Quiz 3: Lecture/Reading Info Flashcards
Fibrinogen protein
Fibrinogen floats around our blood inactively
Thrombin has to cut fibrinogen to make fibrin
Fibrin interacts with other fibrin molecules to form long complexes
These long complexes catch platlettes and form blood clots
Warfarin
blood thinner to stop clots
does this by stopping thrombin from cutting fibrinogen
therefore, there is no fibrin
Hydropathy index
the more positive, the more hydrophobic the molecule is
the less positive, the less hydrophobic the molecule is
The Isoelectric point
the point at which a molecule has a net charge of zero
What happens at pH below the isoelectric point?
the molecule has a positive charge
What happens at pH above the isoelectric point?
the molecule has a negative charge
What happens to proteins at their pI point?
they may be insoluble due to the lack of charge
Cysteine and the disulfide bridge
Cysteine can ionize and this negatively charged sulfur will interact with another cysteine to form a disulfide bridge
Cysteine reduced form
Is not in a disulfide bridge and has negative charge on separate cysteines
Cysteine oxidized form
Is in disulfide bridge which removes the negative charge/electrons
Are disulfide bonds normally found in intracellular proteins?
the cytosol is a reducing environment, so disulfide bridges are often broken down within the cell and not found there
Proline
R side chain attaches itself to the amino group
Has a constricted ring like structure
Ability to break secondary structures
What is peptide bond formation catalyzed by?
ribosomes
ribosomes bring amino acids together and make a polypeptide sequence
How are amino acid residues different from amino acids?
Amino acid residues are found within the polypeptide and this can modify them chemically
they have lost a water molecule through dehydration synthesis when being added to the polypeptide
what molecular weight do we use for amino acids?
110 g/mol
can divide total weight of protein by 110 to figure out roughly how many amino acids are in the protein
Peptide bond rigidity
The C=O and the N-H in the peptide have some resonance
This makes the peptide bond have double bond like qualities and is rigid / planar
Type I B-turn
proline used to add a kink
Type II B-turn
glycine used since it doesn’t have a R-group and is easier to turn
Are there only 20 amino acids?
No! There are many more less common ones
What type of stereoisomers are L and D configurations?
they are enantiomers
What stereochemistry do biological amino acids have?
exclusively the L conformation
What are aromatic side chains?
relatively nonpolar
however, tryptophan and tyrosine are slightly more polar due to their nitrogen and hydroxyl group respectively
What wavelength do proteins absorb light at?
280 nm
use this in lab
Cysteine is what type of molecule
a weak acid due to it’s ability to make weak hydrogen bonds with oxygen or nitrogen
sulfhydryl group is quite modestly polar
What is the only amino acid with a pKa near neutral?
histidine
y-carboxyglutamate
occurs from carboxylation of glutamic acid
this transition from GLU to GLA is essential for the functioning of thrombin and warfarin prevents this transformation from occuring
Zwitterion
amino acids that can act as either an acid or a base
amphoteric
normally, have a positive amino group and a negative carboxyl group and a non-ionizable R group
positive electrode
called the anode
at pH greater than pI, the amino acid will be negatively charged and migrate towards the positive anode
negative electrode
the cathode
at pH less than pI, the amino acid will be positively charged and migrate towards the negative cathode
peptide bond textbook definition
two amino acid molecules can be covalently joined through a substituted amide linkage
How does peptide bond happen even though thermondynamically unfavorable?
the carboxyl group must be chemically activated or modified so that the hydroxyl group is more easily eliminated in dehydration synthesis
Why does hydrolysis of peptide bond not occur even though energetically favorable?
the activation energy barrier is too high
kinetically unfavored
simple proteins
contain only amino acid residues and no other chemical constituents
conjugated proteins
contain permanently associated chemical components other than amino acids
this other components are known as prosthetic group
ex: lipoproteins have lipids, glycoproteins have sugar
Edman degradation
labels and removes only the amino-terminal residue from a peptide, leaving all other peptides in tact
proteases
catalyze the hydrolytic cleavage of peptide bonds
can be down in a predictable and controllable way
conformation
the spatial arrangement of atoms in a protein or any part of a protein
conformations that naturally occur are normally the most thermodynamically stable
native proteins
functional, folded conformation
stability
the tendency to maintain a native conformation of proteins
How do proteins fold energetically?
most of the free energy is derived from weak interactions within a protein and the increased entropy in the surrounding aqueous solution
Are there normally lots of unpaired hydrogen-bonding and ionic groups?
No! These are highly destabilizing
Proteins maximize hydrogen bonds and ionic interactions
Phi angle
between the N-Ca bond
Psi angle
between the Ca-Carbonyl bond
What values can phi and psi be?
anything between -180 and +180
secondary structure
refers to any chosen segment of a polypeptide chain and describes the local spatial arrangement of its main chain atoms
Most prominent secondary structures?
a-helicies, b-turn, and b-conformations
A-helix
the simplest arrangement the polypeptide chain can assume that maximizes the use of internal hydrogen bonding
Where are the R-groups in A-helix?
they protrude outwards from the backbone
Why does the a-helix occur so frequently (roughly 25% of all amino acid residues)?
it maximizes the amount of hydrogen bonding
Hydrogen bonds in A-helix?
occur between the hydrogen atom attached to the electronegative nitrogen atom and the electronegative carbonyl oxygen atom on every fourth amino acid
Amino acid with greatest tendency to form alpha helix
Alanine
Why do long strains of Glu not make an alpha helix?
the negative charged carboxyl groups repel each other
this is the same with positively charged R-groups
Why does Gly not make an alpha helix?
too much flexibility
Why does Pro not make an alpha helix?
introduces a kink in the helix
B sheet
more of a zig zag structure than the alpha helix
where are beta turns normally found?
near the surface of the protein since the turn needs hydrogen bonds from the aqueous environment to stabilize it
Where is B sheet and A helix in a Ramachandran plot?
B sheet is above near the +120-180
A helix is below near the 0- -60
Ion exchange chromatography
exploits differences in the sign and magnitude of the net electric charge
for example, in cation exchange chromatography, the solid matrix has negatively charged groups, so proteins with more positive charge take longer to migrate
Size exclusion chromatography
separates proteins according to size
large proteins emerge quicker than smaller proteins
Why do large proteins emerge quicker than smaller proteins in the size exlcusion chromatography?
the smaller proteins get stuck in little pores in the matrix that the larger proteins cannot
Affinity chromatography
based on the binding affinity
the beads in the column have a covalently attached ligand and proteins with an affinity for this ligand bind to the beads
Sodiym dodecyl sulfate (SDS)
SDS binds to amino acids and gives them a negative charge and similar rod-like shape
Since amino acids now have a similar charge:mass ratio and shape, electrophoresis separates them strictly by size
Catalytic effect of enzyme
the increase in the rate at which substrate is converted to reaction products when the enzyme is present
activity
the total units of enzyme in a solution
specific activity
the number of enzyme units per miligram of total protein
increases during purification
A-Helix dipole
Tend to have more a negative charge near the carboxyl group and a positive charge near the amino group (this creates a dipole)
therefore, to stabilize want negatively charged amino acids near the positive amino end and want positively charged amino acids near the negative carboxyl end
Difference between cystine and cysteine?
Cystine is when 2 cysteine molecules are in a disulfide bridge
How can you measure UV light absorption?
with aromatic rings
When can amino acids without ionizable R groups act as zwitterions?
in neutral conditions
will have +NH3 and -COO
When looking at a titration curve of an amino acid how do you know if it has an ionizable R group?
There are 3 buffering regions
Hydrogen bonds in alpha helix
are roughly parallel to the backbone axis
Why are antiparallel B sheets more stable than parallel B sheets?
antiparallel B sheets can line hydrogen bonds up more directly and therefore, have stronger H-bonds
Right handed alpha helix
Goes in clockwise spiral
What does a buffer zone between 2-3 indicate on a titration curve?
the carboxyl group is ionizing
What does a buffer zone between 9-10 indicate on a titration curve?
the amino group is ionizing
Gel filtration column
another name for size exclusion chromatography
The first protein to elute from the gel filtration column will be the ____ protein in an SDS page
slowest since it is the largest
How would you isolate a molecule of interest from cation-exchange chromatography?
in this case, we want the cations
cations are currently attached to negatively charged beads
so we raise the pH above the pI
Now the cations will be more negative and elute
Intramolecular
within the molecule
A-helicies have intramolecular bonds
Intermolecular
between the molecule
B-pleated sheets have intermolecular bonds
What is a problem with Edman degradation?
5% does not react
We will have 95% of the protein of interest and 5% of the protein before it
This builds up overtime
Can only use Edman degradation for small proteins
CORN rule
counterclockise = L
clockwise = D
make sure H group is in the back
R versus S configuration
counterclockwise = S
clocwise =R
Heterodimer with no cysteines on the SDS-page
Will see 2 bands on the gel
Homodimer with no cysteines on the SDS-page
1 band on the gel
Multimeric protein made of 3 differently sized polypeptides, 2 of which are connected by disulfide bonds?
See 2 bands on the gel
Had 3 peptides, but 2 are now connected as 1. So have a total of 2 separate bands on the page
