Corti Flashcards
The peptide bond is kinetically stable
From a thermodynamic pov, the hydrolysis of the peptide bond is favored, however proteins in our body keep getting synthesized but they are not immediately degraded:
hydrolysis reaction is spontaneous but SLOW
( some proteins are still degraded very rapidly thanks to enzymes)
Rotation around the peptide bond
peptide bonds cannot rotate because they have C=O double bonds
However they still allow rotation around all the remaining bonds
Different types of peptides
- Oligopeptides: when the chain contains a few residues (n lower than 25\30)
- Polypeptides: when the number of residues is higher than in oligopeptides
- Proteins: number of residues very high
Ionizable groups in the peptide
ONLY the alpha-amino group of the N-terminal and the alpha-carboxyl group of the C-terminal may be present in a protonated form
We can control the change of charge by changing the pH
We need to take into account that ionizable groups may cancel each other out
Hydrogen bonds in biological systems
Hydrogen bonds can be formed also between molecules that contain carbonyl or other hydroxyl groups
H bonds are directional: strength may vary depending on the geometry of the bond
Water
Asymmetric molecule
H-O-H bond angle is 104.5 degrees
Oxygen nucleus attracts electrons more strongly (=unequal sharing of electrons= electrostatic attraction between O of one molecule and H of another= hydrogen bond)
Polar solvent because of the possibility to form H bonds
Hydrogen bond
- Depend on the structure of water
- Longer than 1.77A
- Much weaker than the covalent bond
- Short life of 10^-9 s
Bond dissociation energy
The amount of energy necessary to break the bond
Enthalpy (H)
The measure of the total energy in the thermodynamic system
The change in enthalpy accounts for the type and number of bonds broken and formed
Endothermic and exothermic reactions
Endothermic reactions absorb energy in the form of heat (H>0)
Exothermic reactions release energy in the form of heat ( H<0)
Endergonic and exergonic reactions
Exergonic: spontaneous
Endergonic: non spontaneous
Ice melting is both: breaking of H bonds (energy) increases the movement so it increases the entropy of the system. Considering ΔG = ΔH- TΔS, entropy is higher than ΔH and because ΔS has a minus sign in front of it ΔG becomes negative (ΔG<0). Which leads to a spontaneous (exergonic) reaction.
Gibbs free energy (G)
The amount of energy capable of doing work during a reaction at constant temperature and pressure
G<0 ( system releases free energy), the reaction is exergonic
G>0 ( system gains free energy), the reaction is endergonic
Entropy S
A quantitative expression for the randomness or disorder in a system
Hydrogen bonds in biological systems
H bonds can be formed also between molecules that contain carbonyl or hydroxyl groups
H bonds are directional, so the strength of the bond may vary depending on the geometry ( in proteins the strength of the bond depends on the conformation of the protein)
Can be seen between:
- neutral groups
- peptide bonds
Hydrophilic compounds
Are polar, can dissolve in water, because they contain several hydroxyl groups ( + and - charged)
ex.
- Glucose
- Glycine
- Aspartate
- Lactate
- Glycerol
Hydrophobic compounds
Non polar, insoluble or poorly soluble
Contain long aliphatic chains or phenyl groups
Amphipathic molecules
Chemical compounds that have both polar and non polar regions ( they have hydrophilic and lipophilic properties )
Fatty acid
Example of amphipathic molecule
Carboxylic group: polar part
Alkyl chain: non polar
Micelles are formed when we increase the concentration of the alkyl chain: micellization releases water molecules which increases entropy
Ionic interactions
Very important to determine the structure of the protein
We can see repulsion and attraction between:
- positively charged groups and negatively charged groups ( ex. amino groups and carboxyl groups)
- two positively or two negatively charged groups close to each other
Hydrophobic interactions
It’s not a true interaction
Exists because water keeps two hydrophobic groups close to each other
Van der vaals interactions
Very weak bonds They are the sum of the attractive or repulsive forces between molecules include forces between: - permanent dipoles - two induced dipoles - permanent dipole and induced dipole
Self ionization of water
An ionization reaction in pure water or in an aqueous solution, in which a water molecule, H2O, deprotonates (loses the nucleus of one of its hydrogen atoms) to become a hydroxide ion, OH
Molarity of water 55.5 M
Self ionization of water
An ionization reaction in pure water or in an aqueous solution, in which a water molecule, H2O, deprotonates (loses the nucleus of one of its hydrogen atoms) to become a hydroxide ion, OH
Molarity of water 55.5 M
Acid dissociation constant (Ka)
Quantitative measure of the strength of an acid
- the lower the pH, the higher the concentration of H+ ions
- the lower the pKa, the stronger the acid
Is a better measure of the strength of an acid because pH depends on the concentration of the acid
pH
A measure of the concentration of hydrogen ions in an aqueous solution
( a weak acid could have a lower pH than a diluted strong acid)
pKa
Is the pH value at which a chemical species will accept or donate a proton
The lower the pkA, the stronger the acid and the greater the ability to donate a proton
Acid base titration
A method of quantitative analysis for determining the concentration of an acid or base by neutralizing it with a standard solution of base or acid having known concentration
Buffer
A solution usually containing a weak base and its conjugate acid ( or a weak acid and its conjugate base, or a salt, that tends to maintain a constant hydrogen ion concentration
Proteins are important physiological buffers because they contain histidine that has a pH value of 6, being really close to neutrality.
Titration curve
The plot of the pH of the solution versus the volume of the titrant added as the titration progresses
Can be used to determine the equivalence point of an acid-base reaction ( the point at which the amount of acid and of base complete neutralization)
Equivalence point
Point in titration at which the amount of titrant added is just enough to completely neutralize the analyte solution
The pH of a solution at equivalence point is dependent on the strength of the acid and of the base
- strong acid-base ph=7 at equivalence
- weak acid-strong base pH>7
- strong acid-weak base ph<7
Carbonyl functional group
C=O The carbon atom of this group has two remaining bonds that may be occupied by hydrogen/alkyl/aryl substituents. If at least one hydrogen= aldehyde If neither is hydrogen= ketone The general formula is: CnH2n-20
Hydroxyl group
-OH
One oxygen atom covalently bonded to one hydrogen atom
Alcohols ad carboxylic acids contain one or more hydroxyl groups
Carboxyl group
COOH
A carbon atom that’s double-bonded to an oxygen atom and singly bonded to a hydroxyl group
Can be present in the protonated and not protonated form
Methyl group
- CH3
An alkyl derived from methane
Ethyl group
C2H6 o CH2-CH3
An alkyl substituent derived from ethane
Amino group
- NH2
-NH3+
A nitrogen atom attached by single bonds to hydrogens.
Organic compound that contains an amino group: amine
Polar ( N is more electronegative
Phenyl group
The functional group of C6H5
derived by the removal of an hydrogen from the benzene
Amide
An organic compound that contains a functional group consisting of an acyl group (derived by the removal of one or more hydroxyl groups from an oxoacid) linked to a nitrogen atom.
Simplest amides are derivatives of ammonia in which one hydrogen atom has been replaced by a acyl group
R-C-NH2=O
Urea
(NH2)2CO
A nitrogenous compound containing a carbonyl group attached to two amine group with osmotic duretic activity
Soluble in:
- water
- glycerol
- ethanol
Neither acidic nor basic when dissolved in water
Sulfhydryl group
R-SH
A family of organic compounds that contains an R group bound to a sulfur atom and a hydrogen atom.
Disulfide bonds
R2S2
R-S-S-R
Function to stabilize the tertiary and quaternary structures of proteins
Phosphoryl group
-PO3 charge -2
Takes part in phosphoryl transfer: the group is transferred from a phosphate ester to a nucleophile
Phosphate group
PO4 ( one double bond and three single bonds)
Polar covalent bond ( O more electronegative)
Ester
RCOOR′
R may be an hydrogen, an alkyl or an aryl
R’ may be an alkyl or an aryl ( if it where an H, it would be a carboxylic acid)
Any class of organic compounds that react with water to produce alcohols and organic or inorganic acids
Esters derived from carboxylic acid are the most commons
Proteins
Polymers of amino acids
Each amino acid is connected to its neighbour through a specific type of covalent bond
Amino acids
Each amino acid contains
- Central C ( alpha-Carbon)
- Carboxyl group
- Amino group
- Variable R group ( specifies which class of amino acids it belongs)
alpha-Carbon of amino acids
Is a chirality center (it is asymmetric and not superimposable on its mirror image)
Because of the tetrahedral arrangement of the bonding orbitals around the alpha carbon atom, the four different groups can occupy two unique spatial arrangements.
Stereoisomers of aminoacids
- D-stereoisomer: the carboxyl group on the top, side chain bottom, alpha amino group on the right and the hydrogen on the left
- L-stereoisomer: same but amino group and hydrogen are inverted
Non-polar aliphatic R groups
- Glycine
- Alanine
- Valine
- Leucine
- Isoleucine
- Methionine
- Proline
The R groups are non polar and hydrophobic
Glycine: smallest, simplest structure, because the side chain is just an atom of H ( no chirality because it has 2 H atoms attached to the alpha carbon)
Leucine, Valine, Isoleucine: branched amino acids
Methionine: contains S on its side chain
Proline: non polar because the side chain forms a ring that does not contain a NH3+ but instead has a NH2+.
Unique aminoacid that forms a cycle ( not aromatic because no double bonds)
Aromatic R groups
- Phenylalanine
- Tyrosine
- Tryptophan
All three amino acids contains rings with double bonds
Phenylalanine: phenyl group
Tyrosine: phenyl group+ hydroxyl group ( can form hydrogen bonds, can be phosphorylated to form esters)
Tryptophan: Indol group, it’s the biggest amino acids
Polar uncharged groups
- Serine
- Threonine
- Cysteine
- Asparagine
- Glutamine
The R groups are more soluble in water
Asparagine and glutamine are the amides of two other amino acids: aspartate and glutamate, they are also both hydrophilic and unstable
Positively charged R groups
- Lysine
- Histidine
- Arginine
Arginine: contains a positively charged guanidinic group
Histidine: contains an imidazole group ( an heterocyclic compound with two N atoms) present in protonated or deprotonated depending on the pH
They have pKa values that ranges from 6-12.14 ( basic)
Negatively charged R groups
- Aspartate
- Glutamate
These can also be called as aspartic acid an glutamic acid
They have lower pKa values ( acidic)
Absorbance spectrum
Property of aromatic amino acids
-Tryptophan
- Tyrosine
- Phenylalanine
They have the capability to absorb light in the UV spectrum
This property is important because it allows us to measure and quantify proteins in solutions
Spectrophotometer
It’s a standard and inexpensive technique to measure light absorption or the amount of chemicals in a solution
Mechanism: source of light connected to a device that allows you to select a particular length of the light, the light is then reflected and goes through the cuvette that contains the sample you want to test
Lambert-beer law
A wildly used method for measuring the protein concentration, especially when proteins are pure
Absorbance (A)= epsilon/C l
epsilon: molar absorption coefficient
I: length of the cuvette
C: concentration
Nonstandard amino acids
We don’t have the corresponding codon in our DNA, but they are present in proteins ( usually because of postranslational modifications) Can be found in: - Collagen - Thrombin - Myosin - Elastin Two most important non standard amino acids: - Ornithine - Citrulline
Ornithine
Made of 5 carbon atoms with an amino group on the C5
We can find it in a neutral or positive form because the amino group can be protonated
Citrulline
6 carbons + ureidic group ( urea attached to a carbon)
IsoAspartate
Example of how non standard amino acids are formed
Spontaneous reaction
Starting from asparagine, there is a deamidation reaction, which can form aspartate or isoaspartate ( called like this because the side chain is attached to the carbon beta)
This reaction occurs in fibroactin ( protein in the extracellular matrix of our tissues)
Cysteine and Cystine
In proteins it’s possible to find two cysteine close to each other, which can oxidate and form a disulfide brige, we then obtain a Cystine
Amphoteric
Amino acids are amphoteric: they can act either as a base or an acid
Nonionic- Zwitterionic form
Different structures of amino acids
Nonionic: the carboxylic group is represented in a protonated form and the amino group in a non protonated form
Zwitterionic: Carboxylic group is deprotonated, alpha amino group protonated
Isoelectric point
Point at which the charge of the amino acid is 0 and if you put the amino acid at this pH in an electric field there is no migration because there is no charge
It’s the average of the two pKa values in the buffering
The pKa may be affected by the environment Ex. pKa value of the carboxyl group of acetic acid is lower than the one of glycine: the carboxyl group of glycine is more acidic because the carboxyl group of glycine is close to the alpha amino group (which is positively charged), creating a repulsion force ( between NH2 and the departing proton) lowering the pKa.
It is possible to change the charge of an amino acid by changing the pH
pH>pI: charge is negative
pH
Peptide bond
Sort of an amide bond formed by the condensation of the alpha amino group and the alpha carboxyl group
Thermodynamically unfavourable, in our cells it occurs because the endergonic (non- spontaneous) reaction is coupled with an exergonic (spontaneous) reaction
HOW?
Our cells use an “activated” form of amino acids:
aminoacyl-tRNA.
Amino acids are first coupled with tRNA (transfer RNA) in a reaction that needs a large amount of energy, provided by ATP hydrolysis, and which gives as a product aminoacyl-tRNA.
Afterwards, the formation of peptide bonds between aminoacyl-tRNA molecules can occur, as it is a thermodynamically spontaneous reaction
Aminoacid+ tRNA+ ATP=> aminoacyl-tRNA + AMP + 2Pi
Metabolism
Includes all the reactions that take place in our body
can be divided into:
- catabolism: includes all the degradation reaction that starts from store nutrients ( releases energy as ATP)
- anabolism: includes all the reactions that synthesize complex biomolecules (uses ATP)
ATP connects catabolic and anabolic reactions
Biological functions of peptides
- hormones: made of peptides ( oxytocin, bradykinin…) or polypeptides ( insulin, glucagon, vasostatin)
- poisons: made of cyclic peptides
- antibiotics: composed of glycopeptides
- aspartame: artificial sweetened, dipeptide made of aspartyl, phenylalanine, methyl ester
Levels of protein structure
Primary structure: represents the sequence of amino acid residue
Secondary structure: alpha-helix or beta-sheet
Tertiary structure: tridimensional structure of a polypeptide
Quaternary structure: presence of different assembled subunits
Multimeric proteins
They are made of a few polypeptide subunits
cam be differentiated as:
- dimer
-trimer
- tetramer
or as:
- homomeric ( made of 2 identical subunits)
-heteromeric ( made of 2 different subunits)
ex. Protomer: a particular oligomeric protein formed by one alpha-amino acid and one beta
Amino acid composition
A table in which it is indicated the number of every amino acid residue present in a certain protein molecule
Different from the sequence
The 20 amino acids are typically present in different concentrations in different proteins
Prosthetic groups
Chemical groups acquired by proteins in post-translational modifications
- lipids ( lipoproteins)
- carbohydrates ( glycoproteins)
- phosphate groups (phosphoproteins)
- heme (hemoproteins)
- flavin nucleotides ( flavoproteins)
- metals ( metalloproteins)
Protein purification
- Source identification: the protein of interest must be isolated. The source may be natural ( biological fluids, organs, tissues), or recombinant ( derived from genetically engineered cells)
- Preparation of crude extract: you have to disaggregate and disintegrate the source by using ultrasound or homogenizer. After disintegration we can proceed with extraction by performing protein precipitation with salts, solvents and polymers
- Fractionation: Used to purify more the protein extracted. Consists in fractionating all the proteins in the sample and try to isolate each different polypeptide ( with chromatography, electrophoresis, filtration, ultrafiltration, dialysis)
Characterization of proteins
After isolating a protein, you need to carry out an “ identity test”.
First you need to characterize the purity of the protein ( using HPLC or gel electrophoresis) and also the biological activity of a protein ( might have been denatured during the process).
The parameters we may check to verify a proteins identity:
- molecular weight ( SDS-PAGE, gel filtration or mass spectrometry)
- isoelectric point (isoelectrofocusing)
- concentration
-amino acid composition and sequence
- protein structure
Column chromatography
Simplest and cheapest chromatographic system
We have:
-a tube with a porous support containing a solid porous matrix (stationary phase) in the middle
- A reservoir that contains the eluent ( mobile phase), which is usually a buffer
- The eluent is liquid, while the porous matrix is solid
- Using a pipet we start loading the tube on top of the column with the sample, then we start eluting the sample
- Depending on the type of interactions, the proteins are eluted at different rates
FPLC ( Fast protein liquid chromatography)
HPLC ( High pressure liquid chromatography)
Both examples of chromatography, but in these devices there is also pressured caused by pumps ( the column is formed by very small beads, the smaller the better resolution)
We have:
- Pump connected to the reservoir
- Detector connected to the column, which allows to report whatever is coming out from the column
- Fraction collector linked to the detector, in order to separate the components in exit
Size excluclusion chromatography
The stationary solid phase is made by beads with a lot of small canaliculi.
Small molecules can enter the canaliculi ( they will be slowed down), while big molecules will pass more freely.
This procedure allows to separate molecules according to their hydrodynamic size or volume
Ion exchange chromatography
A lab technique that utilizes the ion interaction between proteins and charged polymer beads to separate and select the polar components of a mixture
We have:
- column with + or - charged polymer beads
So that they can form ion interactions with the proteins that present an opposite charge, while the un bonded protein will elute out
Solid matrixes that can be used:
- CM ( carboxyl-methyl group): cellulose or agarose functionalised with a carboxyl group ( negative). CATION EXCHANGE
- DEAE (diethylaminoethyl group): cellulose or agarose functionalised with a positive group. ANION EXCHANGE
How to detach proteins from the Ion Exchange chromatography
- Changing the pH to stop the ionic interactions
- By increasing the concentration of the salt (ex. sodium chloride),. Since salts contain ions, they can actively compete with the proteins for the binding to the resin if at a sufficiently high concentration
How to detach proteins from the Ion Exchange chromatography
- Changing the pH to stop the ionic interactions
- By increasing the concentration of the salt (ex. sodium chloride),. Since salts contain ions, they can actively compete with the proteins for the binding to the resin if at a sufficiently high concentration
Affinity chromatography
Much more powerful than ion-exchange chromatography or size-exclusion chromatography since it is not based on molecular charges or shapes but on bio-selective recognition
The solid beads are functionalised with a bio ligand ( a molecule that can recognise the protein of interest, it could be enzyme inhibitors, hormones, hormone receptors, antibodies and antigens)
The ligand is covalently linked to the beads and it will stop only the proteins that it bio-selectively recognise from being washed away.
Detachment methods for proteins in affinity chromatography
- pH change: By adjusting the pH, we can change the charge of the molecule or the charge of a certain residue, resulting in a conformational change ( we then lose the bio-selective recognition between the molecules)
- salt concentration changes: The addition of ions will lead to other conformational change which will influence the complementarity between the protein and the beads.
- addition of denaturing agents (if the denaturation is not reversible it will cause the protein to be unusable)
- Addition of free ligands: by adding an excess of free ligands in the solution the protein will be bound to the ligand in the solution and successively elute from the column. However, this does not isolate the protein which is now just bound to a different substance.
Hydrophobic interaction chromatography
This technique works thanks to the interactions of the hydrophobic regions of a protein
The beads in the column have been functionalised with hydrophobic groups, to enable hydrophobic interactions
These interactions are favoured in presence of high salt concentration ( lowering the salt c elutes the protein of interest)
Dialysis method
Used to remove salts from proteins
A protein sample which contains a salt is inserted in a dialysis bag. This dialysis bag is made by a porous membrane that allows the passage of only small molecules (proteins>salt molecules). The bag is then inserted in a low concentration of salt solution so that the salt will establish a new equilibrium moving outside. This process is repeated multiple times so to eliminate all the salt in the dialysis bag.
( gel filtration and size exclusion chromatography can also be used)
Polyacrymalide gel electrophoresis
Analytical method based on the migration of charged proteins in an electric field.
You are using 2 glasses close to each other, you leave to polymerize in between the 2 glasses a gel.
Different samples are loaded in wells or depressions at the top of the SDS polyacrylamide gel, so when you apply an electrical field, the proteins start to migrate depending on their charge but also depending on their size.
The force moving the macromolecule is called the electrical potential, E.
Proteins are then separated depending on the charge, molecular size and molecular shape, and you can see whether your protein is pure or not.
It is possible to visualize the bands using stains, one of the most common stains used is the blue Coomassie stain which binds to the proteins but not to the gel itself.
Proteins have migrated as a function of charge and as a function of size. Thus, in order to have an analytical method that allows us to separate electrophoretically only as a function of size, we use Sodium Dodecyl Sulfate which is abbreviated to SDS.
SDS
Sodium Dodecyl Sulfate is an ionic detergent.
When proteins are mixed with SDS and boiled, they become negatively charged irrespectively of their original charge.
The advantage of this system is that all the proteins are negatively charged. Thus, if you run a gel and boil the samples in the presence of SDS, you can have the protein which migrates only as a function of size, because all proteins will have the same
charge
Determination of the amino acidic sequence
Nowadays we are using two methods for determining the sequence.
1. Edman Degradation Method
2. Mass spectrometry
3 steps:
- after you have purified the protein and determined the amino acidic composition, you must recognize the first amino acid residue. To do so, it is sufficient to label the protein with the reagent 2-4-Dinitro-fluoro-benzene. The fluorine atom is very reactive with the alpha amino group so it reacts rapidly with the reagent, labelling the protein (the first amino acid residue). With this step, it is also possible to know how many chains are present.
If the chains are two or three, you have to separate the chains and sequence each one - We use a different reagent called phenyl isothiocyanate (still binds to the alpha amino group of the first residue). The advantage of this new reagent is that if you analyze the protein under mild condition, only the first amino acid will be detached. Therefore, you have a chain, you label the first one and you detach it from the chain.
Now you have a shortened chain and you can repeat the same reaction until the end of the sequence
How to generate fragments of proteins
You can use enzymes: ex. Trypsin, type of protease, enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products, is an important example. We have trypsin in our gut, and it helps us to hydrolyze/digest proteins and generate fragments. Another example is Chymotrypsin. This one is also in the gut. Also, Pepsin is very important. We have this last one in the stomach.
Electrospray ionization mass spectrometry
In an electrospray mass spectrometer, there is a glass capillary in which you put your protein which is vaporized to generate ions in the presence of protons. The presence of proton makes each molecule of the protein to be covered by protons, so it becomes overall positively charged in this manner. Depending on the number of protons and the mass, you have a certain proton to mass ratio that can be analyzed by a detector. After this, you obtain a very complicated spectrum which can be deconvoluted by a computer and the computer can calculate the molecular mass in a very precise and accurate manner.
Merrified method
Synthesizing a protein with a short amino acid sequence,
occurs on solid phase (when one solid reagent is linked to something solid while the other reagent is in solution9
Bits are used to react with the carboxyl group of the first amino acid, this one must have the alpha amino group blocked, in this case with the compound Fmoc (fluorenyl-metoxy-carbonyl).
Fmoc= is a reagent that can block the alpha amino group to prevent unwanted reaction, like polymerization.
So, in amino acids only the carboxyl group is free to react with the resin.
This not actually the first step but the last, because the chemical synthesis occurs from C- term to N-term. So, the C-term is the first to be synthesized but the last in the sequence.
In a sequence the next amino acid must be add (condensation reaction) in an inactivated form, in other words it must be deprotected: Fmoc, that
protects the amino group, has to be removed and this
can be easily done by flushing with a solution containing
a mild organic base. You have to imagine that you have many bits and you flush all of them with mild base and the protecting group is removed. So at this point you have only the last amino acid chemically attached to the bits.
The third step is to activate the carboxyl group in order to react with the alpha amino group otherwise the reaction will not occur because it is not spontaneous. In this case the activating group is the DCC deyelohexylcarbodiiamide, this reagent can activate the carboxyl group. But to prevent polymerization of this reagent you have to block again the end-terminal group.
All the reagents (amino acids with activated carboxyl group or deprotected group) are commercially available.
All these steps can be automatized with peptide synthesizers. You only have to set the sequence that you want and fill bottles with all these reagents and then the machine automatically synthetizes the peptide.
But at the end when you will obtain finally the chain that you desire, you have to detach the peptide form, the solid phase, the bits and it can be easily done by flushing the resin with the hydrofluoric HF and this way, the peptide goes into solution.
Isoelectrofocusing (IEF)
Isoelectrofocusing is an electrophoretic technique which separates proteins as a function of their isoelectric points.
Isoelectric points are like the markers of specific proteins. For isoelectrofocusing, you have to prepare a polyacrylamide gel (not as a slabbed gel) as a column. You use a glass tube and you make the gel polymerize inside the tube. After polymerization, you can add a mixture of ampholytes (mixture of several amphoteric substances that behaves as an acid or as a base).
Suppose a mixture is made by many different ampholytes, each one with different pKa value.
The protein may start to migrate and it will move along the gel until it reaches the point in the gel corresponding to the pH of its isoelectric point. Then, the charge becomes zero and the protein stop to migrate.
Two dimensional gel electrophoresis
This technique combines the SDS page with Isoelectrofocusing and it is a very powerful technique. After running an IEF, you can get the gel and then use it for an SDS page. Then, if you apply an electric field, it is possible to elute the proteins from the first gel and leave the protein entering the second gel which is a gel for the SDS page analysis. So, in this case, the protein re-migrates as a function of their molecular weight. .
Mass spectroscopy
Mass spectrometry is a very powerful technique that provides you information regarding the molecular weight in a very precise and accurate manner. A mass spectrometer is not inexpensive, it costs a lot of money. A mass spectrometer, first provides the molecular weight precisely, second it can help in determining the sequence of the protein. In a few hours, with this technique, you have identified the protein, you can sequence it and you know what your protein is, and it is a technique really widely used in our laboratories. And if afterwards you want to analyze the protein more in depth, you can use protein purification to separate the protein from the rest and study this protein. This is what the laboratory usually does. With mass spectrometry, however, there is the problem of determination of the amino acid sequence. The best way to check for the protein identity is to have the
amino acid sequence at hand.
Collagen
Molecule made by three chains but NOT by alpha helixes, the formation starts with a coil-coil three chain that forms protocollagen that consequently forms Collagen.
the peculiarity of this molecule is that it contains a lot of glycine and alanine ( two of the smallest amino acids).
There are 30 variants of collagen
Globular proteins
Include Enzymes, hormones, antibodies, receptors, transport and regulatory proteins
Hemoglobin
A protein used for the transport of oxygen from lungs to the tissues
It is a tetrameter (4 subunits), made of two identical alpha-subunits and two identical beta-subunits ( a dimer of alpha and beta protomers)
We have 4 porphyrin rings and 4 heme groups, so it is important to say that hemoglobin is a tetramer with 4 iron atoms
Structure of the heme group
6 coordination bonds, 1 bond with a nitrogen atom of his, 4 bonds with nitrogen atoms of the porphyrin ring and 1 bond free to bind oxygen
Transport of Oxygen from lung to tissues and oxygen binding proteins. What is the problem about them? Oxygen is poorly soluble in water, since it is a symmetrical molecule, and you don’t have partial positive or partial negative charges on it: it’s a non-polar molecule. Also, none of the amino acids can bind oxygen. Iron and copper can bind oxygen; however, they cannot be used as they can promote the formation of reactive oxygen species, that are toxic. What is the solution that years and years of evolution brought us? The incorporation of iron into the HEME group of a protein (myoglobin, hemoglobin). In that case, iron is less reactive as it remains in its reduced ferrous Fe++ state. If iron remains in this state in can’t be oxidize and can’t generate toxic radicals.
The key point is that hemoglobin can exist in two different conformational states depending on whether the oxygen is bound or not: the T state (tense) and the R state (relaxed).
X-Ray diffraction method
Used to determine the 3D structure of a protein
First step: Preparing crystals, taking a highly concentrated protein in presence of salt and making the solution evaporate, obtaining super saturated solution= crystal.
At this point, you use the x ray to obtain a refraction that generates an electro density map so that it is possible to identify full density regions
X-Ray diffraction method
Used to determine the 3D structure of a protein
First step: Preparing crystals, taking a highly concentrated protein in presence of salt and making the solution evaporate, obtaining super saturated solution= crystal.
At this point, you use the x ray to obtain a refraction that generates an electro density map so that it is possible to identify full density regions
Proteins can be divided into classes
- all alpha
- all beta
- alpha/beta
- alpha+beta
Symmetries of proteins with quaternary structure
- Cyclic symmetry: Rotation around a central point
- Dihedral symmetry
- Icosahedral symmetry: typical in viruses
- Helical symmetry: the subunits can be superimposed by helical rotation
Protein denaturation
Denaturation: the loss of the 3d structure
We can denature proteins by using denaturing agents such as:
- heat: affects hydrogen bonds
- pH: affects ionic interactions
- solvents: affects hydrophobic interaction
-Urea: affects hydrophobic interaction
- Guanidinium chloride: affects hydrophobic interaction
- Detergents: affects hydrophobic interaction
Urea and Guanidium chloride: Chaotropic agents ( perturb the structure of water)
Renaturation
The regain of the native structure and the biological activity of a protein
Spontaneous process
Chaperones
Proteins that bind partially to hydrophobic residues of unfolded/ unproperly folded or denatured proteins, favoring the correct folding
Heat-Shock proteins
Protein Disulfide Isomerase (PDI)
Enzyme that catalyzes the cleavage of wrong SS bridges and promotes SS shuffling until the correct bonds are formed
Ligand
Molecule that can bind a protein in a reversible manner
Binding site
Site recognized by the ligand
Induced fit
Conformational change in the binding site induced by the ligand
Identification of Enzymes
By a 4-digit code number , the Enzyme Commission number (EC). The first digit denotes the class, the second and the third one denote subclass and sub-subclass ( chemical group involved, type of bond...) the fourth digit indicates the progressive number in the sub-subclass.
Types of enzymes
1- Oxidoreductases (transfer of electrons)
2- Transferases (Group transfer reactions)
3- Hydrolases (Hydrolysis reactions)
4- Lyases (Addition of groups to double bonds)
5- Isomerases ( transfer of groups within molecules to yield isomeric forms)
6- Ligases ( formation of C-C, C-S, C-O, C-N bonds)
Enzymes
Most enzymes are proteins ( some can be ribosomes)
They have the capacity of catalysing reactions.
Some enzymes need cofactors, that are inorganic ions; other may need coenzyme, which are complex organic molecules usually made from vitamins
The enzyme splits the reactions in 3
1. Formation of the enzyme-substrate complex
2. Formation of the enzyme-product complex
3. Detachment
The enzyme is complementary to the transition state of the substrate, and not the substrate per se ( bent stick)
Some enzymes need: cofactors (inorganic ions) and coenzymes (complex organic molecules)
Factors that increase the activation energy barrier
- Entropy: Two substances that need to bind or break have to collide in an oriented manner, enzymes can reduce the entropy and make the substance bind by orienting them
- Substrate solvatation: Enzymes promote substrate desolvation
Km in enzymes
Michaelis constant
The concentration that gives half of maximum velocity
Kcat
Turnover number
Gives you an idea on the efficiency of the enzyme
The number of S molecules converted to product in 1 second by 1 enzyme molecule, when the enzyme is saturated with S.
Specificity constant
the ratio between the turnover number and the Michaelis-Menten constant K(cat)/ K(m)
Specific activity
The higher the specific activity, the purer is the enzyme, and it is characterised by U/mg (units per milligrams of protein)
the higher the specific activity, the purer is the enzyme, and it is characterised by U/mg (units per milligrams of protein).
Enzyme inhibitors
- Reversible inhibitors: competitive and uncompetitive inhibitors
- Irreversible inhibitors
Competitive inhibitors
They bind to the active site of the enzyme, competing with the substrate
we have a new equilibrium made of the EI complex ( an excess of substrate can displace the inhibitor)
we have a new dissociation constant, Ki=inibitory constant
The competitive inhibitor can affect Km, because the inhibitor can subtract a certain amount of enzyme from the equation to form the product and the equilibrium moves to the left
Km increased, Vmax not affected
Uncompetitive inhibitor
The inhibitor binds to the ES complex
this kind of inhibition, decreases both K(m) and V(max)
Mixed-type inhibitors
Bind to both E and ES, but
their affinities for these two forms of the enzyme are different (Ki ≠ Ki’).
Thus, mixed-type inhibitors interfere with substrate binding (increase Km) and hamper catalysis in the ES complex (decrease Vmax).
Non-competitive inhibitors
(a particular, rare case of
mixed inhibitors) have identical affinities for
E and ES (Ki = Ki’). Non-competitive inhibition does not change Km (i.e., it does not affect substrate binding)
but it decreases Vmax (i.e., inhibitor binding hampers catalysis)
Suicide inhibitors
It’s a group of irreversible inhibitors, they are molecules that can bind to the active site of E, modified by the enzyme, to produce molecules that reacts irreversibly with the enzyme. It is similar to the substrate (as the enzyme can convert it into product), but then the product reacts with the enzyme and blocks it (that’s why they are called suicide inhibitors).
Enzyme activity is modified by the pH
Each enzyme has its optimal pH
If we increase the ph, the activity decreases until it is completely lost
Serine proteases
are enzymes that cleave peptide bonds in proteins
- trypsin
- chymotrypsin
- plasmin
- urokinase
- elastase
Chymotrypsin
catalytic triad: Serine, Histidine, Aspartate
They are distanced in the sequence but brought together with the enzyme folding process
They cleave peptide chain at the carboxyl side of aromatic residues
Cysteine proteases
Catalytic site: Cysteine and Histidine
ex. Papain, can be used to cleave antibodies
Regulatory enzymes
It’s an enzyme of a biochemical pathway which regulates the pathway’s activity
Their activity may depend on the presence of creatine molecules ( modulators)
1. Allosteric enzymes
2. Covalently modified enzymes
3. Enzymes affected by regulatory proteins
4. Enzymes activated by proteolytic cleavage , they exist in inactive forms ( zymogens or proenzymes)
Allosteric modulator
A group of substances that bind to a receptor to change that receptor’s response to stimulus
Heterotropic
modulator is different from the substrate.
Homotropic
the enzyme is regulated by the substrate itself
Positive modulator
increase the response of the receptor by increasing the probability that an agonist will bind to a receptor
Negative modulator
reduces the affinity and/or the efficacy of an agonist for a receptor
3 amino acids that can be phosphorylated
serine, threonine and tyrosine
trypsinogen and chymotrypsinogen.
zymogens produced in the pancreas
Trypsinogen activation
Trypsinogen is activated by enterokinase ( proteolytic enzyme, produced by intestinal cells)
Trypsinogen becomes trypsin ( which activates other enzymes)
Cymotrypsinogen
Inactive because this is a pro-enzyme.
It is produced by pancreas as a single chain molecule inactive. But with trypsin it becomes chymotrypsin which is active
Fibrinogen
large molecule composed of different subunits called alpha, beta and gamma.
Thrombin converts fibrinogen to fibrin to generate ends which can interact with gamma subunits.
When these peptides are removed, this generates Gly-His-Arg sequences which can recognise the gamma subunits in this manner.
Immune response
the response of an organism to invade in pathogens and foreign compounds
- Humoral response: mediated by secreted antibodies
- Cellular response: mediated by cells such as lymphocytes, macrophages…
Immunogens
compounds that are capable of inducing an immune response
Antigen
any compound recognized by a product of the immune response
Haptens
compounds usually with a low molecular weight that can induce an immune system only when they are coupled to a carrier protein
Antigenic determinant or epitope
antibody binding site of an antigen Epitopes in proteins tend to correspond to: Accessible regions Hydrophilic regions Flexible regions Non conserved regions
Paratope
antigen binding site of an antibody
Immunoglobulins
y shaped molecule consisting of two heavy chains linked together by disulfide bridges and two light chains again linked by disulfide bridges
Both heavy and light chains are organized in different foldign domains ( VH, CH, VL…)
V: variable
C: constant
The paratope is located at the VH and VL domains
Antibodies are flexible molecules
Affinity
The strength of the interaction between ligand and protein
Avidity
is something which is related to how many kind of interaction can occur.
Isotype
antigenic determinats of constant regions
Idiotype
antigenic determinats of variable regions
Monoclonal antibodies
An antibody produced by a single clone of cells or cell line and consisting of identical antibody molecules.
Each monoclonal antibody is made so that it binds to only one antigen
Prepare monoclonal antibodies
1- You have to inject the antigen into ( for example) a mouse, for one/two months
2- Then you can test the blood of the animal to see if it has generated antibodies
3- If you see a good response, then you can isolate the spleen, since in spleen cells you have lymphocytes, which are the cells that can produce the antibodies.
hey fused the spleen cells with cancer cells, in particular Myeloma cells (not to be confused with Melanoma cells ).
Myeloma is a tumor of plasma cells, this means that they decided to use malignant cells, capable of producing antibodies, and fuse them with the linfocites obtained from the animal that was immunized with the antigen. To achieve this, it’s sufficient to put into a test tube both cells, add polyethylene glycol and then mix. You will then obtain some hybrid cells, made by linfocites and by myeloma cells, called Hybridomas.
how we can select the suitable hybridoma, meaning the one that produces the antibodies that we want?
This can be done by putting the mixture in the presence of peculiar medium, called HAT medium (hypoxanthine,aminopterine,thymidine).
Non hybridized spleen cells can be easily eliminated from the mixture since they’ll spontaneously die after their 10 days life span, while getting rid of the myeloma cells it’s more hard, since they are immortal.
To discard these myeloma cells, scientists genetically manipulate them before the experiment in order to destroy their HGPRT gene, which is important for the synthesis of DNA.
Cells that do not have this gene cannot produce DNA molecules in the presence of aminopterine, meaning that this drug can kill myeloma cells that have no HGPRT.
Therefore, If you wait three weeks after culturing all these cells in this mixture, only hybridomas will survive.
The multiplication of the hybridoma that produces the antibody that we want can be obtained by cloning.
PURIFICATION OF MONOCLONAL ANTIBODIES
You start by making the antibodies precipitate with ammonium sulfate, then, to get rid of other impurities, you may use affinity chromatography (you use the antigen as ligand, if you know it). Next, you add an ion exchange chromatographic column followed by gel filtration.
(For each antibody you have to develop a specific purification method).
Scientist are trying to develop a method to produce antibodies which can avoid the use of animals. For example by the screening of phage display libraries.
A phage is a viral particle which can infect bacteria, so it’s a virus of a bacteria.
The advantage is that you can propagate this viral particle just by infecting bacterial cells, and then you cultivate the bacterial cells to obtain a larger amount of phages. You now have millions and millions of phages which each of them as a different DNA molecule containing the sequence of one antibody, so you have a library, a huge repertoire, of different phages which express on the surface different antibody molecules.
How can we fish out from this library the antibody capable of recognizing the antigen of interest? The selection can be done, for example, by preparing an antigen column with the antigen immobilized through which you flow the phage library.
The phage with the antibody of interest will stick to the column.
We can then elute the phage just by changing the PH of the buffer and we then amplify them. The advantage is that:
1. you don’t use animals
2. you can clone in this library the entire human repertoire of antibodies, also meaning that you
don’t have to humanize them.
3. This is also a technique that may bypass the problem of immunogenicity.
But still, the 90% of antibodies are being produced using the first method.
SANDWICH ELISA
Used to detect antibodies in the blood or in the biological fluid. It’s called sandwich because it involves two antibodies that form a complex with the antigen, like in a sandwich.
The two antibodies have to be different and they have to recognize two distinct epitopes.
If the antigen is present, it will bind to the first antibody, then the second antibody labelled with an enzyme is added.
The advantage is that if a molecular sandwich has been formed it can be easily detected because when you add the substrate for the enzyme the solution will become colored.
If the antigen is absent, the second antibody would be washed away because it could’t form a molecular sandwich.
color= antigen is present
no color = antigen is absent.
WESTERN BLOTTING WOTH RADIO LABELED ANTIBODIES
First you have to separate a complex mixture containing the antigen that you want to test, then you transfer the proteins that have been separated onto a polymer sheet to immobilize them. This polymer sheet is added into a bag containing the antibody specific for the antigen of interest. The antibody will bind with the antigen of interest.
You then wash everything and you expose the filter to a photogenic film to finally see a band where the antibody was bound.
IMMUNOHISTOCHEMICAL ANALYSIS OF TISSUE SECTIONS
Used to detect cancer cells in a tissue.
You can take sections of the bioptic material and stain them with antibodies that will bind to cancer cells. You then reveal the antibodies that binded with secondary antibodies labeled again with an enzyme.
To expose the results, you just need to a
MMUNOFLUORESCENSE MICROSCOPY
In this case antibodies are labelled with fluorescence compounds.
You can use different colors so you can stain different antibodies clearly.
MMUNOFLUORESCENSE MICROSCOPY
In this case antibodies are labelled with fluorescence compounds.
You can use different colors so you can stain different antibodies clearly.
Heme group
the structure of the heme group which consists of a protoporphyrin ring, where you have four pyrrole rings bind together by four methene bridges, and we have here in the centre an iron atom.
Iron can form 6 coordination bonds and 4 of these bonds are formed with the nitrogen atoms of the heme group, while the other two remain free.
Kd
the particular concentration of the ligand that gives the 50% of binding sites occupied.
Hemoglobin and CO2
CO2 binds with more affinity to the heme group then the O2
Evolution has solved this problem by inserting the heme group into the pocket of a protein like myoglobin; in this case the general architecture of the protein generates a sort of small cavity with an optimal geometry for oxygen, but not for carbon monoxide.
Myoglobin
Reserve of oxygen in the tissue
Glycogen phosphorylase
Regulation by phosphorylation
an enzyme which involved in the glycogen breakdown and this breakdown of glycogen which is a polysaccharide present in the liver occurs by phosphorlitic reaction. There is an enzyme capable to remove one by one all the monomers, glycogen phosphatase.
This enzyme may exist in two different forms. One less active this is called phosphorylase B. Other one is more active and that is called phosphorylase a. The difference between two is just
phosphorylation series
Protein kinases
enzymes that catalyze the transfer of phosphoryl groups from ATP to substrates. And this substrate could be a protein
Consensus sequence is a sequence is recognised by the kinase. There are three amino acids which are serine, threonine and tyrosine. Only these three amino acids can be phosphorylated (not all of them some certain ones). Protein kinase A is a very important protein kinase which has a role in the signal transportation
Carboxypeptidases
Carboxypeptidase are enzymes which removes one by one amino acid residues from the C terminus of the protein
Amino peptidases
Enzymes which removes one by one amino acid residues from N terminus of the protein.
Blood coagulation steps
- Formation of the prothrombin activator Xa (read as factor 10 A)
- Thrombin activation (proteolytic enzyme which cleaves fibrinogen to fibrin)
- Fibrin formation
Coagulation cascade
2 parts
1) Contact activation pathway (intrinsic),
called like that because it can be activated by the contact of blood with the surface, for example if you put blood into patient and put it into a vessel into a test tube it starts to coagulate why because it gets in contact with the plastic or glass, whatever you put the blood inside. It is a consequence of a cascade reaction.
2) Tissue factor pathway (extrinsic), called like that because it’s caused by a damage to the endothelium,
