MCAT information Flashcards
all amino acids have what terminus
All amino acids have a N-terminus and a C-terminus
peptide bonds and what direction are they formed in?
Peptide bonds are dehydration reactions (and produce water and the chain of amino acids) and are formed in the N-C direction
The resulting covalent bond is called an amide bond
cysteine can form additional covalent bonds (aka disulfide bridge)
what determines an amino acid’s properties?
their R-group side chain
what are 3 side chain properties?
aromatic
aliphatic
charged
aromatic side chain property definition and 2 examples
aromatic compounds: organic compounds that have a cyclic structure with alternating single and double bonds (aka conjugated double bonds)
- aromatic compounds are often more stable than aliphatic compounds
ie. phenylalanine, tryptophan
aliphatic side chain property definition and 2 examples
aliphatic: organic compounds that do NOT contain aromatic rings and instead consists of carbon chains or branched structures
- can be saturated (alkanes) or unsaturated (alkenes, alkynes)
ie. valine, leucine
charged side chain property
has either a positive or negative charge (result of an unequal number of protons and electrons)
what are the components of a general amino acid?
amino group (NH2)
carboxyl group (COOH)
R group side chain
alpha carbon (aka central carbon)
hydrogen atom (H): a single hydrogen atom bonded to the central carbon)
Amino acid sequences are listed in what direction?
Amino acid sequences are listed in the N-terminus to the C-terminus direction
Kinases
the enzymes that transfers phosphate groups onto amino acids (addition of phosphate groups PO4 3-)
what type of amino acids can MIMIC phosphorylation?
Amino acids with a net negative charge can MIMIC phosphorylation
any amino acid with ____ can be phosphorylated
Any amino acid with an R-group possessing a hydroxyl OH group can be phosphorylated
The phosphate groups on phosphorylated proteins/amino acids are negatively or positively charged?
The phosphate groups on phosphorylated proteins/amino acids are negatively charged
relation between any R group being charged and the entire amino acid?
Any R-group with a net charge will give the amino acid a net charge
3 examples of positively charged amino acids
Arginine (R), Lysine (K), and Histidine (H)
2 examples of negatively charged amino acids
Glutamate (E) and Aspartate (D)
5 examples of hydrophobic amino acids
A, I, L V, F
7 examples of hydrophilic amino acids
H, R, K, D, E, N, Q
what type of side chains do hydrophobic amino acids have?
nonpolar, long alkyl side chains
what type of side chains do hydrophilic amino acids have?
polar, charged side chains
where are hydrophobic regions/residues found in the protein’s structure?
Hydrophobic residues will be found in the protein’s INTERIOR since this conformation is entropically favorable
secondary structure consists of what?
Consists of alpha-helices and beta-pleated sheets stabilized by hydrogen bonds between amides
which 2 amino acids can disrupt a protein’s secondary structure of alpha helices and beta-pleated sheets?
Proline (P) can disrupt these structures due to its sterically hindered ring Gly (G) due to its very small methyl R group that allows for increased flexibility
P and G are usually found at ends of alpha-helices or within creases of beta-pleated sheets
how is tertiary structure formed and what breaks tertiary structure?
Tertiary structure is formed by side chain interactions: salt bridges, disulfide bonds, and hydrogen bonds
Denaturation breaks tertiary structure
3 types of side chain interactions
salt bridges (a noncovalent interaction between 2 OPPOSITELY charged chemical groups/atoms that combines hydrogen bonding and ionic bonding
disulfide bonds
hydrogen bonds
disulfide bonds
a type of side chain interactions (COVALENT BOND) and are formed by oxidation of the thiol (SH) side chains of 2 cysteine (C) residues
aka cystines
2Cysteine(–SH) –(oxidation) –> Cystine(–S–S–) + 2H+ + 2e-
Quaternary structure consists of what?
Quaternary structure consists of multiple subunits or polypeptides sometimes with a prosthetic groupe (ie. heme in hemoglobin)
isoelectric focusing
a lab technique used to separate proteins based on their isoelectric point (pI) - the pH at which a molecule has no net electric charge (aka the number of positively charged groups = number of negatively charged groups)
a protein is applied to the gel that contains a continuous pH gradient and the protein will migrate through the pH gradient due to an electric field until it finds its isoelectric point where the molecule has no net charge (aka reach the point where their pH = pI)
- At pH values below its pI, the molecule has a positive charge and moves toward the negative electrode
- At pH values above its pI, the molecule has a negative charge and moves toward the positive electrode
- At its isoelectric point (pI), the molecule has no net charge and will stop migrating because it is no longer influenced by the electric field
Basic amino acids (RKH) have a higher pI; acidic amino acids (ED) have a lower pI
isoelectric point (pI)
pI: average of all pKa values of functional groups
also the pH at which a molecule has no net electric charge (aka the number of positively charged groups = number of negatively charged groups) ==> aka zwitterion
basic amino acids have higher or lower pI; acidic amino acids have higher or lower pI
Basic amino acids (RKH) have a higher pI; acidic amino acids (ED) have a lower pI
SDS page: what does SDS stand for and PAGE stand for?
SDS page: a lab technique to separate proteins based on their MOLECULAR WEIGHT only (shape does not matter)
PAGE stands for polyacrylamide gel electrophoresis: essentially a spongy network that blocks protein fragments based on SIZE and CHARGE
SDS stands for sodium dodecyl sulfate: a chemical that essentially coats everything with a NEGATIVE charge and DENATURES proteins
→ once a protein is denatured, their movement through the gel is not affected by their SHAPE and now only depends on their MASS
what does a reducing agent do in SDS page?
A reducing agent (ie. tBME) adds hydrogens (to the protein)
- results in the reduction of disulfide bonds (S-S) which results in SH and HS → efficiently breaks the disulfide bond
what are the 3 types of SDS page?
native page
nonreducing SDS page
reducing SDS page
native page
no SDS, no reducing agent
→ protein is not denatured & the disulfide bond S-S remains → protein will stay together and the movement of proteins through the polyacrylamide gel depends on both their size and shape → will see one single band
nonreducing SDS page
yes SDS, no reducing agent
–> coats everything with a negative charge and disrupts the electrostatic interactions → the 50kDa component is no longer attached to the protein → will see two separate bands in the gel
what does SDS do to the protein?
what does the reducing agent do to the protein?
SDS breaks up the electrostatic interactions of the protein since it coats everything with a negative charge
reducing agent breaks the covalent disulfide bond (S-S)
reducing SDS page
yes SDS, yes reducing agent
→ everything is coated with a negative charge and there’s a reducing agent → electrostatic interactions and S-S bond are disrupted → protein separates into 3 different components → will see 3 separate bands in the gel
Allows researchers to determine how many SUBUNITS there are in a protein and what their connections are
blotting technique mnemonic SNOW DROP
Southern blot detects DNA
Northern blot detects RNA
O stands for nothing
Western blot detects proteins
what are the 6 types of chromatography?
Column chromatography
Size-exclusion chromatography
Ion-exchange chromatography
Affinity chromatography
Gas chromatography
Thin layer chromatography (TLC)
chromatography techniques are used for what?
A method of separating and identifying proteins of interest from a sample (ie. a mixture of substances)
Stationary phase (adsorbent)
Mobile phase (eluent)
Stationary phase (adsorbent): the solid medium (that remains fixed in place and provides a medium/surface with which the components of the mixture can interact)
Mobile phase (eluent): the fluid medium (that carries the components of the mixture with it)
Retention time
Retention factor (Rf)
Retention time: the amount of time a given protein spends on the adsorbent
Retention factor (Rf): the distance moved by the protein / the distance moved by the solvent front
relationship between a protein’s affinity for the stationary phase and the speed at which it migrates at in chromatography
Proteins with greater affinity for the stationary phase will migrate slower
column chromatography
separate by polarity (more polar, migrate slower)
size-exclusion chromatography
separate by size (smaller proteins migrate slower)
Ion-exchange chromatography
separate by charge (more charged migrate slower)
- subcategories: Cation-exchange & anion-exchange
Affinity chromatography
separate by affinity (higher affinity migrate slower)
Gas chromatography
separate by boiling point (higher boiling point migrate slower)
The mobile phase is a gas, NOT a liquid in this case
Thin layer chromatography (TLC)
glass plate, separate by polarity (usually adsorbent is more polar, but for reverse phase TLC, the eluent is more polar)
The simplest chromatography; the glass plate acts as the stationary phase
–> add a small quantity of sample to the stationary phase and then observe upon the addition of the mobile phase how fast does the sample migrate along the stationary phase
we can analyze what 4 things about a protein?
We can analyze protein structure, activity, amino acid composition, and concentration
what do we use to analyze a protein’s structure?
NMR or X-ray crystallography
NMR
a powerful analytical technique used to determine the structure of molecules, particularly organic compounds
- NMR exploits the magnetic properties of atomic nuclei and their interactions with an external magnetic field to provide detailed information about the chemical environment and arrangement of atoms within a molecule
ie. proton NMR, carbon-13 NMR
X-ray crystallography
X-ray crystallography: crystallize protein → irradiate with X-rays to produce diffraction pattern → convert diffraction pattern to electron density map which is interpreted to reveal the molecule’s 3D structure
how do we analyze a protein’s activity?
monitor a known reaction with a given concentration of substrate and compare to a standard
–> gives a relative measurement of protein activity
what do we use to analyze a protein’s amino acid composition?
Edman degradation and mass spectrometry if sequence is less than 70 amino acids
otherwise, digest with trypsin or cyanogen bromide first and then run it again
Edman degradation
a way to remove amino acids from the N-terminus of a protein
→ once they are removed, use mass spectrometry to identify them
Mass spectrometry
ionize the compound of interest with a high energy electron beam → accelerate these ions to all have the same kinetic energy → deflect them with a magnetic field → these ions hit a detector and different levels of deflection can be converted into a mass spectrum
what do we use to analyze a protein’s concentration?
UV spectroscopy or colorimetric assay
UV spectroscopy
shine light onto your sample and vary the wavelength of the light → measure how much light is absorbed at the different wavelengths: more absorption of one sample compared to another sample at the same wavelength ⇒ tells us that sample is more concentrated with the molecule of interest
However, a problem with UV spectroscopy is that it is quite susceptible to sample contamination
colorimetric assay
uses color changes to measure the concentration of a substance in a sample
–> The assay typically involves a chemical reaction that results in the formation of a colored product, with the intensity of the color being directly related to the concentration of the substance being measured
–> The color change can be detected using a spectrophotometer, which measures the absorbance of light at specific wavelengths
ie. BCA assay, Lowry reagent essay, Bradford protein assay - green dye turns blue after nucleophilic attack by amino acid
what is a way to break proteins down so that we can analyze them at a smaller resolution?
use hydrolytic agents which cleave peptide bonds via the addition of water
3 examples of hydrolytic agents
trypsin
chymotrypsin
cyanogen bromide
which hydrolytic agents are biologics and which are synthetic compounds: trypsin, chymotrypsin, cyanogen bromide
Trypsin and chymotrypsin are biologics; cyanogen bromide is a synthetic compound
trypsin cleaves what
Trypsin cleaves at C-terminal side of Lys and Arg residues
chymotrypsin cleaves what
Chymotrypsin cleaves at N-terminal side of Trp, Phe, and Tyr (aromatic residues)
cyanogen bromide cleaves what
Cyanogen bromide cleaves at C-terminal side of Met
michaelis menten kinetics definition and Vmax, Km, Kcat
Provides a model for how enzyme reaction rate changes as a function of substrate concentration
Vmax = max velocity of enzyme’s reaction rate
Km = substrate concentration at which the enzyme’s reaction rate is half of its maximum value (Vmax)
- lower Km ==> higher enzyme affinity
Kcat = turnover number (the number of substrate molecules that a single enzyme molecule can convert into product per unit of time when the enzyme is fully saturated with substrate)
- a measure of enzyme’s catalytic efficiency under optimal conditions
Enzyme + substrate –> enzyme-substrate complex –> enzyme + product
lineweaver-burke plot: x-axis, y-axis, x-intercept, y-intercept, slope
a linearization of MM kinetics plot and can change in the presence of different inhibitors
x-axis: 1/[S]
y-axis: 1/v
x-intercept: -1/Km
y-intercept: 1/Vmax
slope = Km/Vmax
michaelis-menten kinetics 4 assumptions
assumptions
1. Only measuring initial velocity
2. Concentration of enzyme-substrate complex is constant
3. Substrate concentration is much higher than enzyme concentration
4. Forward reaction rate is much higher than reverse reaction rate
3 different types of inhibitors
competitive
uncompetitive
noncompetitive
competitive inhibitor: Km and Vmax
Km increases; Vmax does not change
- the two lines intercept at y-axis on lineweaver-burke plot
uncompetitive inhibitor: Km and Vmax
Km decreases; Vmax decreases
Uncompetitive inhibitor binds the the enzyme-substrate COMPLEX
noncompetitive inhibitor: Km and Vmax
Km unaffected; Vmax decreases
Binds to allosteric site on enzyme
- the two lines intercept at x-axis on lineweaver-burke plot
