Midterm 1 Flashcards
what is the central dogma of biology
DNA–> RNA –> Protein
explain what the central dogma describes
information processing in any cell
what words are associated with DNA in the central dogma
replication
4 bases (dA, dT, dG, dC)
what words are associated between DNA and RNA in the central dogma
transcription
RNA uses what 4 bases
A, U, G, C
what word is associated between RNA and Protein in the central dogma
translation
what are the building blocks of proteins?
20 amino acids
what are the building blocks of DNA
4 bases ( dA, dT, dG, dC)
what are the types of proteins
enzymes
transport proteins
receptors
structural proteins
enzymes?
catalyze all chemical reactions
transport proteins?
move molecules
receptors proteins?
transduce signals –> means converting a stimulus into a different kind of signal
structural proteins ?
architecture of cells
No water =
NO LIFE
most living organisms contain about –>
65% water
describe the partial charges on a water molecule
2 Hydrogens have partial positive
2 pairs of lone pairs on oxygen have partial negative charge (making oxygen have partial neg charge)
WHat are the unusual and important properties of water
- only substance that exists on earth in all three physical states : solid, liquid, gas
- high boiling and melting point for such a small molecule
- Density of liquid water is greater than density of ice –> hence ice floats
- Very polar - metabolites and ions are soluble but lipids are not
structure of ice
crystalline structure where oxygen makes 4 bonds with other hydrogens from other water molecules
what do we compare water with to show that it has such a high boiling and melting points for such small molecules…..
compare water and methanol ( mass of 18 vs 32 Da)
ice vs liquid water structure
ice is much more organized less dense and crystalline
water forms….
weak bonds because they constant break and reform and not permanent rather are very DYNAMIC
what are electrolytes
molecules that can dissociate to form ions
hyrdrophobic
water fearing
hydrophilic
water loving
what happens to a hydrophobic molecule in water
- Aliphatic side chain disrupts water structure
- Water cannot H-bond with hydrocarbon
- Water must order itself around the hydrocarbon without optimal H-bonds. Such ordering is entropically not favorable.
- Energetically more favorable for hydrocarbon to separate from water. That’s why oil and water don’t mix
describe how water would interact with a cluster of lipid molecules
only lipid portion at the edge of the cluster force the ordering of water. Fewer h20 molecules are ordered and entropy is increased
amphi=
2 sides
pathic =
coming together
amphipathic=
both hydrophilic anad hydrophobic
SDS is a _________ molecule
detergent
detergent molecules are usully
amphipathic –> have a polar head group and a hydrocarbon tail
describe SDS
sodium dodecyl sulfate is an amphipathic molecule with a polar head group and a hydrocarbon tail
In water, SDS molecules self assemble into ….
micelles
what is a micelle structure
the hydrophobic tails aggregate in the center away from water
the hydrophilic heads form the outer surface, interaction with water
in micelles, al hydrophobic groups are
sequestered from water; ordered shell of H20 molecules is minimized
list the strong and weak forces
- covalent bonds–> STRONG, 250-800 KJ/mol
- non-covalent bonds: relativey weak, 0.4-250 kJ/mol
calorie? chemistry def
1cal= energy required to raise temperature of
1 gm water by 1.0 °C
calorie (dietetics)?
1 Cal = 1,000 calories = 1 kcal
Joule? (physics)
1 J = 0.239 cal and 1 cal = 4.18 J
1 kJ = 1,000 J = 239 cal = 0.239 kcal`
non covalent interactions are …
easily changed or modified, more dynamic than covalent bonds
what are examples of non covalent interections
base pairing of DNA double helix
RNA-DNA interactions in transcription
Folding of proteins
binding of metabolites to enzymes
what are the types of noncovalent interactions
- charge-charge IONIC interactions
- Hydrogen Bonds
- Van der Waals forces
- Hydrophobic interactions
descrive ionic interactions
non-directional
distance dependent
attractive or repulsive
energy 6-250 kJ mol-1
describe hydrogen bonds
highly directional
fixed length (2.5A-3.5A)
Most H-bonds require 2-10 kJ to break which are weaker than those of water
what is the range of fixed length for hydrogen bonds
2.5 A -3.5 A
what are the donors and acceptors in an Hydrogen Bond
Donors: R-NH, R-OH, R-SH
Acceptors: O: or N: via an unshared pair of electrons
angular dependence of hydrogen bonds
refers to the fact that the strength of a hydrogen bond is highly dependent on the angle between the donor atom (the atom with the hydrogen), the hydrogen atom itself, and the acceptor atom (the atom receiving the hydrogen bond), with the strongest hydrogen bonds occurring when this angle is close to 180 degrees
describe Van der Waals forces
due to permanent, transient or induces dipoles that occur in all molecules
which is the weakest by far of the weak forces and give the range
Van der Waals forces
between 0.4- 4 kJ mol -1`
what is the optimal distance for carbon atoms in Van der waals forces for most stability
3 A = 10^-8cm= 0.3 nm
What is the optimal distance between c-c bonod in a typical convalent c-c bond
1.5 A
What is hydrophobic interaction
coalescence of non-ppolar, “water-fearing” molecules in an aqueous environment
the force of coalescense in hydrophobic interaction is provided by
mainly stability of the H-bonding network of surrounding water- very little by the inherent attraction of the non-polar molecules for each other
Rank the strength of hydrophobic interactions and Van der Waal forces
Hydrophobic interactions are stronger than
Van der Waals forces - 3-10 kJ mol-1
what are the two laws of bioenergetics
- energy can not be destroyed or created
- entropy of the universe increases
Keq=?
keq= [products]/[reactants]
if a process is spontaneous then which side of the equation is favored and what does keq look like
if process is spontaneous, then the reaction moving to the right is favored and Keq > 1.0
reactants and products have ….
a free energy
How do obsereve the change in free energy
we observe as we go from reactants to products
give the delta G equation
ΔG°(reaction) = G°(products) - G° (reactants )
describe the spontaneous process for a delta G reaction
G°reactants > G° products SOOOO
ΔG°(reaction) <0
describe ΔG°
is the standard Gibbs Free energy change for a given reaction uncder standard conditions of pressure (1atm), temperature (25C or 298K) and [H+]( ph 7.0 or 0.1uM
Under standard conditions the concentration of every reactant and product is
1M and WATER is 55<
give a easy def of ΔG°(reaction)
tells is is at equilibrium a reaction lies to the right of left
What is special about ΔG°(reaction) and living system reactions
living system reactions are seldom at equilibrium so a more useful quantity is ΔG under non- equilibrium conditions
textbook def of Gibbs free energy ΔG
free energy needed to convert reactants to products under a defined set of conditions
ΔG°(reaction) textbook bdefinition
STANDARD free energy of a reaction. Depends on free energies inherent in the structures of the chemicals
Give the equation of Gibbs Free Energy
Look this one up
why does the gibbs free energy equation include the lnQ portion?
ΔG due to differences in the concentrations of reactants and products
what happens in the Gibbs Free energy equation when all the concentrations are equal to 1
then ΔG= ΔG°
what happens in the Gibbs Free energy equation when Q is a very small number
tends to make ΔG more negative –> reaction is favorable –> SPONTANEOUS
what happens in the Gibbs Free energy equation when Q is a very large number
Tends to make ΔG more positive–> reaction is unfavorable –> NOT SPONTANEOUS
at equilibrium what is Gibbs Free energy
ΔG= 0 and [C]^c[D]^d/[A]^a[B]^b= Keq
SO Keq= e^(-ΔG°/RT)
what is energy coupling
an important role for enzymes and for high energy compounds like ATP
Why does energy coupling happen
by coupling (1) and (2) an enzyme can tap the energy of a highly favorable reaction to drive an otherwise unfavorable reaction
ATP hydrolysis is highly…
EXERGONIC and FAVORABLE–> ΔG°= -31 kJ/mol
why is ATP hydrolysis so highly exergonic
- electrostatic repulsion- less with products
- resonance forms - more with products
- Proton release
- Hydration stabilization- more with products
describe proton release in ATP hydrolysis in more depth
H +] is kept very low in the cell ( ~10-7 M)
* This favors reactions that release protons (mass action effect)
APT IS ENERGY…
currency
what are some things that ATP hydrolysis can do
couple ATP hydrolysis with non-spontaneous reactions
can also transder energy to ATP–>For example: creatine kinase converts ADP and phosphcreatine
to ATP and creatine ∆G° = -12.5 kJ/mol
∆G is INDEPENDENT of …
reaction path –> doesn’t matter what road you take only the destination
what is the alternative equation for delta g that includes heat…
∆G = ∆H - T∆S
what is ∆H
enthalpy, which is the heat given off in a reaction
when ∆H is <0 what does this mean
favorable spontaneous
when a reaction goes from two molecule to one molecule what is the sign of entropy
negative ∆S<0
every enzyme has its own….
optimal pH range–> if outside of pH range, it may not work as efficiently
pure water is slightly .,,
ionized
________ do not exist in solution, they are immediately hydrated to _________ ___
free protons do not exist in solution, they are immediately hydrated to hydronium ions
describe protons in hydronium ions
they can create a proton hop chain
where hydronium ion gives up a proton to OH which turns it into hydronium then that hydronium donates a proton…. etc
In pure water, the concentrations of [H+] and [OH-] are
Equal = 10^-7M= 0.1 uM
Kw=?
Kx= Keq[H2o]
what is the concentration of water in water
55.5 M
what is Keq of water
1.8 x 10^-16 M
equation for pH
pH=-log[H+]
what is the neutral pH of water where [OH-]=[H+]=10^-7M
pH= -log(10^-7)= 7
strong acids and bases…
completely dissociate
life is _____ in a very ____ pH range near pH=7
life is compatible in a very narrow pH range near pH=7
what is Ka and what is the equation
ka is the acid dissociation constant
ka= [H+][A-]/[HA]
what is pka… how can we contextualize it
at that pH there is an equal amount of conj acid and base
at the pka there is
an equal amount of conj. acid and base
ther pka is a measure of …
acid strength of a particular compound
when the pka = pH, the concentrations of …
[Ac-] and [HAc] are equal
when [Ac-] and [HAc] are equal what is ka equal too
ka= [H+]
Henderson-Hasselbach Equation
pH= pka + log[A-]/[HA]
why are phosphates a good buffer for neutral pHs?
bc their chemical structure alllows it to exist in multiple forms near-neutral ph–>
in phosphate buffering what does the first midpoint look like
[H3PO4]=[H2PO4-]
pka=2.2
in phosphate buffering what does the second midpoint look like
[H2PO4-]= [HPO4^2-]
pka= 7.2
in phosphate buffering what does the third midpoint look like
[HPO4^2-]=[PO4^-3]
pka= 12.7
true or false:
phosphate a good buffer to use around neutral ph
True
true or false:
A reaction with ∆G= -17 kJ/mol coupled to a reaction with ∆G= 15 kJ/mol will be favored
TRUE
true or false:
The K eq of a reaction with ∆G= -17 kJ/mol coupled to a reaction with ∆G= 15 kJ/mol will be >1
TRUE
True or False
Strong Acids partially dissociate in solution
FALSE
what are the building blocks of all proteins
amino acids
how many amino acids do we have
20
proteins are ….. (complicated name lol)
linear heteropolymers of AA
what are some uses of proteins
neurotransmitters
metabolic energy
precursors to other molecules
What are essential vs non-essential amino acids
essential amino acids are the ones that we cannot synthesize, so they are the ones that we must get through our diet
nonessential are the ones that we can synthesize
in humans how many amino acids are essential and nonessential
we have 11 non essential and 9 essential amino acids
give me a statement regarding amino acids and bacteria
some bacteria are able to synthesize all 20 amino acids
what is the general form of the amino acid
carbonyl, hydrogen, and amino group attached to an alpha carbon, with a R group or side chain attached to the alpha carbon as well
amino acids are ….. zz
ZWITTERIONS
WHAT ARE zwitterions
they are when the amino acid is in its IONIC form, so the carbonyl has a negative change and the amino has a positive SOO they cancel out–> NEUTRAL CHARge
how at least How many pkas does every amino acid have and why
at least 2 amino acids bc the Coo- can grab a proton and bc the amino Nh3+ can lose a proton
describe the peptide bond formation
coming together of the c- terminus to the n- terminus. The carbonyl loses the oxygen and the NH3 loses 2 H+–> creating a byproduct of water
When two amino acids are joined together to form a peptide bond there is a net …
Loss of one water molecule
Gain of one water molecule
Loss of one amino group
Gain of one carboxyl group
LOSE OF ONE WATER MOLECULE
–> water molecule is removed as a byproduct. This means the water that was part of the reactants is no longer present in the product (the peptide), so the system has lost water overall
in a final peptide bond (it is already formed) what two atoms are next to each other
the C from the carbonyl and the N from the amino group
oligopeptide?
a peptide of a few amino acids
what are examples of oligopeptides?
dipeptide
tripeptide
tetrapeptide
what is a polypeptide
many amino acids, no formal definition with regard to size
what is the definition of a protein
Hundreds of thousand chain of amino acids–> applicable when molecular weight is > 5000 Da
In what direction are peptide sequences written and read
N-terminus to C-terminus
LEFT TO RIGHT
what ph is a zwitterion amino acid usually at
around pH 7
What is a feature of nonpolar, aliphatic R groups
they usually have a net charge of 0
What are the types of amino acids
- nonpolar, aliphatic R group
- Polar, uncharged R groups
- aromatic R groups
- positively charged R groups
- negatively charged R groups
describe disulfide bonds in a specific amino acid
cysteines can form disulfide bonds between each other
like for example in between two cytsteines on different chains or even on the same chain
what do aromatic R groups usually have
A RINg (no way :))
Hydrophobic amino acid: pK 1 = 3.1, pK 2= 11.1 Which statement is BEST?
C. At pH 7, charge is -1, pI = 7.1
D. At pH 7, charge is 0, pI = 7.1
E. At pH 7, charge is +1, pI = 7.1
A. At pH 7, charge is +1, pI = 4.5
B. At pH 7, charge is 0, pI = 4.5
D
what are the levels of proteins structure
- primary
- secondary
- tertiary
- quaternary
what is primary structure
amino acid sequence
what is secondary structure
alpha helix and the beta strand–> a repeating pattern of bond angles in the polypeptide
backbone.
what is tertiary structure
Overall 3D structure of a polypeptide. Includes regions of secondary structure, turns and regions with random structure
(disorder).
what is quaternary structure
3d arrangement of MULTIPLE (more than 1) polypeptide
What is the highest level of protein structure in human
insulin, which has two polypeptides of different mass
linked by several disulfide bonds?
A. Primary
B. Secondary
C. Tertiary
D. Quaternary
QUaternary!
what are the four assumptions of secondary structure
- bond length and angles in proteins- should be the same as in free amino acids and small peptides
- Van der Waals radii must be respected
- amide bond is planar and trans –> no rotation at amide bone
- repeating structures will be stabilized by non-covalent forces–> especially the H-bond
disulfide bonds are usually….
PERMANENT bc its covalent meaning that it takes too much energy to break so its usually permanent
The 3D structure of the polypeptide backbone is completely defined by the
phi and psi angles
what is the phi angle
Phi angles are bond between N and Cα in the carbon backbone
what is the psi angle ?
angle around the bond between the alpha carbon (Cα) and the carbonyl carbon (C) in the protein backbone
what do the phi and psi angle create
they create an amide plane
what is an amide plane
the flat plane formed by the atoms involved in a peptide bond (amide linkage) between two amino acids, where the carbon of the carbonyl group, the nitrogen of the amine group, and the attached oxygen and hydrogen atoms all lie in the same plane due to the partial double bond character of the peptide bond
what character of the peptide bond makes it ______
the partial double bond character of the peptide bond makes it planar.
contrast the alpha and beta sheets
alpha helix: non planar backbone, and H-bonding neigbors are close
beta sheet: planar backbone, H-bonding neighbors are distant
what are similarities between the alpha and the beta sheet
both involve H-bonding between a backbone carnonyl oxygen and a backhone -NH group. Side chains are not involved
what are the most common helix found in proteins
right-handed alpha helix
in an alpha helix
Carbonyl O atom of amino acid ______
accepts a H-bond from the peptide
NH group of amino acid ______.
Carbonyl O atom of amino acid 1
accepts a H-bond from the peptide
NH group of amino acid 5.
the alpha helix has what residue per turn
3.6 residue
what is the pitch for an alpha helix per turn
5.4 A per turn
in alpha sheet - all the C=O point in which direction and to what
they all point up in the same direction to the helix axis
in alpha sheet - - where do the -NH point to and in what direction
they all DOWN up in the same direction to the helix axis
in alpha sheet - R groups point to what and to where
The R groups always point awya from the helix at about the same angle
in alpha sheet - the phi and psi angle ________-
for each residue is the same
explain the helical wheel diagram
based on the polarity and hydrophilicness of the amino acids you can determine the position of the protein’s 3d structures 2
in a beta sheet describe the way the c=o and -Nh groups are situated
the c=0 and -NH groups alternate left or right H-bonds
describe the R groups on a beta sheet
R groups alternate up and down out of the plane and sheet
what are the two types of Beta sheets
parallel or anti-parallel
when looking at the phi and psi angles in beta sheets what can you say about them
the phi and psi angle for each residue is the same
what is the difference between the antiparalle and parallel beta sheets
the antiparallel sheets have hydrogen bonds that are in the planar position meaning that they are stronger than the hydogen bonds in the parallel strand since those hydrogen bond are not planar
additionally the antiparallel sheet has a
C–>
N <–
C–>
Parallel
C–>
C–>
C–>
what can we say about the bends and loops in proteins?
just because they are unstructured doesn’t mean they are unimportant rather they help with the stability of the polypeptide
What are the two types of tertiary structure
fibrous proteins
globular proteins
what are fibrous proteins?
-twisted double alpha helices (keratin-nails, hair)
- twisted alpha helicies (collagen- tendons)
- super beta sheets (silk)
what are globular proteins
- most contain multiple regions of alpha and beta structures
- some are predominantly one or the othe r
- hydorphilic side chains tend to reside on the surface; alphatic/hydrophobic side chains tend to be buried inside
what is a domain? (think protein)
compact, locally folded region of 3-D structure with a specific function
what is an important difference between secondary structure and tertiary structure?
secondary structure is hydrogen bonding while tertiary structure is hydrophobic bc of the 3D structure
what are the three landmarks of quaternary structure
a. only relevant to multi-subunit proteins; refers to relative arrangement of the subunits
b. subunits can be identical or not
c. several types of symmetry exist with regard to the arrangement of subunits
symmetry is a landmark to what protein structure
QUATERNAry
describe the types of symmetry in proteins
cyclic: single axis of symmetry
dihedral: two types of rotational symmetry axes
tetrahedral
octahedral
icosahedral –> covid 19 also protein ferritin
describe the virus assembly from subunits
180 identical subunits with 3 slightly different conformations in a icosahedron formation
describe the formation of active sites on proteins
Different colors highlight specific amino acids that contribute to the formation of the active site. These residues might interact with a substrate or participate directly in catalysis with the enzyme .
is the 3D shape of the protein important
YES–> very important to the function of the protein
describe the reaction where we found out that protein folding is very important for the protein function
Native state (top):
–> The protein is in its folded, catalytically active form.
Disulfide bonds between specific cysteine residues stabilize the structure.
Denaturation (middle):
–> Addition of urea and mercaptoethanol breaks non-covalent interactions and reduces disulfide bonds to free sulfhydryl (–SH) groups, leading to an unfolded, inactive state.
Renaturation (bottom):
–> Removal of urea and mercaptoethanol allows the protein to refold spontaneously.
Correct reformation of disulfide bonds restores the native, catalytically active structure, demonstrating the role of primary sequence in guiding proper folding.
can we renature proteins after denaturation?
most of the time no, must be a specific protein like ribonuclease
describe Sequential stabilization of intermediates
some 3D folding structures may have better free energy values than other folding structures
death by misfolding…. what is this referring too
an incorrectly folded protein can be deadly and even cause disease like alzeihmers –> if chunk of protein is cut then it causes the protein to re-fold which can cause MAJOR problems
describe the molecular weight of a free amino acid vs amino acid residue in protein
the free amino acid is heavier by 18 Da which is the mw of water
the mw of the amino acid in the protein is the free amino - 18 Da
amino acid residue masses are ___ mass
units (Da) less than the free amino acid
18
each peptide bond eliminates one
H2O
the molecular weight of a peptide =
sum of all residue masses + 18 Da
OR
sum of all free AA masses - (NAA-1) * 18 Da
average residue mass of all 20 AAs is
118.9 Da
for a protein of average composition,
average residue mass is
≈ 110 Da, because
some amino acids are observed more
frequently than others in proteins
To estimate the mass of a 150 AA protein…
150 x 110 = 16,500 Da = 16.5 kDa (kiloDaltons)
The isoelectric point (pI) is
the pH at which a particular molecule
or surface carries no net electrical charge
At pH above pI…1
, proteins or amino acid carry a net negative charge
At pH below pI…
proteins or amino acids carry a net positive charge
what types of amino acids absorb UV light at higher wavelengths than other amino acids
aromatic amino acids
which specific amino acids absorb UV light at higher wavelengths than other amino acids
Tryptophan and Tyrosine
does Phenylalanine absorb UV light
YES but not as useful as Tryptophan and tyrosinde in determining the concentration or absorption
what is the lambert Beer law
log Io/I= Abs= Ecl
what is E in the lambert beer law equation
extinction coefficient is the optical density of a material at a given concentration,
what is the l in the lambert beer law equation
pathway length of the cuvette
absorption should bee….
less than one
what is Etotal=
(number of proteins)(extinction coefficient) +(number of proteins)(extinction coefficient)…..
what is the unit of concentration
M
Favored conformation of a peptide bond is….
the sequential alpha carbons in the TRANS position
peptide bonds tend to form…
a planar structure
water is released during the formation of ….
peptides bonds
at equilibrium, the peptide bond forming reaction favors …
hydrolysis
Which of the following best describes the state of a
protein whose pI = 9, when in a solution whose pH =
7?
C. The protein will have a net positive charge.
steps in the purification of a typical soluble protein
1.homogenization
2. centrifugation
3. salting in and salting out
4. column chromatography
what is homogenization
is a step in the process of preparing a cell-free extract (CFE). The process involves disrupting cells to release their contents, and then removing the cell debris and other insoluble components
what is centrifugation
to remove membrane, nuclei , large organelles (mitochondira)
cell debris is usually at the bottom
what is salting in/out
Salting in:
The solubility of a substance increases when salt is added to a solution. This happens because the ions in the solution shield the substance molecules from each other’s charge.
Salting out:
The solubility of a substance decreases when salt is added to a solution. This happens when the salt and water compete for the substance
describe how salting out can help with purification
Salting out can be used to separate proteins by precipitating them out of solution. This is because the solubility of proteins decreases at high salt concentrations
what are the main types of chromotography
-ion-exchange
- gel filtration
- affinity
what is ion exchange chromatography based on
uses charge to separate molecules
describe which rate at which molecules would move in an ion exchange
- non charged ions move first bc they are not attracted to the bead
- the most charged ions will move the least bc they are highly attracted to the bead
what is gel filtration based off of
separated based off of a molecules size bc the beads have cracks
describe the rate of movement for molecules in gel filtration
the small molecules get stuck in the cracks so they move less
the larger molecules do not get stuck in the cracks so they move more
describe the affinity column
a ligand is on the bead that has a tag for a specific protein. The target protein binds to the ligand while the rest of the proteins filter out
–> then the target protein is eluted from the beads using a compound that is more attracted to the ligand the protein
how does one monitor protein purification??
SDS-PAGE
what factor does SDS page separate molecules
based on molecular weight
SDS- page is a type of
GELL
about how many SDS binds per _ amino acid
about 1 SDS binds per 2 amino acids
in the elecvtric field of a gel the proteins migrate to the
Positive (anode ) side
in a sd sgel describe the rate of movement based on molecule size
large proteins are retained in the continous gel and move slower, small proteins move faster
Facts about GEL
- proteins can be visualized by gel
- if protein is pure, we should only see one band
what can we estimate using gel if we know the molecular weight
By using proteins of known molecular weight, we can estimate the molecular weight of our protein
describe the way the SDS surrounds the protein
the negatively charged molecules fully surround the protein so they unfold it HOWEVer they cannot break disulfide bonds
what do we need to add to a gel to break disfulfide bonds
mercaptoethanol
what is the equation for specific activity
units/ mg
imagine two beakers
beaker a: has 10,000 proteins –> 7 different types
beaker B: has 1000 proteins–> 7 different types
which has the higher specific activity and activity
Both beakers have the same activity through B has a higher specific activity as there are less mgs of proteins
in a gel, the intensity of the band can tell us about
the concentration of the reaction
why do we want to identify proteins
proteins associated with cancer and other disease states, protein modification and interactions
What are the typical approaches to protein identification
- 2-D gel electrophoresis
- digest protein of interest with protease
- determine masses of resulting peptides match masses to parent protein
what is the first dimension of 2D Gel electrophoresis
stops protein migration at the proteins pI
what is the second dimension of 2D Gel electrophoresis
separation by molecular weight–> the smaller/lighter the molecule the further down it travels
what are the 5 steps to sequencing proteins
- break and block disulfide bonds
- cleave proteins into smaller peptides
- separate the peptides
- sequence the peptides
- align the peptide sequences
describe the first step of protein sequencing –> breaking and blocking disulfide bonds
reduction and alkylation
1) reduction: break disulfide bonds in the proteins (using 2-Mercaptoethanol)
2) blocking step aka alkylation: where we introduce simething that covalently binds to prevent the formation of disulfide bonds
describe the cleavage step in protein sequencing
enzymatic cleavage using protease enzymes –> cuts peptide bonds at SPECIFIC locations
describe what a reagent is? (think protein sequencing)
specific compound that you can use to preform selective cleavage
ex: trypsin’s specificity is that it cuts at lys, and arg c termnius
describe the separation and purification of proteins (in protein sequencing)
USE HPCL
–> uses reverse phase columns for separating peptides ; binding of protein to bead is VIA HYDROPHOBIC interaction, not charge
–> fragments that are least hydrophobic come out first –> need something to elude hydrophobic molecules so you need to increase solvents
what is HPLC
high preformance liquid chromatography
–> a form a liquid chromatography but smaller column size, smaller beads inside column and higher pressures
What are the methods that we can use to sequence a polypeptide
a) Edman degradation
b) MS or tandem mass spectrometry
describe Edman Degredation
a chemical reaction that is executed in a series of steps and labels and removes one N-terminal amino acids at a time from the amino end of a peptide
what is the primary reagent used to sequence proteins during the edman degredation & what type of bond does it form
phenyl isothiocyanate–> forms covalent bond with the released amino acid
describe sequencing a polypeptide via MS/MS
The separated peptides are introduced into the mass spectrometer and ionized using a technique like electrospray ionization (ESI) to create charged ions.
The mass spectrometer measures the mass-to-charge ratio (m/z) of each peptide ion, allowing for selection of a specific peptide for further fragmentation.
The selected peptide ion is then fragmented in a collision cell by colliding with inert gas molecules, causing peptide bonds to break at various sites, generating a set of smaller fragment ions.
The resulting fragment ions are analyzed by the mass spectrometer to generate a tandem mass spectrum, which shows the masses of all the fragment ions produced from the selected peptide
what type of molecule will hit the detector in MS/MS
uncharged molecules never hit the detector
Smaller molecules hit detector first
what is the last step in protein sequencing
aligning the peptides to establish final sequence –> placing the sequencing in the correct order
myoglobin has what type of structure
tertiary
hemoglobin has what type of structure
quaternary
how many heme groups do myoglobin and hemoglobin have
myoglobin= 1
hemoglobin=4
what is so special about the heme group
it is the part of both hemoglobin and myoglobin that has an iron and allows the oxygenic bond
what type of symmetry does heme group have & why is it important
octahedral symmetry allows the oxygen to bind and leave
what are the similarities between hemoglobin and myoglobin
1) polypeptide length and sequences are similar
2) presence of heme, mechanism of Oxygen binding
what are the differences between hemoglobin and myoglobin LOCATION
location:
Mb= muscle
HB= blood
what are the differences between hemoglobin and myoglobin subunits
Mb= 1
HB= 4
what are the differences between hemoglobin and myoglobin FUNCTION
MB: O2 reserve
HB: O2 transport
what are the differences between hemoglobin and myoglobin O2 SOURCE
MB: blood
HB: lung
what are the differences between hemoglobin and myoglobin O2 DESTINATION
MB: mitochondria
HB: periphery
what are the differences between hemoglobin and myoglobin BINDING
MB: hyperbolic
HB: sigmoidial ( needs to be very effective)
these words refer to the graph’s of the O2 binding btw
when thinking about O2 binding curves what is Y equivalent too
Y= bound MB/ total MB
y= [O2]/ kd + [O2]
y=pO2/p50+pO2
when deos Kd= [O2] 0.5
when [Mb]= [Mb:O2 ]
what equation describes a hyperbolic curve
y= x/(x+z)
Buffering of O2 equation
MbO2 –> <—Mb + O2
what are thinks to keep in mind for HB’s O2 binding curve
- Hb needs to pick up O 2 efficiently at the lung where pO 2 is high
- Hb needs to drop off O 2 efficiently in the tissues where pO 2 is somewhat lower (30 mmHg = 4 kPa).
- Normal or hyperbolic binding won’t suffice; not enough difference in saturation when pO 2 drops to
only 1/3 the higher value.
What binding suits a transport
protein well –> like HB
Sigmoidal (cooperative)
describe what creates the Sigmoidal binding of HB
Change in conformation of heme as Hb
goes from T (tense) -> R (relaxed) state
The tense state in HB has a
lower affinity for O2, deoxy state
meaning usually doesnt carry oxygen
the Relaxed state for HB has a
higher affinity for O2, oxy state
usually carries oxygen
how do we go from a tense to a relaxed state HB
heme group becomes more planar–> conformation change in R group moves Val 68 away
Hb is an allosteric protein whose properties are affected by changes….
in structure, which are mediated by interactions with small molecules like O2 (causes change from T- R state)
What are the models of cooperativity
a) A simple 2- subunit model
b) 4-subunit model
what are the types of 4 subunit models
1) concerted model
2) sequential model
describe the 2- subunit model of cooperative ligand binding
model used to explain how a protein with two subunits can bind ligands cooperatively, meaning that the binding of a ligand to one subunit increases the affinity of the other subunit for the same ligand, leading to a sigmoidal binding curve where ligand binding occurs more readily once the first ligand is bound
–> STill don’t understand that well
describe concerted model for the cooperative transition of HB subunits
model where all subunits of a protein molecule simultaneously switch between low and high affinity states upon ligand binding, meaning that when one ligand binds, it triggers a conformational change affecting all other binding sites at once, essentially acting as an “all-or-none” transition
*** ALL OR NONE
describe sequential model for the cooperative transition of HB subunits
a model where the binding of a ligand to one site on a protein molecule induces a conformational change that increases the affinity of neighboring binding sites for the same ligand, leading to a sequential increase in binding as more ligands attach, resulting in a cooperative binding effect
what is the Bohr effect
the higher the concentration of CO2 (LOWER PH) the easier it is to transfer oxygen from hemoglobin to a tissue
**essentialy the affinity of hemoglobin for oxygen depends on the pH environment as the concentration of co2
in the bohr effect what are the allosteric effectors
H+ and Co2 because they both bind to HB and stabilize the T state
enzymed are _____ by the reaction
UNCHANGED–> hence can be reused
enzymes are ______ ______ and _____ of metabolic reactions
selective catalysis and regulation
what do some enzymes require to work
cofactors (small organic molecules and or metals such as Ca, Zn, Cu, or Fe)`
most enzymes are ____ but some are _____
most enzymes are PROTEINS but some can be RNAs
what is an example of extreme catalytic power
catalase
what is delta G
overall rxn free energy change
what does delta G determine
determines where equilibrium lies
what does delta G T mean?
transition state free energy aka Activation energy
what does Delta G T determine
determines the rate at which equilibrium is achieved
how does an enzyme affect delta G
do not alder the equilibrium of delta G
how does enzymes affect delta G T
they accelerate the reaction by decreasing Delta G T (activation eneryg)w
what is another word for Delta G T
activation energy
essentially–> what do enzymes do
they stabilize the transition state
Describe the LOCK AND KEY model of enzyme substrate interaction
lock and key model” describes an enzyme’s active site as a rigid structure that perfectly matches the shape of a specific substrate, like a key fitting into a lock
Describe the INDUCED model of enzyme substrate interaction
the active site of an enzyme changes shape slightly upon substrate binding to better accommodate it, essentially “molding” around the substrate to create a better fit
what is the difference between an induced model and a lock and key model (for enzymes)
key difference is that the lock and key model assumes a static active site, while the induced fit model allows for conformational changes in the enzyme upon substrate binding
describe the concept of stickase enzyme
the concept that enzymes are designed to best fit and stabilize the transition state of a reaction, rather than the initial substrate–> this is because if tje enzyme perfectly fit the reactant and not the intermediate it would stay in that conformation as it is very stable
What are the factors contributing to rate enhancement by Enzymes
- concentration
- orientation
- strain
- chemical catalysis
describe how concentration will effect the rate of an enzyme
“effective molarity in the active site- proximity effect”
–»>the more enzyme molecules present, the more active sites are available to bind with substrate molecules, leading to a higher rate of reaction and increased enzyme activity–> also the PROXIMITY EFFECT
what is the proximity effect
Enhancing the proximity of reactants increases their collision frequency, thus causing the reaction to proceed at a faster rate
describe how orientation can affect rate enhancement by enzymes
orientation increases the probability of correct bond or orbital alignment
describe how STRAIN can affect rate enhancement by enzymes
–> weakening of bonds by distortion–> essentially the stickase model
refers to the physical distortion or stress applied to a substrate molecule when it binds to an enzyme’s active site, which can significantly increase the rate of a chemical reaction by stabilizing the transition state, effectively lowering the activation energy needed for the reaction to occur; essentially, the enzyme “strains” the substrate molecule to make it more reactive.
describe how chemical catalysis can affect rate enhancement in enzymes
lowering the activation energy of a chemical reaction, essentially creating an alternative pathway that requires less energy for the reaction to proceed, thereby allowing the enzyme to significantly speed up the process without being consumed itself
what are the major types of chemical catalysis
acid-base, covalent, metal ions
A given enzyme may use several types of ______ ____ in its mechanism
chemical catalysis
what is a major factor for most enzyme’s rate
chemical catalysis
enzymes are HIGHLY …
specific
True or false:
Enzyme catalysts bind to substrates but are never covalently attached to substrate or product.
False
True or False:
Enzyme catalysis increases the equilibrium constant for a reaction, thus favoring product formation
FALS–> remember its doesnt change Delta G
True or false:
Enzyme catalysts increase the stability of the product of a desired
reaction by allowing ionizations, resonance, and
isomerizations not normally available to substrates.
FALSE- they stabilize the transition state
TRUE OR FALSE:
Enzyme catalysts lower the activation energy for the conversion of substrate to product
TRUE
True or false:
To be effect enzyme catalysts must be present at the same concentration as their substrates
FALSE
Enzyme reactions have at least ____ steps
TWO
describe the first step of an enzyme reaction
binding step rapid and reversible
describe the second step of enzyme reactions
catalytic step slower and irrevisible (often)
E+S means
FREE enzyme and substrate compound
ES means
enzyme and substrate complex (binded together)
what are the assumptions for the steady state kinetics
1) [ES] remains constant over time
2) presteady state, the build up of the ES is complex- is microseconds
3) Usually have nM enzyme, mM substrate in reaction
what is the physical meaning of K2
of reactions a single enzyme molecule can catalyze per unit time
Therefore, 1/k2 is:
Time for conversion of 1 molecule S → P
what is the physical meaning of Km
- a measure of how tightly an enzyme binds its substrate
what is the definition of Km in terms of S
it is the value of S at which half of the enzyme molecule have their active sites occupied with S
Lower Km means
more effect binding–> higher affinity
LOOK AT LECTURE 8 equations and graphs –> didnt include in this
what is enzyme efficiency
efficiency= kcat/Km
In a cell, [S] for most reactions is most likely:
A. Very low – don’t want too much S –> P
B. Saturating – maximal reaction rate
C. Around Km
D. Irrelevant since DG0 doesn’t change
E. Very high to minimize activation energy
C Around Km
what is the difference between reversible and irreversible inhibition
Reversible inhibitors bind to enzymes in a way that can be reversed, while irreversible inhibitors bind in a way that is permanent
how can you recognize competitive inhibition on graph
V max remains unchanged, km increases with increases [I]
what is competitive inhibition
an inhibitor that resembles the normal substrate binds to the enzyme, usually at the active site, and prevents the substrate from binding
what is alpha in terms of competitive inhibition
alpha= 1+ [I]/ki
what is Ki in terms of competitive inhibition
ki= [E][I]/[EI]
what are the modes of enzyme regulation
1) allosteric control/regulation
2) covalent modification
what are the types of allosteric control/regulation
1) homotropic allostery
2) heterotropic allostery
what is homotropic allostery
binds in the active site and only occurs with multisubunit proteins
what is heterotropic allostery
bind in a different shape
what are the types of covalent modification
1) group addition
2) proteolysis
describe group addition
often reversible phosphorylation, and many other types
describe proteolysis
irrevesible
describe why we need regulation of enzyme activity
It would be wasteful to continue to turn substrate into product if
enough is available for proper cellular function. Therefore, enzymes
often are highly regulated by binding small molecules that can either
decrease or increase activity
describe heterotropic allosteric activation in depth
The enzyme consists of a catalytic (C) subunit, which binds the substrate, and a regulatory (R) subunit, which responds to modulators. In its less-active state, the enzyme has low substrate affinity. When a positive modulator (M) binds to the regulatory subunit, it induces a conformational change, shifting the enzyme to a more active form with higher substrate affinity. This allows efficient substrate binding, forming an active enzyme-substrate complex and increasing reaction efficiency. Such allosteric regulation is crucial in metabolic pathways, ensuring precise enzymatic control based on cellular conditions.
..
describe the effect of inhibitor or activation on Vo vs [S] plot
activator with decreas Km
inhibitor with increase Km
most allosteric enzymes are…
multi-subunit enzyme
subunit structure and mechanism of activation can lead to ____; also what is an example
post translational modification ; example: Cyclic: AMP
what is an example of phosphorylations reversible covalent modification
ATP to ADP and vice versa–> Phosphorylation is reversible
and is used in many
pathways to control
activity. Enzymes that add a
phosphate to a hydroxyl side
chain are commonly called
kinases. Enzymes that
remove a phosphate from a
phosphorylated side chain
are called phosphatases.
What are the defining features of lipids?
1) major portion of lipid is hydrophobic due to many Ch2 groups
2) minor portion is often hydrophylic
3) do not exist as large polymers (unlike nucleic acids, proteins and polysaccharides)
what are the major functions of lipids
1) energy storage
2) membranes
3) specialized roles
lipids for energy storage in animals is stored in
fat `
lipids for energy storage in plants is stored as
oils
what are some examples of specialized roles of lipids
hormones, vitamins, signaling molecules
triacylglycerols=
triglycerides - fats
glycerol is acylated to form
an ester of a fatty acid
free fatty acids in bloods are mostly bound to
serum albumin, an abundant blood protein
KNOW fatty acid nomenclature
which FAs are found in triglycerides
-up to 3 different FAs in a given triglyceride
-mix of unsaturated and saturated FAs
- FA structure determined melting point
difference between saturated and unsaturated fatty acids
saturated fats don’t have alkenes –> so they don’t have kinks
Fatty acids have kinks
which type of fatty acids can stack
saturated acids and they can’t have other compounds in between them
tay saches disease is cause by
impaired degradation of brain gangliosides
what are the types of membrane lipids
Glycerophospholipids
sphingolipids
describe glycerophospholipids
Glycerol Backbone: The molecule is derived from glycerol, with two fatty acids (FAs) and a phosphate group attached.
Fatty Acid Chains (Apolar Region, Pink): These can be saturated or unsaturated, typically C16 or C18 in length. They are hydrophobic, forming the apolar (nonpolar) tails.
Phosphate Group (Polar Region, Blue): The phosphate group, along with an attached head group, is hydrophilic, forming the polar head.
describe spingolipids
Sphingosine Backbone (Blue Region): Unlike glycerophospholipids, sphingolipids are based on sphingosine, a long-chain amino alcohol.
Fatty Acid Tail: Attached via an amide bond to the sphingosine backbone, forming ceramide when R = H.
Head Group (R Group): Determines the specific type of sphingolipid.
what is the name of the R group -H
ceramide
what is the name for the r group phosphocholine
sphingomyelin
what is the name for the r group monosaccharides
cerebrosides
what is the name of the r group for oligosaccharides
gangliosides
types of glycerophospholipids
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Cholesterol is a
STEROL
cholesterol is common
in animal membranes
cholesterol is a precursor to
several steroid hormones
describe the composition of RBC membranes
49% protein
carbohydrates 8%
Lipid 43%
–> for lipid
- glyceophospholipids 48%
-sphingolipids 27%
-cholesterol 25%
how does cholesterol change for transport and storage
hydroxyl group condenses with a fatty acid to form a sterol ester
amphipathic lipids types of aggregations
micelle
bolayer
liposome
what is inside the liposome
the cell material
inside is like a cavity
why is cholesterol so important in the phosphobilipid layer
helps keep fluidity of layer at cold or wharm temperatures
what are some of the phase transitions in the membrane
–> At physiological temperature,
bilayer exists as liquid crystal –
but it is still highly organized by
allows for embedded proteins and
other molecules such as sterols.
–>At a specific temperature, chains
are largely frozen and the bilayer
is said to exist as a gel.
many carbohydrates have more complex ..
formulas and contain amino, phosphate, sulfate and other groups
what are some functions of carbohydrates
1) fundemental source of metabolic energy for most life forms
2) components of many important biomolecules
carbohydrates are often…
polymers
what are some example of polymers
monosaccharides, oligocaccharides, polysaccharides
what are some monosaccharides
glucose, ribose, fructose
sucrose is a ….
disaccharide: glucose + fructose
polysaccharides can be
linear or branched
asymmetric is another word for
chiral
chiral carbons allow
many distinct monosaccharides
mannose, glucose, and galactose are all
aldohexoses –> are monosaccharides with six carbon atoms and an aldehyde functional group (-CHO) at one end of the carbon chain.
diastereomers
identical structures except for configuration at one or more carbons
all aldohexoses are …
diastereomers of each other
epimers…
differ in chirality at only one carbon
glucose and galactose are …
epimers at carbon 4
enantiomers are
mirror images
what is designated as D and L
enantiomers
most pentoses and hexoses …
spontaneously form ring cyclized structures in solution
what is a 5 membered ring called
furanose
what is a 6 membered ring called
pyranose
when forming a ring, a new….
asymmetric center is created giving rise to 2 possible anomers–> alpha and beta anomers (think front and backside attack)
in solution rapid ______ occurs
mutarotation–>
what is mutarotation
the process by which a substance changes the way it rotates polarized light
where are disaccharides mainly found
mainly found in plants
what are three common disaccharides
sucrose, lactose, maltose
2 monosaccharides are joined ..
covalently by an O-glycosidic bond
Common disaccharides are produced by
enzyme- catalyzed condensation/dehydration reactions
ploysaccharides are also known as
glycans
what is the difference between homopolysaccharides and
heteropolysaccharides
A homopolysaccharide is classified as a chain that contains only one type of monosaccharide unit, whereas a heteropolysaccharide contains two or more types of monosaccharide units
polysaccharides have what kind of branching
can be branched or unbranched
what is the main use of polysaccharides
used by animals and plants as a compact strorage form of CH2Os
what are common examples of polysaccharides q
starch, glycogen, cellulose
starch is made up of
amylose and amylopectin
amylopectin is made of
a branched for of amylose
glycogen is like
starch but more branched
what is the structure of starch
branched (amylopectin) and unbranched (amylose)
amylose is what type of chain
a linear chain
amylopectin is what type of chain
branched chain
what is protein glycosylation
a post-translational modification process in which sugar molecules (glycans) are attached to specific amino acid residues on proteins
sugars frequently compromise …
50% or
more of the total molecular weight of a
glycoprotein
Most glycosylated proteins are either
secreted or remain membrane-
bound
what is the most abundant dorm of post translational modification
glycosylation
what is important in cell to cell recognition
protein glycosylation
Glycosylation confers resistance to
protease digestion by steric protection
describe how blood groups relate to carbohydrates
the difference between the blood types can be conributed to the differnt sugars that are attached
The O substance is a
tetrasaccharide which is
missing the 5th residue
(monoscacharide) and does not
elicit an antibody response
(non-antigenic)
The A antigen and B antigen
are
pentasaccharides which
differ in composition of the 5th
sugar residue
what is hemiacetyl
alcohol + aldehyde
Creates a molecule where there is one carbon between two oxygens where one of the oxygens is attached to a hydorgen and the other carbon is attached to a r group
what is the difference between acetal and hemiacetyl
acetal oxygens will both be attached to R groups
Hemiacetyl has one oxygen attached to a hydorgen and one oxygen attached to R group
hemiacetyl gives what types of abilities
hemiacetal allows for mutarotation (interconversion between α and β anomers)
starch has what types of ends and what do they mean
nonreducing ends: Multiple ends available for enzymatic activity during starch hydrolysis.
reducing ends: The single end with a free aldehyde group
what is the difference between glucose, sucrose, maltose, and lactose
lactose and sucrose don’t have acetyl or hemiacetyl
glucose has hemiacetyl
maltose has acetyl
everything that can be acetyl or hemiacetyl can have
MUTAROTATIon think optical rotation from ochem
how to convert a hemiacetyl to an acetyl
add R-OH, you will get an acetyl and H20
What does the OG glucose look like
4 chiral carbons –> 2nd chiral compound has OH on the left all the other carbons have OH on the right
Why is sucrose a nonreducable sugar?
NO HEMIACETAL AT THE ENDS, CANNOT ADD ANYMORE
What is not present in nonreducing ends?
hemiacetal groups
a lower P50 value means
higher oxygen affinity
–> less oxygen released
a higher P50 value means
lower oxygen affinity
–> more oxygen released
increased Co2 caused the ph to
DECREASE
increase in H+ causes the PH to
decrease
o-glycosidic bonds form
through condensation reactions –> hemicetal + Oh-R to make acetal
mannose and galactose are …
epimers of glucose
HOW is chymotrypsin specific
Substrate Binding Pocket: Chymotrypsin has a deep, hydrophobic binding pocket that selectively accommodates large, nonpolar, and aromatic residues like phenylalanine, tyrosine, and tryptophan. This structural feature ensures specificity for peptides containing these residues.
Catalytic Triad: The enzyme’s active site contains a catalytic triad (Ser195, His57, and Asp102) that precisely orients and activates the substrate for hydrolysis. This arrangement ensures that only peptides with suitable cleavage sites undergo efficient catalysis.
Oxyanion: During catalysis, the oxyanion hole, formed by the backbone amides of Gly193 and Ser195, stabilizes the negatively charged oxygen of the tetrahedral intermediate through hydrogen bonding. This stabilization lowers the activation energy of the reaction and ensures efficient cleavage of correctly positioned substrates
What does Fe2+ bind to in hemoglobin?
an O2 molecule and His 98 (amino acid atom)
In hemoglobin, what is the role of Val68? The role of His98?
Val68: cause of the steric hindrance present in the T-form
His98: stabilizes the hemoglobin molecule, keeps the Fe in the heme group
When do you use affinity chromatography?
same charge, same MW, and/or 6-His tag
