Chapter 3
If you dialyze a 1 ml sample of protein in 1.0 M (NH4)2SO4 against 1 L of water, what is the final concentration of (NH4)2SO4 in your protein sample?
1 mM
What are the 3 steps for Edman Degredation?
1. Treat the peptide with phenylisothiocyanate (PITC) at ph 9.0, which reacts with the N-terminus to form a phenythiocarbonyl (PTC) peptide 2. Treat the PTC-peptide with trifluroracetic acid (TFA) to selectively cleave N-terminal peptide bond to form a thiazolinone derivative 3. Rearrange to a phenylthiohydantoin (PTH)-amino acid with aq HCl then chromatograph and identify the PTH amino acid
What does it mean to have a protein solution that is half folded?
50% of the proteins in the solution are folded and half are unfolded
What does SDS do in SDS PAGE?
Binds to many proteins at a constant weight-weight ratio. This means that there is a defined number of bound SDS molecules per amino acid residue. Thus, the charge contributed by SDS will outweigh the intrinsic charge of the protein and the charge contributed by SDS is proportional to protein molecular weight
What is the basis of separation and characteristic for affinity chromatography?
Biological or engineered interaction; Superior resolution, high capacity
What is Nickel-Affinity chromatography?
Creates a His tag because extra amino acids can be added to a protein of interest, which can then be readily trapped
What do hunderds of off-diagonal peakes in a NOESY specrum of a real protein indicate?
Stable structures
What does Rosetta do?
Stimulates the protein folding process to predict protein structure
What type of information does NOE provide?
Structural information
What type of information does gel filtration provide?
Structural information. Because GFC separates based on size, the volume of elution can indiciate the presence of quaternary structure
When is protein sequence most manageable?
With small polypeptides, so in order to sequence a large protein, it must be cleaved into smaller pieces
What does a specific and sensitive array do?
allows you track your protein and analyze purification efficiency
What can protein NMR be performed in ?
aqueous solutions under in vivo conditions with protein concentrations of 1 mM
What happens to proteins during low salt concentration?
increasing the ionic strength shield unfavorable electrostatic interactions, thereby increasing protein solubility
What does NMR provide?
inter-atomic distances, not absolute positions of atoms
Bragg's law for diffraction equation
n*lambda=2dsin(theta)
What is MS/MS carried out on and what happens in it?
nanospray/electrospray mass spectrometers as opposed to spotting on a target plate. The sample is introduced through an inlet from a capillary. Peptides are further fragmented inside the mass spectrometer and the resultant "daughter" ions oberseved
Is SDS positive or negative?
negative
What happens during anion exchange chromatography?
proteins with weak negative charge will elute before those with strong negative charge
What does O-Iodosobenzoate cleave?
the Carboxyl side of tryptophan residues
What Cyanogen bromide cleave?
the carboxyl side of methionine reisdues
What is migration in SDS page proportional to?
the log of the molecular weight of the protein
What do denaturants do?
Alter the equilibrium between the native (folded) and denatured (unfolded) states of the protein
What does Carboxypeptidase A cleave?
Amino side of C-terminal amino acid
What does 2-Nitro-5-thiocyanobenzoate cleave?
Amino side of cysteine residues
What is the most common salt used for salting out?
Ammonium sulfate
What are the 4 things that happen during Edman Degradation?
(Round 1) Labeling -> Release -> (Round 2) Labeling -> Release
What is specific activity?
A measure of enzyme purity. Increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure
What type of process if folding?
A nucleated process that proceeds through discrete intermediates
What happens to the bonds in proteins when temperature is increased?
A number of bonds are weakened
What is the principle of affinity chromatography?
A protein of interest must contain an affinity tag that binds to the column material
What type of array do we want for efficient purification?
A specific and sensitive Array
What is NMR?
Absorption of radiofrequecy that leads to excitation of nuclear spin-states in target molecules. Needs high magnetic fields so nuclear energy levels are sufficiently differentiated for a detectable NMR response
What are some ways to denature a protein?
Acid/Base treatment, heat, Detergent, Reducing agents, Urea/Guanidinium Chloride
What is the most discriminating of techniques studied thus far?
Affinity chromatography
On the graphy of entropy, vs energy/% residues in native conformation, where do the folding intermediates lie?
Along the funnel walls
Why does high temperature denature proteins?
Because it provides greater thermal energy than the strength of the weak interactions (Hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces), thereby breaking these interactions
Why are the limitations with NMR?
Because every atom in a protein can generate an NMR signal, there is an over crowded spectra (i.e. a 100 amino acid protein can yield sbout 1000 NMR lines); limited to small proteins; provides only local distance
What does Hydroxylamine cleave?
Asparagine-glycine bonds
What side chains does acidic pH cause protonation of?
Aspartate and Glutamate
How do slight pH changes affect activity and solubility of a protein?
At a pH above or below the isoelectric pH, the molecuels have a net negative or positive charge, respectively. Thus when protein molecules approach each other, they have the same overall charge and repulse each other. This prevents coalescnece and precipitaion
What does Thrombin cleave?
Carboxyl side of arginine
What does Clostripain cleave?
Carboxyl side of arginine residues
What does Staphylococcal protease cleave?
Carboxyl side of aspartate and glutamate residues (glutamate only under certain conditions)
When does Trypsin cleave?
Carboxyl side of lysine and arginine
What dose Chymotrypsin cleave?
Carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine
What are the 2 types of ion-exchange chromatography, and whatl do they use to separate?
Cation and anion exchange chromatography and they exploit variations in charge density on the surface of proteins to achieve separation
Native PAGE (non denaturing conditions, no SDS nor reductant) separate proteins by ______
Charge, Size, Shape, and Oligermic state
What is the basis of separation and characterisitc for Ion exchange chromatography?
Charge; Good resolution and high capacity
What is gel filtration?
Chromatography based on molecular size; sometimes called a sizing column. Small molecules can enter porous beads, but large molecules cannot. Large molecules therfore flow more freely over the gel bed and emerge first from the column
Describe how detergents denature proteins
Detergents have amphipathic nature, which means they have both hydrophobic and hydrophilic parts. the hydrophobic part associates with the hydrophobic side parts of the protein. the hydrophillic part associates with water. The net result is that the hydrophobic part of the protein no longer cluster together away from the water
What is Edman Degradation?
Determining one residue at a time from the N-terminus
What do urea/Guanidinium chloridy break do to proteins?
Disrupt protein structures by competing for noncovalent interactions, specifically hydrogen bonds, which are required for stability, and alter water structure and dyanmics, thereby diminishing the hydrophobic effect and facilitating exposure of hydrophobic groups
What is affinity chromatography?
Exploits specific interactions between chemical group and your protein of interest. i.e. A glucose-binding protein attaches to glucose residues on beads
What is centrifugation used for?
Follwing lysis, the homogenate can be fractioned by centrifugation. As the homogenate is spun, dense heavy material sediments at the tube bottom, forming a "pellet"
What does NOE do?
Identifies pairss of protons that are less than 5 Angstroms apart; off diagonal peaks are produced by nearby protons because they perturb each other
In phase vs out of phase
In phase adds; out of phase subtracts
What is SDS-PAGE?
Separates proteins and stains with Coomaise blue. Relies on the Electrophoretic mobility, which is the function of the ratio of its charge to its frictional coefficient aka shape, to separate proteins
SDS PAGE separates proteins by _______
Size
What happens to entropy as folding progresses? Why does this not matter?
It decreases, but is offset by increasing interactions
What happens if SDS + heat + reducing agent are added to a folded protein with intrinsic charge ?
It denatures and has a uniform negative charge
What happens if you reduce a native folded protein and add urea? and then remove the urea and oxidize?
It unfolds and is denatured; Then it either folds back to its native conformation (mainly what happens) or misfolds to a "scrambeled" version
What does Peptide Mass Fingerprinting determine about a protein?
Its mass
What do organic solvents do to proteins?
Lower the dielectric constant of the environment, which increases the strength of all electrostatic interactions, which weakens the hydrophobic interactions within the protein
What side chains does alkalkine pH cause deprotonation of?
Lysine and Arginine
How do extreme pH denature proteins?
Many, but not all proteins may be denatured. Extremes can change the charges on acidic and basic groups, and thus disrupt salt bridges
What does MALDI stand for?
Matrix Associated Laser Desorption Ionisation
What is the basis of separation and characteristic for Fel filtration
Molecular size; moderate resolution and low capacity
What happens to proteins during high salt concentrations?
Much of th water that would solvate the protein is taken up in the hydration shells of the ions. Without the water shall, the protein precipitates out of the solution
Our protein of interest forms dimers, and we analyze it by gel filtration chromatography. What do we expect compared to monomer?
Our protein will elute earlier than expected for the monomer.
What happens as heating continues after long range interactions are weakened/broken?
Some of the cooperative H-bonds that stabilize the helical structure will begin to break and then the short range interactions that initially direct protein folding are lost
What happens during MALDI Mass Spectrometer
Peptides are mixed with matrix and then applied to wells on a target plate. Peptide ions are generated by LASER firing at the target plate. The time of firing of the LASER and the arrival time of the ions at the detector are known; the relative masses can then be calculated
What does PAGE stand for in SDS-PAGE
Polyacrylamide gel electrophoresis. Acylamide mixed bisacrylamide form a cross-linked polymer network when polymerized
What diseases can misfolded proteins cause?
Prion diseases, such as Kreutzfeld-Jakob disease; Amyloid fibers cause Alzheimer's disease Can cause cancer because excess copies of chromosomes can lead to overexpression and misfolding of porteins
What is generally used to cleave proteins?
Proteases
What is the premier technique for studying protein dynamics?
Protein NMR
What is Salting Out?
Proteins are less soluble at high salt concentrations, so increasing the salt concentration can cause preciptation of the protein
What happens during cation exchange chromatography?
Proteins with low positive surface charge will emerge first from the column, highly charged proteins will elute last
What software preditcts the 3D protein structure from a sequence?
Rosetta developed by David Baker
What is the basis of separation and characteristic for Hydrophobic interactions?
Surface hydrophobicity; good resolutoin and high capacity
What do sharp transition on a Denaturant vs % protein unfolded graph suggest?
That unfolding is a cooperative process, which says that conditions that lead to the disruption of any part of a protein structure are likely to unravel the protein completely
What does Bragg's law imply?
The diffraction patters is determined by the spacing between the atoms and therefore we can calculate the structure from the diffraction pattern
What are the first bonds interactions to weaken as the temperature increases? What happens if heating is then ceased?
The long range interactions that are necessary for the presence of tertiary structure. As these bonds are weakened and broken, the protein obtains a more flexible structure and the groups are exposed to solvent. If heating ceases at this stage, the protein may be able to readily refold to the native structure
What structure contains all information for native folding?
The primary sequence
What happens during Peptide Mass Fingerprinting?
The protein is digested by distinct proteases and then separated and purified resulting in proteolytic peptides (HPLC). The mass of each individual peptide is determined. Produce theoreteical digest of all proteins in a database. Compare theoretical masses with experimentally observed masses for the digested peptides. Assign a score to matching peptides/protein
What happens as a protein is folded?
The segments of the chain folded correctly are retained, leading to increasing stabilization
What gives the NMR line?
The transition between spin states gives NMR line
What happens to proteins during SDS-PAGE?
They are denatured and are separated according to their mass
What do reducing agents do to proteins?
They break disulfide bonds which are needed for stabilizing protein structure
What must be done to proteins before they are purified?
They must be released from their cell. There are both mechanical and chemical means to do this
How is salt removed from a solution of protein?
Through Dialysis
What is the approach purification?
To always minimize the number of purification steps and maximize the product yield in the most cost effective way
What is CD-spectroscopy used for?
To assess secondary structure content
What does specific mean in terms of specific array?
To identify protein of interest within a complete mixture
What is the goal of purification?
To provide an aqueous sample containing only type of molecule- the protein of interest
