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Biochem Lecture 6
Terms in this set (61)
What are the most prominent post-translational modifications?
Phosphorylation, acetylation, and methylation
Takes place on S,T,Y,H. Uses ATP, kinases, phosphatases, and adds negative charge (depends on pH)
Takes place on K. Uses Acetyl-CoA, acetylases, and deacetylases to neutralize positive charge.
Takes place on K, R. Uses methyl donors (ex SAM). Mono/di/tri for K, Mono/di for R. Uses methylases, and demethylases. Does NOT neutralize positive charge.
What is GFP?
GFP is a naturally fluorescent protein due to a GFP chromophore lying in the center of a beta-barrel protein structure. The chromosome of GFP is an oxidative product of the sequence -FSYGVQ-. SYG found in other proteins does not form a chromophore. No enzymes. Needs oxygen.
What is YFP?
Amino acid substitutions in GFP can tune the color of emitted light. YFP is fused to alpha-tubulin, a cytoskeleton protein.
How can fluorescence be used?
Genetic engineering techniques can used to "tag" virtually any protein, structure, or organelle in a cell.
Expression of combinations of three different fluorescent proteins produces ten different colorations of neurons. This allows many distinct cells within a brain circuit to be viewed at one time. Facilitates analysis of neuronal circuits on a large scale.
What is the Edman reagent?
The Edman Reagent (phenylisothiocyanate) reacts with the alpha amino group of an amino acid or peptide to produce a PTH derivative that can be detected by uv absorbance at 254 nm.
What is HPLC?
High Performance/Pressure Liquid Chromatography. Gradient separation of common PTH amino acids. The areas under peaks are proportional to the moles of each amino acid. Used for quantification.
What is ninhydrin used for?
Ninhydrin degrades amino acids into aldehydes, ammonia and CO2. Ninhydrin is altered to produce an intensely blue or purple pigment. Used for quantification.
How do cysteine residues react?
They react with each other to form disulfides. A cysteine involved in a disulfide bond is then named 'cystine'. More common in extracellular proteins than cystolic proteins (ex. growth factors and antibodies). In the lab this is prevented with reducing agents such as TCEP, beta-mercaptoethanol, and DTT (dithiothreitol). NEM and Iodoacetamide can be used to block the reactivity of cysteine.
What are the other two amino acids?
+ Selenocysteine (Sec, U) in a few proteins in many organisms.
+ Pyrrolysine (Pyl, O) in several archaeal species & one bacterium.
True or False? There are other types of amino acids that act as neurotransmitters and hormones, and are not found in proteins.
True- GABA, Epinephrine, Histamine, and Serotonin.
What are the PT-Modified Amino Acids?
They are incredibly rare- Hydroxylysine and hydroxyproline are found in connective-tissue proteins; carboxy-glutamate is found in blood clotting proteins, and pyroglutamate is found in bacteriorhodopsin.
What is the repeating sequence of the peptide backbone?
"N" is the amide nitrogen of the amino acid
"Cα" is the alpha-C of the amino acid
"Co" is the carbonyl carbon of the amino acid
How many human proteins are there?
A protein is defined by its sequence of amino acids. Each cell type has a distinct set of proteins. The entire complement of proteins is called the proteome.
~20 000 protein-coding genes (Human Genome Project)
~18 000 have been detected in at least one tissue
~10 000 are present in ALL tissues
~2000 are essential
The proteome is actually much larger due to post-translational modification, synthesis intermediates or precoursors, alternative splicing.
Proteins have diverse functions- how might they enhance their protein functionality?
Protein functionality is enhanced through ligand binding
Protein co-factors .... Etc.
What is the terminology for proteins and peptides?
Peptides are short polymers of amino acids
Each unit is called a residue
2 residues - dipeptide
3 residues - tripeptide
12-20 residues - oligopeptide
> 20 residues - polypeptide
> 50 residues - protein
Sequence is a unique characteristic of every protein and it is read from the amino terminus to the carboxyl terminus
One polypeptide chain - a monomeric protein
More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
What are the two types of distinct protein shapes?
Fibrous- Extended and somewhat simple structure. Structural proteins such as keratin and collagen. Rarely crystallize to form as fibers.
Globular- Compact, sphere-like molecules. Most cystolic enzymes. Structural information from X-ray crystallography and NMR.
Rules for protein solubility:
For soluble proteins, the most hydrophobic aa are on the inside and vice versa.
For insoluble proteins, the hydrophobic aa are on the outside, soluble in detergents.
Sequence of aa
Local regular structures e.g. helices, sheets
Overall 3-dimensional shape as secondary structures pack together
Subunit or chain interactions into oligomers
Proteins move in solution and undergo conformational change. These don't typically require covalent bonds to be broken
How do we know about proteins?
Purification is the separation of a protein from the thousands of other proteins in a cell
Cells may be tissue, blood, cell culture, bacteria etc.
Recombinant DNA technology allows us to take the gene for a protein of interest and express it in a non-native organism
Can purify a specific protein or an 'activity'
Typically purify mg amounts
Approaches relevant to other biomolecules
Preparative approaches result in protein to 'work with'
Analytical approaches reveal something about the protein
What is the first step in protein purification?
If the protein is cytoplasmic, you just need to lyse the cell. If membrane or organelle-bound, may purify specific 'part' of cell (centrifugation). Be careful not to denature your protein.
Buffers (20-100 mM) prevent changes in pH
Control the temperature (most stable close to 0°C or 'on ice')
Inhibit proteases especially in early steps (chemical inhibitors available)
Keep concentrated and avoid frothing (proteins can adsorp to surfaces)
Cysteine oxidation (add reducing agents)
Techniques to lyse cells
[Solutes outside] < [Solutes inside]
Cells swell and burst as water moves into the cell
Works for animal cells, but not plant cells or bacteria
Enzyme that degrades the cell wall
Detergents, organic solvents etc.
Be careful not to denature the protein
Freeze-thaw cycles, grinding with beads, high-speed blender
Homogenizer that crushes tissue using a piston
French press that squirts cells through a small hole at high pressure
Sonicator that uses ultrasonic vibration
Proteins have many acid-base groups and thus their solubility can be influenced by salt type, salt concentration, solvent polarity, and solvent pH. Identifying conditions where some proteins will precipitate while others won't allows one to purify their proteins.
Note: Salt type trends are shared between different proteins and is likely related to ion size and hydration
I = 1/2(sum of [protein]*Z²
Z is ionic charge
Protein solubility at low or moderate ionic strength increases with salt concentration. Occurs because additional counter-ions shield protein charges, thereby preventing charge-charge interactions that lead to precipitation. Also notice that proteins are LEAST soluble at their pI (+ and - charges are balanced.
Protein solubility at high ionic strength decreases with salt concentration. Occurs because salt ions compete with proteins for water molecules. Normally perform salting out at the protein's pI.
How are salting in and salting out used for purification?
Adjust salt concentration to just below the precipitation point so many unwanted proteins will precipitate; remove precipitate by centrifugation; precipitate the protein of interest. It used to be the common step to use ammonium sulfate precipitation (soluble at 3.9 M in water at 0° C)
What is chromatography?
A group of techniques for the separation of mixtures. Paper, gas, and liquid.
Use a column that has a mobile phase that is liquid
The mobile phase is percolated through a stationary phase that is a porous solid matrix
The stationary phase often has bound liquid
Solutes interact with the stationary phase according to their properties, retarding their progress through the column
Mixtures start as a narrow band and separate into bands of pure substances
What is the stationary phase in liquid chromatography?
Hydrated polymers or polysaccharides (medium pressure)
Silica-based resins (high pressure)
Physical support for chemical groups
Amylose, Cellulose, or agarose
Ion Exchange Chromatography
Separation based on charge
Proteins are electrostatically bound to the resin and then competed off by adding ions to the mobile phase
Have to consider salt concentration and pI
Low affinities move through the column faster; or come off at low salt
High affinities are eluted using a high salt elution buffer
Gradient elutions are performed by loading in low salt and using a gradient of increasing salt to elute. Separates based on charge with stronger interactions requiring more salt to compete.
In ion exchange chromatography, what do the exchangers have?
The anion exchanger has positively charged groups on resin. The cation exchanger has negatively charged groups on resin. The resin is most commonly cellulose.
What are the examples of ion exchangers you need to know?
For cation exchange (negative charge, acidic):
Work below pKa
For anion exchange
(positive charge, basic):
Work above pKa
Gel Filtration/Size Exclusion Chromatography
Separation is based on size (and shape)
Resin is made of hydrated, porous beads
The pores span a narrow range of molecular dimensions.
Large molecules are excluded and elute early. Small molecules enter the pores and elute late.
Elongated proteins elute earlier than expected as they have a high radius of hydration. Load a small volume on top of the resin and then flow buffer through the column.
What is the exclusion limit in gel filtration?
The smallest molecule unable to enter the pores
What is column volume in gel filtration?
Measured using a very small molecule such as acetone. Load less than 3 % of the column volume
What is the void volume in gel filtration?
The volume around the beads is 35% of Vt. Measured using a protein above the exclusion limit.
The elution volume is the volume eluted.
Used to determine the apparent molecular weight of the protein. Plot logMW versus Ve/Vo for standards.
Used to change the solvent of a sample. Desalting is low resolution gel filtration as the protein is much larger than the solvent components.
Used to change the solvent of a sample.
Involves placing a sample in a bag made of a semipermeable membrane with defined pore size and immersing this bag in a bath solution. Diffusible solutes in the dialysis bag, but membrane pores should be smaller than the size of the protein. Change buffer or use a large volume for efficiency.
Used to change the solvent of a sample. Ultrafiltration forces a solution by pressure (vacuum) or centrifugation through a semipermeable membranous disk with a precise MWCO.
The solution is frozen and the solvent is sublimed away under vacuum. Long-term storage.
The resin carries a ligand that binds to the target protein. The ligand is covalently attached to an inert and porous matrix. The protein binds specifically and tightly, but non-covalently.
High specificity so that greater than 95% purity in a single step. Elute with a solution that disrupts the protein-ligand interaction. Typically an excess of competing ligand. Could also be pH, ionic strength, etc.
Matrix is often agarose-based as it has a large number of hydroxyl groups that are capable of forming covalent linkages to ligand. Use linkers (epoxy-based) to prevent steric hindrance. Binding must not be too strong or catalytic.
Technology allows the addition of tags onto recombinant proteins. These tags can be cleaved off with proteases in vitro.
What are agarose beads linked to?
Agarose beads are covalently linked to a metal-chealating group such as NTA. The NTA group can coordiante Ni2+, Mn2+, Zn2+, etc. Protein is engineered to have 6-8 consecutive His residues. These his residues coordinate the cation. Elute protein by adding imidazole in solution.
Separation based on hydrophobicity. Stationary phase is silica substituted with non-polar alkyl chains (C4, C8, C18)
Longer chain is more hydrophobic. The mobile phase a gradient from polar substance (water) to nonpolar substance (acetonitrile). Used to separate lipids and peptides or proteins with exposed nonpolar parts (i.e. denatured). Common in the liquid chromatography part of mass spectrometry.
Hydrophobic interaction chromatography
Separation based on surface hydrophobicity. Hydrophobic groups (octyl or phenyl) covalently linked to polar resin (agarose). Weaker hydrophobic interactions than reverse phase chromatography (not denatured). Load in high salt to enhance hydrophobic interactions, and use a gradient of decreasing salt to elute.
High Performance Liquid Chromatography
Improved separation using high-resolution columns and shorter retention times. Narrow, long columns packed with fine and non-compressible silica beads (modified as previous). Up to 15000 psi.
How do protein purifications typically work?
A typical protein purification scheme uses a series of separation methods. Note the dramatic increase in specific activity of the enzyme through a series of five different purification procedures. The term "specific activity" refers to the activity of the enzyme per mg of protein.
The migration of ions in an electric field. Analytical. Charge promotes migration, while friction opposes migration. Frictional coefficient is dependent on size, shape, solvation, and solution viscosity. Most frequently done in a gel.
Polyacrylamide or agarose gels have controllable pore sizes so separation is based on gel filtration as well as electrophoretic mobility. Large molecules are retarded relative to smaller ones. Monitor sample migration through a gel from the cathode (-) to the anode (+) as a current is applied. You can see the sample by using a stain.
How are PAGE gels created?
Parallel lanes facilitate comparison. Buffer is usually pH~9 (net negative charge); commonly glycine. Sample mixed with dense glycerol or sucrose solution to prevent it mixing with buffer. Band narrowness influenced by length of sample column.
Use a stacking gel on top of the running/separating gel. The stacking gel has larger pores and is pH~7. The glycine is no longer charged so there is an ion deficiency; sample anions narrowly stack according to their mobilities; improves resolution.
What is the SDS is PAGE gels for?
SDS is an amphipathic detergent that denatures proteins and causes them to adopt a rod-like shape (1 SDS per 2 amino acid residues). Uniformly coats proteins with negative charge (constant charge to mass ratio)
Makes separation based almost entirely on mass (not charge). 5-10% accuracy in mass determination. Disrupts all non-covalent interactions; loading dye often includes reducing agents too.
What can SDS Page dels be used to determine?
The molecular weight of an unknown protein, by creating a standard plot of logMW vs distance migrated.
Native PAGE (No SDS)
Protein remains folded. Size, charge and shape. Can't estimate molecular weight. Lower % of acrylamide.
Isoelectric Focusing (IEF)
Separates proteins based on their pI. Very accurate (can separate <0.1 pI difference). Often performed in 6 M Urea (denatures protein without influencing charge).
1) Add ampholyte (containing both acidic and basic groups) solution to an electrophoresis gel.
2) Apply electric field to establish a pH gradient
3) Add protein sample to well
4) Apply electric field
5) Protein will migrate until they reach their pI (no charge, diffusion)
6) Stain to 'see' protein
Two-Dimensional gel electrophoresis
Macromolecules are first separated according to charge by isoelectric focusing in a tube gel. The gel containing separated molecules is then placed on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS-PAGE, where they are separated according to size. Can essentially separate all proteins from one another.
Sand in water. Macromolecules sediment when subject to large accelerations by centrifugal force. Rate of sedimentation relates to mass, frictional coefficient, and solution viscosity. Sedimentation coefficient measured in Svedbergs (S).
No density gradient. Particles pellet from solution at different speeds. Larger and denser particles sediment faster.
Used for harvesting cells, or separating organelles using a series of spins, or cell fractionation.
Can use a density gradient (concentration gradient) of homogenous and inert medium (ex. sucrose or CsCl).
Two types- Zonal + Equilibrium Density Gradient
Zonal Centrifugation (Non-equilibrium)
Sample is layered on top of a buffered solution of higher density or on a preformed density gradient. Speed of centrifugation depends on mass and shape (not density). If centrifuged long enough, the entire sample will pellet.
Equilibrium (Isopycnic) Density Gradient
Particles are mixed with a substance that will form a gradient DURING centrifugation such as with CsCl (for DNA), NaCl (for lipoprotein), sucrose (for organelles), Percol (for organelles)... etc.
These materials form a steep gradient. Particles band where their density is the same where their density is the same as the gradient media and this banding is independent of time spun. Not useful for separating protein mixtures as most proteins have similar densities.
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