Unit 2 Question Bank
It is determined by the nucleotide sequence of the gene, and it dictates every higher order of structure and therefore the protein's function. Clinically it serves as a molecular fingerprint identifying the protein and as a route back to the gene that encodes it.Harper's ch.4, p.25
Proteins whose Stokes radius is too large to enter the pores are excluded, remain in the flowing mobile phase and elute first; smaller proteins enter the pores, are retarded, and elute later. Proteins therefore emerge in descending order of Stokes radius.Harper's ch.4, p.27 · Practical Exp 1
It is a function of both molecular mass and shape: a rapidly tumbling elongated protein sweeps out a larger effective volume than a spherical protein of the same mass, and so behaves as the larger molecule during size-exclusion chromatography.Harper's ch.4, p.27
In theory only proteins that interact with the immobilised ligand adhere; all others wash through. Bound protein is eluted by competition with free soluble ligand, or less selectively with urea, guanidine hydrochloride, mildly acidic pH or high salt. Recombinant examples: a Ni²⁺ matrix binding a polyhistidine tag, a glutathione matrix binding a GST fusion.Harper's ch.4, p.28
SDS binds at about one molecule per two peptide bonds, denaturing the polypeptide and conferring an approximately uniform charge-to-mass ratio, so that migration depends on relative molecular mass (Mr) alone. Used with 2-mercaptoethanol or dithiothreitol to reduce disulfide bonds, it resolves the individual subunits of a multimeric protein; bands are visualised with Coomassie Blue. It is the standard method for assessing protein purity.Harper's ch.4, p.28
Each protein migrates until it reaches the region where the pH equals its isoelectric point (pI) — the pH at which its net charge is zero — and there it stops. Combined with SDS-PAGE it gives two-dimensional electrophoresis, separating by pI in one dimension and Mr in the other.Harper's ch.4, p.29
Treatment with acid in a non-hydroxylic solvent then releases a phenylthiohydantoin (PTH) amino acid, identified by its chromatographic mobility, together with a peptide one residue shorter. Because the remaining peptide bonds are untouched, the process can be repeated on the newly exposed N-terminus — typically for 5–30 cycles.Harper's ch.4, pp. 29–30
The first spectrometer separates individual peptides by mass and directs one selected peptide into the second, where it is fragmented and the masses of the fragments determined. Clinically it is used to screen newborn blood for amino acids, fatty acids and other metabolites, giving diagnostic indicators of disorders such as phenylketonuria.Harper's ch.4, p.33
Unlike the genome it is not fixed: genes are switched on and off, different cell types express different proteins, expression changes with growth, differentiation and external stimuli, and many proteins are modified post-translationally. The proteome is therefore described as a moving target. Proteomics is the study of it, aiming to identify proteins whose expression correlates with medically significant events.Harper's ch.4, p.33
The overall strategy
Determination of primary structure proceeds in four stages: purify the protein; assess its purity and dissociate it into individual chains; cleave those chains into peptides short enough to sequence; sequence the peptides and reassemble them using overlaps.
1 · Purification
Classic methods exploit differences in relative solubility: isoelectric precipitation (at its pI a protein has no net charge and aggregates), precipitation with ethanol or acetone, and salting out with ammonium sulfate.
Higher resolution is obtained by column chromatography, in which proteins partition between a stationary and a mobile phase:
| Method | Separates by |
|---|---|
| Size exclusion (gel filtration) | Stokes radius — large proteins excluded from the pores elute first |
| Ion exchange | Net charge; elute by raising ionic strength |
| Hydrophobic interaction | Exposed hydrophobic surface; elute by lowering salt |
| Affinity | Specific binding to an immobilised ligand — the most selective step |
| Reversed-phase HPLC | Hydrophobicity at high pressure; used to purify peptides |
2 · Assessment of purity and dissociation of chains
Purity is assessed by SDS-PAGE, in which SDS confers a uniform charge-to-mass ratio so that separation depends on Mr alone; bands are stained with Coomassie Blue. Disulfide bonds must be cleaved before sequencing — reductively with 2-mercaptoethanol or dithiothreitol, or oxidatively with performic acid — so that the constituent polypeptides separate. Isoelectric focusing, and two-dimensional IEF/SDS-PAGE, give further resolution.
3 · Cleavage into peptides
Since sequencing methods read only a limited number of residues, long chains are cleaved into smaller peptides. Cleavage also circumvents a blocked N-terminal α-amino group produced by post-translational modification.
| Reagent | Cleaves on the C-side of |
|---|---|
| Trypsin | Arg, Lys |
| Chymotrypsin | Phe, Trp, Tyr |
| Cyanogen bromide | Met |
| S. aureus V8 protease | Glu |
More than one method of cleavage must be used, so that the two sets of peptides have overlapping sequences; the overlaps establish the order of the fragments in the parent chain. The peptides are purified by reversed-phase HPLC.
4 · Sequencing the peptides
Edman degradation. Phenylisothiocyanate derivatises the N-terminal residue as a phenylthiohydantoic acid; acid in a non-hydroxylic solvent releases a phenylthiohydantoin, identified by chromatographic mobility, plus a peptide one residue shorter. The cycle repeats, in an automated sequenator, for about 5–30 residues.
Mass spectrometry, now the method of choice. Peptides are volatilised by electrospray ionisation or MALDI and their masses determined; fragmentation within the instrument by collision-induced dissociation breaks the labile peptide bonds preferentially, so successive fragments differ by one residue and the mass difference identifies it. Since each amino acid has a unique mass — except leucine and isoleucine — the sequence can be reconstructed. Tandem MS permits analysis of mixtures without prior purification.
The hybrid approach. A short stretch of the real protein is sequenced chemically, and this information is used to identify and clone the gene; the remainder of the sequence is then obtained by DNA sequencing, which is faster and cheaper. Note, however, that DNA gives no information about post-translational modification.
The common principle
All chromatography partitions molecules between a stationary phase — a column of beads of modified cellulose, acrylamide or silica — and a mobile phase percolating through it. Association between protein and matrix is weak and transient; proteins that interact more strongly with the stationary phase are retained longer. Optimal separation is achieved by manipulating the composition of both phases. Fractions of eluant are collected as they emerge.
Size-exclusion (gel filtration) chromatography
Porous beads separate proteins by Stokes radius, the radius of the sphere occupied by the tumbling molecule, which depends on both mass and shape. Proteins too large to enter the pores are excluded and travel with the mobile phase; smaller proteins enter the pores and are retarded. Proteins elute in descending order of Stokes radius.
Ion-exchange chromatography
Separation by charge–charge interaction. Cation exchangers carry negative groups (carboxylate, sulfate) and bind net-positive proteins; anion exchangers carry positive groups (tertiary or quaternary amines, e.g. DEAE-cellulose) and bind net-negative proteins. Bound proteins are displaced by gradually raising the ionic strength, and elute in inverse order of the strength of their interaction.
Hydrophobic interaction chromatography
The matrix is coated with hydrophobic groups such as phenyl- or octyl-Sepharose. Proteins with exposed hydrophobic surfaces adhere, and the interaction is enhanced by high ionic strength. Elution is achieved by lowering the salt concentration — the opposite of ion exchange — and if necessary by adding ethanol or glycerol to reduce polarity.
Affinity chromatography
Exploits the biological specificity of a protein for its ligand: an immobilised substrate, product, coenzyme or inhibitor. In theory only proteins that recognise the ligand adhere, making this the most selective method. Elution is by competition with free ligand, or less selectively with urea, guanidine hydrochloride, acidic pH or high salt.
Reversed-phase HPLC
Uses incompressible silica or alumina microbeads at pressures of up to a few thousand psi, permitting high flow rates and greatly enhanced resolution. The stationary phase is an aliphatic polymer 3–18 carbons long; peptides are eluted with a gradient of a water-miscible organic solvent such as acetonitrile or methanol. It is the standard method for purifying peptides after cleavage.
The problem electrophoresis must solve
Electrophoresis separates charged biomolecules by the rate at which they migrate in an applied electric field. That rate normally depends on both charge and size — two variables at once, which makes the result uninterpretable. Each of the two methods below solves this by fixing one variable.
SDS-PAGE — eliminating charge as a variable
Acrylamide is polymerised and cross-linked to form a porous matrix. The anionic detergent sodium dodecyl sulfate binds the polypeptide at a ratio of approximately one SDS molecule per two peptide bonds, causing it to unfold or denature.
Because each SDS molecule carries a charge of −1 and the number bound is proportional to chain length, the large number of anionic SDS molecules overwhelms the charge contributions of the amino acid side chains. Every SDS–polypeptide complex therefore has approximately the same charge-to-mass ratio. Charge has been neutralised as a variable, and the only thing left to distinguish the molecules is the physical resistance each encounters moving through the acrylamide mesh. Large complexes meet greater resistance, so polypeptides separate strictly by relative molecular mass (Mr).
Used together with 2-mercaptoethanol or dithiothreitol, which reduce disulfide bonds, SDS-PAGE resolves the individual subunits of a multimeric protein. Bands are visualised after electrophoresis with a dye such as Coomassie Blue. This is the most widely used method for determining the purity of a protein.
Isoelectric focusing — using charge as the sole variable
Ionic buffers called ampholytes together with an applied field generate a pH gradient within the polyacrylamide matrix. An applied protein migrates through the gradient until it reaches the region where the pH equals its own isoelectric point (pI), the pH at which its net charge is zero. There the field can exert no further force and the protein halts.
The method is self-sharpening: a molecule that diffuses towards lower pH becomes positively charged and is driven back; one that diffuses towards higher pH becomes negatively charged and is likewise returned. Proteins therefore concentrate into narrow bands at their pI.
The advantage of combining them — two-dimensional electrophoresis
The two methods separate on independent properties: pI and Mr. In two-dimensional electrophoresis the sample is first resolved by IEF in one dimension; the IEF gel is then laid horizontally across the top of an SDS gel and the proteins resolved again by SDS-PAGE in the second dimension.
Because two proteins that happen to share a pI will almost certainly differ in Mr, and vice versa, resolution improves dramatically. A crude bacterial extract giving a smear of overlapping bands on a one-dimensional gel resolves into hundreds of discrete spots, and about 1000 proteins can be resolved on a single gel. Two-dimensional electrophoresis is therefore particularly suited to complex mixtures, and formed the basis of first-generation proteomics: spots were excised and identified by Edman sequencing or mass spectrometry, matched against databases by Mr and pI.