Lecture 5 Flashcards
Big Picture Items
- 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
How many proteins are possible?
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
Protein production
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
Protein purification procedures
Salting out
Solubility
Often called “ammonium solphate fractionation”
Protein purification procedures
Selective dialysis
Size
Dialysis through a size-selective membrane
Protein purification procedures
Gel filtration chromotography
size
Percolation through a porous solid matrix
Protein purification procedures
Ion exchange chromatography
Charge
Dependent on the isoelectric point (“pI”) of a protein
Protein purification procedures
Affinity chromatography
Binding Specificity
Based on binding to a known target (e.g. ligand)
Protein purification based on solubility differences
- Mixture of three proteins
2. Salt added, and centrifuged: the red protein is precipitated into the pellet and removed. (RED
Selective dialysis
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.
Gel filtration chromatography
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
Ion exchange chromatography
- 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
Isoelectric Point (pI)
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.
Isoelectric Point (pI)
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.
Principle of ion exchange chromatography
Anion exchange:
- Chemical groups R+ on a resin in a column are equilibrated with
anions A− at low ionic strength
R+A− - Polyanion Pn− (protein P with overall charge −n) is added to the
column, displacing A−:
R+A− + Pn− R+Pn− + A− - Pn− is attached to the column matrix; excess A− flushes out.
- The column is then washed with several volumes of Na+A- at low
concentration to elute weakly bound impurities. - Next the column is washed with Na+A− at higher concentration
which elutes the bound Pn−:
R+Pn− + A− R+A− + Pn- - The Pn− polyanion (the protein) is collected in a fraction collector.
Ion exchange chromatography
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
Affinity chromatography
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
Methods for determining protein concentration
UV spectroscopy
Based on UV spectroscopic absorption of aromatic sidechains (280nm)
Methods for determining protein concentration Coomassie staining (Bradford)
Sensitive measure based on a protein-binding stain
Methods for determining protein concentration
Enzyme-linked Immunosorbent Assay (ELISA)
Check for presence (and concentration) of a specific protein
Protein concentration determination and A280
- 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
Protein concentration determination with Coomassie blue
- 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
Concentration determination of a specific protein
1) Immobilize antibody
2) Incubate with protein
3) Add second antibody, covalently linked to an enzyme
4) Wash; measure enzyme activity
Concentration determination of a specific protein 2
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.
Protein characterization methods
Mass spectrometry
Amino acid sequence
SDS-PAGE
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.
Amino Acid Sequence determination
Amino acid sequence may be determined:
- Chemically
- By Mass Spectroscopy (“Mass Spec”)
In general: based on a “divide-and-conquer” approach
Some proteases and their specificity
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)
Mass spectrometry
• 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
Electron Spray Ionization Mass Spectrometry (ESI)
- 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.
Electron Spray Ionization Mass Spectrometry (ESI)
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).
The use of a tandem mass spectrometer (MS/MS) in amino acid sequencing of a protein
- 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
Protein sequence and protein evolution
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
Sequence alignment
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.
Phylogenetic tree based on cytochrome c
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.
Rates of evolution of four proteins
Different protein families have clearly remarkably different rates of change in amino acid sequence during evolution
(Horizontal axis according to the fossil record
