiocyanate) Rx with N-terminus
  under alkali conditions to yield PTC-amino acid
  that is cleaved with acid to yield a PTH-amino
  acid that is detected by chromatography
• Cleavage is specific for the PTC-adduct thus,
  the reaction can be repeated to cleave single
  a.a.s from the N-terminus
• The cycle can be repeated up to 100 times in
  modern automated sequencing equipment using only
  picomoles of peptides

27
Protein Sequencing

• Step 3
• Reconstructing the Sequence
• Sequenced fragments from separate enzymatic
  cleavages are compared to find overlapping
  sequences
• Disulfide linkages can be found by comparing
  reduced and non-reduced protein fragments by
  Diagonal Electrophoresis or gel filtration

28
Protein Sequencing

• Step 4
• Homology Seeking
• Databases of protein sequences (e.g., Swiss-Prot)
  are maintained and shared worldwide
• Sequence homology is an index of relatedness
  among species and the evolution of certain
  proteins, e.g., cytochrome C
• Invariant residues indicate key reactive sites
  and conservatively substituted
• Hypervariable regions exist in less
  functionally-important regions leading to neutral
  drift

29
Acid Base Properties
H2N CH2 COO -
pK2 9.78
pI 7.24
pI ½ (pKi pKj)
H3N CH2 COO -
pK1 2.35
H3N CH2 COOH
What are the pIs for K, R, D, E, and H?