Biochemistry Test 1 Review

9/21/2014 12:14:00 PM

Bonds
Covalent
Share electrons (single, double, etc..)
VERY strong (~400 kJ/mol)
Non-Covalent
Relatively weak
Ionic/electrostatic
o E=kq1q2/Dr
o D = dielectric constant
A molecules ability to stabilize charge or a solvents

polarity
The higher, the more polar and higher the ability
stabilize charges
o ~1/5 Covalent bond strength
Hydrogen bonds
o Polarity: ability to separate charges
o Between O, N, C or H
o Hydrocarbons are apolar
o 1 HB ~ 20 kJ/mol

Van der Waals

o Repulsive when near, attractive when far
o Effective only in large quantities
Cell volume: ~30% bio-matter, 70% water
Molarity of pure water: 55.5 M
Water
Polar molecule
o EN O: 3.5/ EN H: 2.1
Electric Dipole

Quadrapole resulting from the two lone pairs on O, leading to 4

potential hydrogen bonds
Lone pairs are bond acceptors, and hydrogens are bond donors
When it forms water, it loses 15% of HBs from ice phase
Hydrophobic Effect
Water forms a cage around apolar molecules, leading to limited
number of orientations around the molecule low degeneracy
o VERY unfavorable

The effect occurs when non-polar molecules minimize solute-water

interactions by interacting w/ each other, releasing water molecules
to form more orientations, allowing higher degeneracy
Accessible surface area is drastically reduced in the second
conformation of apolar molecules
Gibbs Free Energy
G = Gibbs Free Energy
H = Enthalpy
S = Entropy
G<0, H<0 S>0 favorable
Vice versa
G = H TS
G = Gbefore G after
G0 = equilibrium free energy
Hydrophobic Effect
Water around apolar molecules is ice-like, more HB
Enthalpy favors the unbound state as there is less kinetic energy in
the system, leading to a lower heat filled environment
S favors the bound state as there is a lot more degeneracy in the
system
The favored H is much smaller than the favored S, leading to a
negative G, leading to B being the more favored state
Thermodynamics
In a reaction, A B, G = G0 + RT ln ([B]/[A])
G > 0: Endergonic (Not Spontaneous)
G < 0: Exergonic (Spontaneous)
Rate
Rate: Kb * Cb, where k = speed of apple collection and c =
concentration of apples
Generically, k = the rate constant of the reaction that controls the
pace of the forward or backward reaction. The higher the
concentration ,the higher the rate
Equilibrium
The forward and backward reaction rates equal each other
K reactant/K product = Keq
C product/ C reactant = Keq

Main Reaction + Manipulations

Delta G = Delta G0 + RT ln ([B]/[A])
At equilibrium, you get the definition of delta G0 by setting delta G
= 0 to get Delta G0 = -RTln([B]/[A])
With multiple reactions and products, you have: delta G = delta G0
+ RT*ln ([B][D]/[A][C])
Hydrophobic Effect is Enthalpically unfavorable and entropically favorable
Viruses and pH
The viruses have proton binding groups in their coats. When these
bind the ligand, they separate, releasing the viral ingredients to be
processed and released by the cell.
pH and water
pH + pOH = 14
pH = -log [H+]; same for pOH
Henderson-Hasselbach Equation
pH = pK + log ([Base]/[Acid])
Proteins
Definition: Long chain of linear polymers of amino acids
Central Dogma: DNA -> Protein (through transcription and
translation) -> Folded Protein
Amino Acids
Building blocks
Primary Structure with Amino terminus (N-terminus) and Carboxyl
terminus (C-terminus)
Usually arranged NC terminus
Amino acids as Zwitterions
Zwitterion a neutral molecule with, both, positive and negative
charges

As the pH changes, the amino acid backbone is deprotonated,

leading to increasingly negative charges.
Below pH 2, the backbone is positively charged, due to the
neutrality of the carboxylic acid being protonated and the extra
hydrogen on the amine group
After being deprotonated at pH 2 and above, the negative charge
from the carboxylic acid neutralizes the positive from the amine
group, rendering the backbone neutral overall

Above pH 9, the amine group is also deprotonated of its positive

proton, leaving it neutral and the entire molecule is made negative
due to the charge from the deprotonated carboxyl group
to remember
Acid/protonated positive form dominates at low pH (pH < pKa)
pKa = pH where [Acid] = [Base]
Every pH unit change changes the base/acid ratio ten-fold
Exact neutrality of the backbone is at 5.5

3 Points

Chirality
All amino acids are chiral, except glycine which has 2 hydrogen
atoms attached to the alpha Carbon
L isomers are made, D isomers are not
R-Groups of the amino acids are the key differentiating factor
Proline is an imino acid, containing a ring bonded to the backbone
Found a lot in turns
Cysteine contains a sulf-hydryl group attached to the backbone
Found in abundance in secreted proteins
Forms disulfide bridges through release of 2 H+ and 2 eo Oxidation

These disulfide bonds are both intra- and interchain bonds

They exist to stabilize secreted proteins
Found a lot in insulin where A and B chains are connected by
disulfide bonds
Aromatic amino acids
Forces acting on these compounds are usually the hydrophobic
effect or Van der Waals Forces
Three are Phenylalanine, Tyrosine, or Tryptophan
Proteins are sequences of Amino acids (Primary structure)

Peptide

The C-terminus of amino acid i connects to the N-terminus of i+1

Layout is usually N terminus to C terminus
bond
Found between carboxyl carbon and the nitrogen of amino acid i+1
Peptide bonds are formed through dehydration synthesis
Not-rotatable bonds that force local planarity
As a consequence of planarity, the carboxyl oxygen and the amino
hydrogen face opposite directions

Carboxyl oxygen is a hydrogen bond acceptor, while the amide

plane is a hydrogen bond donor
There are three bonds in the backbone per amino acids, but only
two of them are rotatable. 2 rotatable = bonds on either sides of
the R-group and the not-rotatable is the peptide bond
Hierarchical structure of proteins
Primary structure is the amino acid sequence
Secondary structure is the alpha/beta structure
Tertiary structure is the fold
Quaternary is the combination of folds
Secondary structure
Direct result of rotatable backbone angles
Result in alpha-helices, beta sheets, and turns
Phi/Psi angles
o Are the result of turning of bonds
o Phi angles rotation of the bond between the alpha carbon
and the nitrogen
o Psi angles rotation of the bond between the alpha carbon
and the carboxyl carbon

Many combinations of these phi-psi angles produced disallowed

collisions; in correcting the correct spacing to minimize collisions,
we get the secondary structures
Ramachandran Plots
Phi on the x-axis; psi on the y-axis
Bottom left quadrant contained the right-handed alpha helices,
while the top right quadrant contained very rare left-handed alpha
helices
White areas of the graph showed collisions which were not allowed

For glycine, there are two vertical strips shown on the plots,
revealing the interactions between identical hydrogen groups
Alpha Helix
Basic unit of structure that appeared with the correct phi-psi angles
There are 5.4 angstroms between each turn in the helix
There are 3.6 amino acids/turn
The carboxyl oxygen of amino acid i hydrogen bonded w/ the amino
hydrogen of amino acid i+4

All the hydrogen bond donors (NH bonds) point backward (down)
into the helix
All hydrogen bond acceptors (OH bonds) point forward (up)
All the hydrogen bonds are satisfied
R groups always point away from the helix and are always i+4 units
away from their hydrogen bonded amino acids
Usually the building blocks of structural proteins (keratin and
collagen)
The disulfide bonds between helices determine the rigidity of the
bonds

Amphiphathic helices
The non-polar amino acids are grouped on one end, while the polar
amino acids are on the other end
Usually, the polar ends are oriented outward, towards the aqueous
media, while the non-polar molecules face inward, towards each
other
Leucine Zipper
There is a leucine amino acid at every 7th amino acid residue
The helix is actually 3.5 amino acids/turn instead of 3.6
This is an example of supercoiling
Beta Strands
Found on the top left portion of a Ramachandran plot
The secondary structure is usually elongated
The R groups are above and below the strand, while the backbone
is in a plane
Each same side R-group is 7 angstroms away
Beta Sheets
Formed by beta strands coagulating

Dominated by hydrogen bonding of carboxyl oxygen to amino

nitrogen
Antiparallel in structure where one strand is oriented NC while the
other is oriented CN to allow for hydrogen bonding
Parallel sheets also found, but are more rare due to lower stability
and stress of the hydrogen bonds
Inter-strand side chains sit right next to each other
Usually have a twist to them.

o The side chains interact w/ each other, causing twisting and

pleating, etc.
Turns/Loops
Usually are conducted through Proline or Glycine
They are necessary for globular proteins and join the secondary
elements
Tertiary Structure
Overall fold of protein
Packing or arrangement of Secondary structural elements
Forms an active site and grants functional relevance to protein of
Protein

Protein

interest
Stability
Conformation degeneracy a.k.a entropy
o Favors the unfolded (U) state
Hydrophobic effect
o Hydrophobic effect will favor the folded state as there is less
accessible surface area
Proteins are actually stabilized into their folded state despite the
lower degeneracy. There is approximately 20-80kJ of delta G.
Delta G = G (Hydrophobic effect) (Positive) + G (Conformational
Effect) (Negative)
The hydrophobic effect is rather large, overwhelming the
conformational entropy by a small portion
Folding
Apolar sides are, MOSTLY, on the inside, while the polar is ALWAYS
outside
Salt bridges stabilize the protein structure
o Amino acids that are distant in sequence are proximal in the

tertiary structure, causing charge stabilization

Different Protein Classes
Soluble Proteins function in water, including the hydrophobic effects
Integral Membrane Proteins (IMP)
Peripheral Membrane Proteins interact w/ IMPs of polar groups on
membrane
Membrane assistant proteins have lipid tails on proteins
Porins

o Apolar structures on the outsides, with polar sides inside ->

VERY different from regular proteins
o Beta barrels
Rhodopsins
o Are membrane spanning, helical proteins
o Compose the vast majority of proteins
Quaternary Structure
Spatial arrangements and interactions of multiple subunits
Their overall function is greater than the sum of the parts:
Cooperativity
Thermodynamics Hypothesis
Amino acid sequence specifies the structure
Demonstrated via the Anfinsen experiment
Usage of Beta-mercaptoethanol and urea
Beta-mercaptoethanol reduces the disulfide bonds, and urea binds
as a ligand to stabilize the unfolded form of the ribonuclease
When Urea and the Mercaptoethanol were removed, the protein
adopted a scrambled shape, with no activity
With trace amounts of mercaptoethanol, the protein was allowed to
form the correct structure again
Levinthals paradox
With the number of possible conformations that a protein could
adopt, it would take forever to arrive at the correct conformation if
they folded based on sampling conformations
This was solved by propsing hierarchical protein folding, suggesting
that there are intermediates for protein folding, which allow
sequential folding from local bondages
Prion Protein is the prototypical form

The first 120 amino acids become disordered, adopting beta strands
that interact with other molecules of PrP
Amyloids- coagulations of misfolded prion proteins
o Early culprit is the intermediate during hierarchical protein
formation that goes on to recruit other proteins for
misfolding; one vector is enough to cause chain reaction
because it acts as a nucleating effect
Isoelectric point

pH at which the molecule is neutrally charged

The average of the pKs of all the functional groups of the amino
acids in the peptide
Centrifugation
Place sample in tube and spin at very high speeds; all large
material remains in the pellet, while the smaller stuff remains in the
supernatant
Salting Out
Increase the salt concentration of a sample so that the POI remains
in the supernatant, but the contaminants are precipitated out
Dialysis
Use a semipermeable membrane to exchange LMW proteins; large
ones cannot pass through the membrane, while small ones can go
into the buffer
Can be used repetitively/iteratively
Chromatography
Separates proteins base on the physio-chemical properties
POI interacts weakly with beads, but contaminants interact strongly
Itll take longer for the contaminant to elute out, while the POI
arrives sooner, and this can be seen via absorbance vs. time graphs
Gel Filtration (Size exclusion) chromatography
Based on the size of proteins and so HMW proteins travel faster,
while LMW proteins are slowed down as they enter carbohydrate
polymer beads
VERY crude method and requires at least a 50% or more difference
in molecule size to be semi-effective
Ion Exchange Chromatography
Isolate based on charges

Use negatively charged beads to capture positively charged POIs

and elute from beads
Vice-versa
Affinity Chromatography
Based on a specific property of the POI a.k.a Binding site

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9/21/2014 12:14:00 PM