Lecture 5

Question 1

Big Picture Items

Answer 1

• Proteins differ tremendously in size and properties
• Several properties are useful for protein purification
• Specific properties are used for protein characterization
• Different proteases have different specificities
• Mass spectrometry is a powerful analytical method
• Protein sequences reveal evolutionary relationships
• The rate of evolution of different proteins differs

Question 2

How many proteins are possible?

Answer 2

The average protein chain is ~ 400 residues in length.

At each position any of the 20 amino acids could occur, so that the number of possibilities is: 20400 = 2.6 x 10520.

The number of atoms in the universe is
estimated as 9 x 1078.

So the number of possible proteins of length 400 residues would exceed the universe in size by many orders of
magnitude.

Protein sizes range from ~ 30 to ~ 35,000 amino acids

Question 3

Protein production

Answer 3

Very often large amounts of proteins are needed for e.g. 3D
structure determination or drug screening: these usually require multi-milligram quantities of pure protein.

One can:
1. Obtain protein from natural sources
2. Clone and overexpress a gene from one species in a rapidly
growing cell of another species: “heterologous expression”.

Popular expression systems include:

• Escherichia coli
• Yeasts like Saccharomyces cerevisiae, Pichia pastoris
• Insect cell lines in culture
• Human cell lines in culture

Question 4

Protein purification procedures

Salting out

Answer 4

Solubility

Often called “ammonium solphate fractionation”

Question 5

Protein purification procedures

Selective dialysis

Answer 5

Size

Dialysis through a size-selective membrane

Question 6

Protein purification procedures

Gel filtration chromotography

Answer 6

size

Percolation through a porous solid matrix

Question 7

Protein purification procedures

Ion exchange chromatography

Answer 7

Charge

Dependent on the isoelectric point (“pI”) of a protein

Question 8

Protein purification procedures

Affinity chromatography

Answer 8

Binding Specificity

Based on binding to a known target (e.g. ligand)

Question 9

Protein purification based on solubility differences

Answer 9

1. Mixture of three proteins

2. Salt added, and centrifuged: the red protein is precipitated into the pellet and removed. (RED

Question 10

Selective dialysis

Answer 10

Dialysis is normally used for buffer exchange, but newer membranes are produced with various size cut-off limits which allow for removal of proteins below a certain molecular weight.

At equilibrium: All the smaller proteins (red) have diffused outside the dialysis bag.

Question 11

Gel filtration chromatography

Answer 11

The gel beads used have cavities which are permeable to smaller molecules and impermeable to larger molecules

larger molecules come first off the column, smaller molecules come later

Question 12

Ion exchange chromatography

Answer 12

• Ion exchange chromatography is used to separate molecules based on their surface charge
• Molecules are passed through an ion exchange matrix. Interaction strength depends on:
• Charge density of protein (modulated by pH)
• Strength of ions that compete with the protein for binding

Question 13

Isoelectric Point (pI)

Answer 13

The isoelectric point of a molecule is the pH at which the net charge of the molecule is zero.

at lower values of pH proteins will carry more positive charge
at higher value of pH proteins will carry more negative charge

If the pH is above the pI the overall charge of the protein is negative.
If the pH is below the pI the overall charge of the protein is positive.

Question 14

Isoelectric Point (pI)

Answer 14

The pI of a protein molecule obviously depends primarily on its amino acid composition.
• However, since the pK’s of individual functional groups in a folded protein depend also on the environment of the group, the pI of a protein depends also on its conformation.
• The precise calculation of the pI of a protein is quite a challenge.
• Proteins with different overall charge run with different speed in an electrical field, which allows for characterization and purification methods based on charge.

Question 15

Principle of ion exchange chromatography

Answer 15

Anion exchange:

1. Chemical groups R+ on a resin in a column are equilibrated with
   anions A− at low ionic strength
   R+A−
2. Polyanion Pn− (protein P with overall charge −n) is added to the
   column, displacing A−:
   R+A− + Pn− R+Pn− + A−
3. Pn− is attached to the column matrix; excess A− flushes out.
4. The column is then washed with several volumes of Na+A- at low
   concentration to elute weakly bound impurities.
5. Next the column is washed with Na+A− at higher concentration
   which elutes the bound Pn−:
   R+Pn− + A− R+A− + Pn-
6. The Pn− polyanion (the protein) is collected in a fraction collector.

Question 16

Ion exchange chromatography

Answer 16

1) positive charges elute first due to repulsion with the matrix
2) negative charges retained on the column due to favorable electrostatics
3) most negative charges elute latest

Question 17

Affinity chromatography

Answer 17

Affinity chromatography separates proteins based on binding some molecule of interest (e.g. a ligand)

1) proteins that don’t bind affinity matrix are washed off
2) after contaminants washed, desired protein is eluted

Question 18

Methods for determining protein concentration

UV spectroscopy

Answer 18

Based on UV spectroscopic absorption of aromatic sidechains (280nm)

Question 19

Methods for determining protein concentration
Coomassie staining (Bradford)

Answer 19

Sensitive measure based on a protein-binding stain

Question 20

Methods for determining protein concentration

Enzyme-linked Immunosorbent Assay (ELISA)

Answer 20

Check for presence (and concentration) of a specific protein

Question 21

Protein concentration determination and A280

Answer 21

• Proteins usually contain several Trp or Tyr or Phe residues. The side chains of Trp and Tyr absorb UV light quite strongly at 280 nm.
• This absorption at 280 nm (A280) is often used for protein concentration determination by absorption spectroscopy
• However:
• Not all proteins contain Trp or Tyr
• UV is not very sensitive, rarely lower than 50 to 100 μg per mL

Question 22

Protein concentration determination with Coomassie blue

Answer 22

• Coomassie brilliant blue binds to proteins.
• In acidic solutions, absorbance shifts from 465 to 595 nm upon binding to proteins.
• Thus, 595 nm absorbance provides a way to measure the total protein concentration.
• Bradford assay uses this absorption shift of Coomassie, enabling detection of protein concentrations as low as 1 μg per mL

Question 23

Concentration determination of a specific protein

Answer 23

1) Immobilize antibody
2) Incubate with protein
3) Add second antibody, covalently linked to an enzyme
4) Wash; measure enzyme activity

Question 24

Concentration determination of a specific protein 2

Answer 24

It is often important to find a rapid & reliable assay to determine the concentration of a specific protein.
A popular method is based on antigenic specificity, and is called:
ELISA (Enzyme-Linked Immunosorbent Assay)
Requires antibodies to the target of interest.
This method, and variations thereof, are key to several “diagnostic kits” which can establish, in a mixture, the presence of particular proteins characteristic of e.g. a cancer cell or a pathogen.

Question 25

Protein characterization methods

Mass spectrometry

Answer 25

Amino acid sequence

Question 26

SDS-PAGE

Answer 26

size

SDS = Sodium dodecyl sulfate
= [CH3- (CH2)10-CH2-O-SO3-]Na+

• SDS denatures proteins & binds to denatured protein with ~one SDS per two amino acids – largely independent of amino acid sequence.
• SDS-treated proteins of similar length have similar, rod-like, shapes, and a charge which is proportional to the length. The larger the protein is, the slower it runs in electrophoresis during SDS-PAGE. (PAGE = Poly-Acrylamide Gel Electrophoresis)
• By using controls, estimates of the molecular mass (within 10 - 20%) can be obtained.

Question 27

Amino Acid Sequence determination

Answer 27

Amino acid sequence may be determined:

1. Chemically
2. By Mass Spectroscopy (“Mass Spec”)

In general: based on a “divide-and-conquer” approach

Question 28

Some proteases and their specificity

Answer 28

Proteases break a peptide bond based on the identities of the adjacent amino acids .

Trypsin Rn-1 = positive residue (Arg/Lys); Rn ≠ Pro

Chymotrypsin Rn-1 = bulky & aromatic (Phe/Trp/Tyr/Leu); Rn ≠ Pro

Thrombin Rn-1 = Arg; Rn ≠ Pro

Thermolysin Rn-1 ≠ Pro; Rn= bulky nonpolar (Leu/Val/Ile/Met)

V-8 Rn-1= negative residue (Asp/Glu)

Question 29

Mass spectrometry

Answer 29

• Mass determination of purified proteins:
Electrospray, MALDI and Fast Atom Bombardment techniques.
• Requires only picomoles (10-12 mole) of material.
• Time required is very short.
• Mass is accurate to ±1 Dalton for proteins up to 300 kD.

• Peptide sequencing using tandem mass spectrometry:
multistep methods
• Proteases fragment the protein into peptides.
• Sequences are determined by matching the masses observed to
expected peptide masses for 1,2,3,… residues

Question 30

Electron Spray Ionization Mass Spectrometry (ESI)

Answer 30

• Dry N2 or some other gas promotes the evaporation of solvent from charged droplets containing the protein of interest, leaving gas-phase ions, whose charge is due to the protonation of Arg and Lys residues, thereby yielding so-called (M + nH)n+ ions
• The mass spectrometer determines the mass-to-charge (m/z) ratio of these ions.
• The resulting mass spectrum consists of a series of peaks corresponding to ions that differ by a single ionic charge and the mass of one proton.

Question 31

Electron Spray Ionization Mass Spectrometry (ESI)

Answer 31

This data is sufficient to determine the mass of the original molecule

• The peaks have shoulders because the polypeptide’s component elements contain small mixtures of heavier isotopes (e.g., naturally abundant carbon consists of 98.9% 12C and 1.1% 13C, and naturally abundant sulfur consists of 0.8% 33S, 4.2% 34S, and 95.0% 35S).

Question 32

The use of a tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein

Answer 32

• Electrospray ionization (ESI) generates gas-phase peptide ions (P1, P2, …) from a digest of the protein to be sequenced.
• Peptides are separated by the first mass spectrometer (MS-1) according to m/z values
• One of them (here P3) is directed into the collision cell, where it collides with helium atoms.
• This treatment breaks the peptide into fragments (F1, F2, etc), directed into a second mass spectrometer (MS-2) for determination of their m/z values,
• From this, the amino acid sequence of the fragment can be determined

This method enabled discovery and determination of post-translational modifications

Question 33

Protein sequence and protein evolution

Answer 33

Sequence information is useful when placed in the broad context of all known protein sequences.

For instance:
• protein fold identification: if > 25% of sequence is conserved between proteins, it is highly likely that two proteins adopt the same overall fold

• creating a family sequence alignment: invariant amino acids in a family are likely to be important for function or for structure
• estimating evolutionary rates of proteins: since not all proteins are subject to the same evolutionary pressure, due to different functions, their rates of change in the course of time is not the same
• constructing phylogenetic trees: these can give profound insight into evolutionary relationships between species

Question 34

Sequence alignment

Answer 34

Alignment of human myoglobin and the human hemoglobin α-chain.

The chains are 27% identical which is sufficient similarity to conclude they are homologous … that is, derived from a common ancestor.

They occur in the same organism, so most likely they are the result of gene duplication & divergence of sequence and function.

Note that it is necessary to insert gaps (so-called “indels”) denoted by “_”, in order to maximize the identities.

Question 35

Phylogenetic tree based on cytochrome c

Answer 35

Each branch point (filled-in circle) represents an organism ancestral to the species connected above it.
• The number beside each branch indicates the number of inferred differences per 100 residues between the cytochromes c of the flanking branch points or species.

Question 36

Rates of evolution of four proteins

Answer 36

Different protein families have clearly remarkably different rates of change in amino acid sequence during evolution

(Horizontal axis according to the fossil record