Amino Acids And Proteins Flashcards
What are the strongest bonds?
Covalent bonds
E.g. Peptide bonds, disulfide bonds
What are covalent bonds?
When two atoms share electrons
What are ionic bonds?
Interactions of ions with one a full positive and the other a full negative
E.g. Salt
What is a hydrogen bond?
A dipole-dipole interaction between a partially positive hydrogen atom and partially negative oxygen (FONS and H)
What are van dear walks interactions?
Dipole dipole interactions
These are the weakest electrostatic bonds
What are hydrophobes?
Nonpolar, hydrophobic molecules
E.g. The inside of the plasma membrane
Why do hydrophobes tend to cluster together?
Nonpolar molecules tend to break the hydrogen bonds of water increasing enthalpy. To counteract this they cluster together in order to achieve to lowest possible enthalpy.
Enthalphy = energy state
What do the core of proteins usually contain? (Usually globular proteins)
They have a hydrophobic center
What is the hydropathy index?
Measure of hydrophobicity of amino acids
What is a glycophorin?
An erythrocyte membrane spanning protein
-used as an example for hydropathy plots
What are the four components of an amino acid?
Amino acid
Carbonyl group
Side chain (-R group)
Hydrogen
-all bound to central carbon
What determines the chemical properties of the protein? E.g. Whether is is nonpolar, charged, polar uncharged
The R group
What are the abbreviations for Alanine, Arginine, Asparagine, Aspartic acid?
Ala, A
Arg, R
Asn, N
Asp, D
What are the abbreviations for Cysteine, glutamine, glutamic acid, glycine?
Cys, C
Gln, Q
Glu, E
Gly, G
What are the abbreviations for Histidine, Isoleuine, leucine, lysine?
His, H
Ile, I
Leu, L
Lys, K
What are the abbreviations for Methionine, phenylalanine, proline and serine?
Met, M
Phe, F
Pro, P
Ser, S
What are the abbreviations of Threonine, Tryptophan, Tyrosine, Valine?
Thr, T
Trp, W
Tyr, Y
Val, V
Which are the essential amino acids?
Phe, Val, Trp
Thr, Ile, Met,
His, Arg, Leu, Lys
“PVT TIM HALL”
Which are the branched chain amino acids?
Leucine, isoleucine and valine
“LIV”
Which amino acids are aliphtic (non-aromatic)?
Pro Ala Val Ile Leu "My friend PAVIL is not charismatic (aromatic)"
Which amino acids are only mildly hydrophobic?
Gly
Ala
Pro
“The reason why there’s a GAP inside proteins”
What is special about glycine?
Only mildly hydrophobic but confers flexility in proteins
What is different about Cys?
It has a polar side chain -SH but acts more hydrophobic than glycine or alanine
It is considered a hydrophobic amino acid
Which amino acids are aromatic?
Tyrosine
-hydrophobic
Tryptophan
Phenylalanine
-Phen/Phan indicate an aromatic
How can proteins be differentiated from nucleic acids using UV light?
Proteins absorbs at 280 nm
Nucleic acids absorb at 260
List the order of absorbance for the aromatic compounds.
Most Trp, Tyr, Phe least
Which amino acids are un charge at physiological pH?
Cysteine Asparagine Threonine Serine Glutamine
“My CATS a G”
Which amino acids are basic ?
Lysine
Arginine
Histidine
Which amino acids are negatively charged? (These are acidic)
Aspartate
Glutamate
What are the kinds of secondary protein structures?
Alpha helix
Pleated sheet
Beta turn
What kind of reactions forms a peptide bond?
Condensation (dehydration)
What is a peptide bond?
A covalent, amide bond between the alpha-carbonyl group of one amino acid and the alpha-amino group of another
(“NCC-NCC-NCC” each NCC is an amino acid)
What are the characteristics of a pepdtide bone?
Rigid (no rotation)
Planar. (Flat)
Has dipole (slightly positive on one side and slightly negative on the other side)
What part of the peptide bond holds a dipole?
Carbonyl oxygen holds a negative dipole
Amide nitrogen holds a positive dipole
What is a primary structure of a protein?
The linear sequence of amino acids in the polypeptide chain
In which order is a protein written in?
From the N-terminus to the C-terminus
“NCC-NCC-NCC”
What is a secondary structure?
Where regions of amino acids fol into a limited number of distinct structures
What kind of bonds form secondary structures?
Hydrogen bonds between carbonyl and amide groups in the peptide bond
-the R groups are usually not involved
What is an alpha helix?
A rigid, right handed helix forming a rod-like structure. (3.6 amino acids per turn)
What forms an alpha helix?
H bonding between peptide bonds (4 residues apart)
Which amino acid destabilizes alpha helixes?
Proline because it is bulky and has a charged R group
What amino acid is to involved in alpha helixes because it allows too much flexibility?
Glycine
What is a beta sheet?
When 2 or more peptide chains are arranged parallel or anti parallel to each other
Form a “pleated sheet”
What forms a beta sheet?
Hydrogen bonds between adjacent peptide chain
What is seen in the R group of beta sheets?
The R group alternate above and below the plane of the sheet
What is a beta turn or reverse turn?
A 180 degree turn in a peptide chain
What do beta turns help form?
Compact globular shapes
How is the 180 degree turn created in a beta turn?
With 4 amino acids usually including proline and glycine
1 hydrogen bond between peptide bonds 2 residues apart
What is a tertiary structure?
An overall 3D arrangement of the entire peptide chain
-a spatial organization of secondary structures
What does the tertiary structure determine in a protein?
The function of the protein
-structural alterations (by mutations) abolish or alter the function
In the tertiary what can happen to the primary structure?
Residues far apart in the primary structure may be brought close together and may act as a functional unit
What is quaternary structure?
The spatial arrangement of polypeptide chains (several)
What does quaternary structure determine?
The function of the protein
What are some examples of quaternary structure?
Hemoglobin: 2 alpha and 2 Beta subunits
Immunoglobulin G: 2 heavy and 2 light chains
Creatine Kinase: a dimer (kinase= add a phosphate?)
Lactate dehydrogenase: a tetramer (involved in the breakdown of sugar)
How are tertiary and Quaternary structures stabilized?
By noncovalent bond interactions involving the atoms of the peptide bon and the side chains (R groups)
Ionic, H-bonds, van dear Walls, hydrophobic
May be further stabilized by covalent bonds (disulfide bonds)
What amino acids can stabilize Tertiary/Quaternary structure of proteins by forming disulfide bonds?
Cysteine
What are domains in proteins?
Discrete areas in a protein that may have different functions
-stable arrangements of several elements of secondary and tertiary structure
How do domains differs in small and large proteins?
Small proteins- one domain, one or few functions
Large proteins- multiple domains, multiple functions
Copying and shuffling of domains during evolution has led to formation of what in proteins?
Protein families
These proteins share common domains
What are super secondary structures?
Relatively large patterns of 3D structures from repeated secondary structures
What are super secondary structures often associated with?
Often associated with specific activity such as ATP binding
What is the Actin Fold?
A cleft between domains for binding and hydrolysis of ATP
Also seen in many ATP-binding and utilizing proteins
What is a told that use to compare amino acid sequence?
BLAST!!!!!
-Basic Local Alignment Search Tool
What can blast be used for?
To compare homologous proteins or DNA
What similarity do you need to highly similar protein structures?
~25%
Even though they may be so different they can have similar functionality
What are orthologs?
Genes in different species that have the same general function
Evolved from a common ancestor
E.g. Human alpha-globin and mouse alpha-globin
What are paralogs?
Imperfect copies of genes within a species (members of gene families)
Generally different but likely related functions
What is column chromatography?
A separation of a crude protein sample in a buffer (mobile phase) through a stationary porous matrix
How does column chromatography separate crude samples?
Based on some property of the protein
E.g. Charge, size, binding properties, hydrophobicity
-“properties of the stationary phase and it’s interaction with the proteins form the basis of separation”
How is purification of a protein accomplished?
By sequential column chromatographies with different stationary phases
What is size exclusion chromatography?
Aka gel filtration
Separation on the basis of the size
How does size exclusion chromatography work?
Solid phase is a porous bead
Smaller molecules enter the bead and migrate slowly
Large molecules are less likely to enter the beads and migrate more quickly
The largest get out the quickest
What is ion-exchange chromatography?
Separation on the basis of charge
At a given pH
How does ion-exchange chromatography work?
Beads are resins carrying a charge
Proteins with opposite charge bind the resin and migrate slowly
Proteins with same charge don’t bind and migrate more quickly
The greater the charge, the greater the effect on migration
What kind of exchangers are seen in ion exchange chromatography?
Cation exchanger: negative charge
Anion exchanger: positive charge
What is affinity chromatography?
Separation on the basis of binding properties
Proteins have different binding properties
How does affinity chromatography work?
Ligand conjugated to beads
-only proteins that bind the ligand are retained on the column
The protein may be elated from the column by competition with free ligand
What is gel electrophoresis?
Separation of molecules in an electric field through a porous gel (sieving medium)
What are some gels used in gel electrophoresis?
Polyacrylamide for protein (amides are used in peptide bonds)
Agarose for DNA/RNA
How does Gel Electrophoresis work?
Migration toward electrode of opposite charge
What are the factores that affect gel electrophoresis ?
Size, conformation and charge affect the rate of migration though the gel
What is SDS-PAGE?
Separation on the basis of size
What does SDS-PAGE require??
Proteins to be unfolded without disulfide bonds
What is SDS?
Sodium dodecyl sulfate (SDS) is a strong ionic detergent that denatures the proteins and coats protein with a negative charge
What breaks disulfide bonds in SDS-PAGE
Mercaptoethnol- a reducing agent break disulfide bonds
What travels faster in SDS-PAGE?
Smaller proteins migrate more quickly
-This is opposite of exclusion chromatography
How is the protein detected in SDS-PAGE? (Stain)
Coomassie Blue
Silver Staining
How can SDS PAGE be used to estimate the size of a protein?
Molecular weight of unknown protein is compared to the standard proteins of known size
How many ionizable groups do all Free amino have as a minimum?
2
What amino acids have at least 3 ionizable groups?
Acidic and Basic Amino acids have 3
What is the Isoelectric charge?
The pH at which an amino acid has no charge
How is the isoelectric point (pi) of a protein determined?
By the total net charge including all charged R groups and the alpha-carboxyl and alpha-amino groups
What is isoelectric focusing of proteins?
Separation of proteins on the basis of their isoelectric proteins
What are ampholytes
A mixture of polyanionic and polycationic molecules with varying pI (isoelectric point)
How do isoelectric focusing work?
Ampholytes in a gel establish a pH gradient in an electric field
Proteins will migrate until they reach their pI
What charge does a protein carry at it’s pI (isoelectric point)?
At pI, no net charge so they stop migrating
What is 2D gel electrophoresis?
A better separation technique for complex mixtures of proteins
Proteins are separated in 2 perpendicular direction using a different basis of separation i each
E.G. Isoelectric focusing and SDS-PAGE
What is proteomics?
The large-scale study of proteins complement of cells
How can the identity of individual proteins be determined in a 2D gel electrophoresis
Through mass spectrometry
What is a ligand?
The small molecule being bound by the protein
What is a receptor protein?
The protein that binds the ligand
E.g. cAMP being bound by a cAMP-binding protein
What are the receptor-ligand interaction mediated by?
Many weak noncovalent bonds
-covalent bonds are extremely rare
How do you determine if ligand receptor binding is of high affinity
The number of bonds between the two molecules
The higher number of bonds, the higher the affinity and a higher likelihood of being bound most of the time
Low affinity binding equilibrium shift will be toward what state?
The unbound state
High affinity binding will have an equilibrium shift toward which state?
The bound state (most of the time)
What is the association rate equation?
Association rate = Kon[A][B]
=association rate constant * concentration of A * concentration of B
What is the equation for equilibrium constant?
K= Kon/Koff =[AB]/[A][B]
What can you use to determine the affinity beside number of bonds?
Dissociation constant (Kd) (units are M)
Association constant (Ka) (units are M-1)
How can you tell if the binding is stronger using the Ka or Kb?
The higher the association constant Ka the stronger the bond
The lower the dissociation constant Kd the stronger the bond
Remember: Kd is equivalent to the concentration of ligand (A) at which receptor (B) is 50% bound
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When Kd is smaller than concentration then it will favor the complex
When Kd is larger than the concentration then the free molecules will be favored
