MARY KIMBERLY L.

ESPALDON

BIOCHEMISTRY FIRST SHIFTING

LEVELS OF ORGANIZATION OF PROTEINS
1. Primary level covalently linked together
Order/sequence of amino acids of the peptide chain
Linking of the amino acids by peptide bonds
Covalently linked together; one dimensional
Defines the 3D structure
2. Secondary level hydrogen-bonded arrangement of the
polypeptide chain
Each amino acid has TWO BONDS THAT SPECIFY
CONFORMATION:
Phi -carbon and amino nitrogen
Psi -carbon and carboxyl carbon
Two types that repeat at regular intervals: -helix, pleated sheet
-HELIX
Only one chain rod-like
Polypeptide chain form hydrogen bonds to itself
forming a helix
C-O hydrogen bonds to N-H group that is four
residues away
All side chains are outside the helix, including
the -carbon
Pitch vertical distance in one turn (5.4)
3.6 residues per turn (13 atoms)
Proline cannot be found causes a bend that
restricts rotation
1. Cyclic in nature
2. Absence of hydrogen bonding
Proximity of side chain causes electrostatic
repulsion strain on the helix
Proximity of bulky side chains causes steric
crowding strain on the helix
-PLEATED SHEET
Peptide bond is almost completely extended
Hydrogen bonding between one or two peptide
chains
Parallel or anti-parallel
1. Parallel C-terminal of the two chains
are going the same direction

2. Anti-parallel C-terminal of the two

chains are going on opposite directions
Irregularity: -bulge
Localized disruptions
Non-repetitive
Irregular motifs in the antiparallel
position
Glycine and proline causes reverse turns
due to steric spatial reasons
Supersecondary structures combination of helices and -pleated sheets
1. 2 parallel and a stretch of
2. unit 2 antiparallel
3. -meander series of tight reverse
turns
4. Greek key doubles back on itself
5. Domain independently folded
structures
6. Motif repeated secondary structures
3. Tertiary level 3D arrangement of ALL ATOMS in the
polypeptide chain
Determined by covalent (H-bonding & disulfide
linkages) and non-covalent interactions with the chain
Hydrogen bonding
Alcohols
Occurs between acids
Electrostatic attractions
and bases
Complete covalent
structure determines
Disulfide linkages
the position of the
disulfide bonds
Hemoglobin and
Metal-ion complexation
myogbloin
Aromatic molecules
complexation
(stacking)
4. Quaternary level
Same interactions occur but between two or more
polypeptide chains
Subunit each chain
Allosteric subtle changes in one site causes drastic
changes at a distant site

MARY KIMBERLY L. ESPALDON

protein are missing

reduces the protein)
PROTEIN CONFORMATION
FIBROUS
Shape
Solubility

Stability

Rod-like
Insoluble
high molecular
weight
Not affected
by change in
pH and
temperature

GLOBULAR
Spherical;
ellipsoid
Soluble
exposed polar
groups
Affected by pH
and
temperature

DISRUPTION OF PROTEIN ORGANIZATION

Proteins in nature have NATIVE CONFORMATION (folded
shape of proteins) biologically active
Ways of disrupting protein organization:
1. Denaturation
process by which a protein loses its natural
conformation by disruption of its structural order
be it quaternary, tertiary or secondary but never
primary
may be reversible or irreversible
Disrupts tertiary
Heat
structure (hydrophobic
interactions)
Sodium dodecyl
sulfate disrupts
Detergents
hydrophobic
interactions
Disrupts hydrogen
bonding (forms hUrea or guanidine
bonds with the protein
that is stronger than
the protein itself)
Reduces disulfide
Mercaptoethanol
bonds
Alter electrostatic
Large changes in pH
attractions (charges in

2. Hydrolysis destruction of primary proteins through

hydrolysis of peptide bonds
Renaturation uses CHAPERONES:
Assists in the folding of proteins
Alphahemoglobin stabilizing protein
prevents -chains from causing
damage to bloods cells and delivering
them to -chains
Post translational modification to fold
correctly
Prevents a protein from associating with
another protein which it should not
associate or keep it from associating
itself in inappropriate ways
Correct folding
Correctly folded proteins are usually
soluble in aqueous cells
Formation of aggregates (misfolded
proteins)
o Hydrophobic regions are
exposed and interact with other
hydrophobic regions
PROTEINS IN VERTEBRATES
1. STRUCTURAL both filamentous proteins
i. Collagen 800 residues
Water insoluble; most abundant in
proteins
Three polypeptide chain wrapped
around like a rope 3.3 residues per
turn
Distinctive peptide chains containing
proline or hydroxyproline (more stable)
Proline converted to hydroxproline by
HYDROXYLASE (catalyzed by vitamin
C)
Each chain containing a repeating
sequence of XPro/HypGly

MARY KIMBERLY L. ESPALDON

Glycine has to be inside every

third because its the only one
small enough to fit
Topocollagen
Triple helical
Crosslink increases with age
ii. Elastin
Found in ligaments and arterial blood
vessels
Non-repetitive coil
Rich in G, A, V, P but not in
hydroxylproline and hydroxylysine
Four lysine residues:
4 allysine residues
2 lysine and 2 allysine residues
Condense to form desmosine or
isodesmosine crosslink
rubbery characteristic
2. TRANSPORT
both allosteric and globular
homologous proteins (same amino acid
residues are in the same position)
bind oxygen reversibly
At any point, myoglobin will have a higher
saturation than oxygen
i. Hemoglobin
One molecule open to 4 molecular of
oxygen
Tetramer (2 -chains and 2 -chains
where the chains are identical)
wrapped around 1 heme
-chain 141 residues long
-chain 146 residues long
Complex of HEME and GLOBIN
Heme iron containing; made of
4 pyrrole rings that form one
porphyrin ring
Globin prevents oxidation of
Fe2+ to Fe3+ Hemoglobin will
not transport O2 in oxidative
state

Positive cooperation/cooperative binding

one oxygen molecule makes it easier
for the next one to bond
Binding curve: Sigmoidal
AT LOW PH, releases oxygen in high
presence of CO2
Affected by H+, CO2, and 2,3biphospoglycerate decreases O2
affinity to hemoglobin
6 coordination sites
1st 4th
Occupied by
nitrogen from
pyrrole rings
5th Histidine 8
Binds strongly to
(proximal
heme
histidine)
6th Histidine 7
Protects iron;
deflects binding
of CO to heme;
allow H+ bonding
of O2 to CO to
molecule
ii. Myglobin
Found mainly on the muscles
153 residues long
Composed of only 1 protein chain with a
heme in the center no -chains
Binds to one molecule of oxygen
Binding curve: hyperbolic
3. REGULATORY produced in the -cells on the Islets
of Langerhans (pancreas)
i. Insulin
2 polypeptide chains (51 amino acid
residues) linked by disulfide linkages
Hypoglycemic hormone
GLYCOLYSIS glucose pyruvate
ATP
GLYCOGENESIS glucose
glycogen
ii. Glucagon

MARY KIMBERLY L. ESPALDON

Single polypeptide chain (29 amino

acids)
Released when blood glucose levels are
below normal hyperglycemic
hormone
GLYCOGENOLYSIS glycogen
glucose
GLUCONEOGENESIS pyruvate
glucose
4. DEFENSE
i. Immunoglobulins aka antibodies
Secereted by -lymphocytes/B-cells
Composed of two lights chains and two
heavy chains with constant and variable
regions
Variable bind to antigens
Constant activate
immunological defenses
Typically Y-shaped
Monomers (IgD, IgG, IgE)
Dimeric (IgA)
Pentameric (IgM)
PROTEIN PURIFICATION
1. SOLUBILITY/POLARITY
i. Isoelectric precipitation
Adjusting the pH of the solution to the pI
of the protein to become insoluble
(create precipitate)
Gel is prepared with a pH gradient
parallel to the electric field gradient
Protein reaches point with no net charge
(pI), no longer migrates
Interaction between solute + solute =
low solubility
Interaction between solute + solvent =
high solubility
The lower the pI, the less precipitate
because the rest has dissolved
ii. Salting out
Removing h2O from proteins by adding
excess amount of salt to make protein
more insoluble and less soluble

When ammonium sulfar is added, some

H2O is removed from proteins to make
ion-dipolar bonds with salts
Without H2O, proteins begin to interact
through hydrophobic bonds
More salt, more precipitate
iii. Chromatography (normal and reverse)
Protein separation based on affinities
with mobile and stationary phase
Mobile phase
moves through stationary
phase
higher interaction with
stationary phase, the
slower
stationary phase
facilitates separation
selectively retards the
flow
based on relative affinity: stationary and
mobile phase
based on molecular polarity: normal and
reverse phase
NORMAL
REVERSE
PHASE
PHASE
o Stationary
o Stationary
non
polar
polar
o Mobile
o Mobile
nonpolar
polar
Polar proteins are Polar proteins are
eluted last
eluted first
iv. High performance liquid chromatography
(HPLC)
Used a pre-packed with stationary
phase and is pressurized
Fast and clear purification
Reverse HPLC for separation of nonpolar molecules
2. MOLECULAR SIZE/WEIGHT

MARY KIMBERLY L. ESPALDON

i. Dialysis
Movement of particles through a semipermeable membrane
Large molecular weight molecules will
remain inside the dialysis bag
Small molecular weight molecules will
pass through
ii. Ultrafiltration
Filtration using a vacuum
Proteins as residues
Faster because vacuum sucks filtrate
iii. Ultracentrifugation
Centrifugating a aolution at various
speeds that separates it main
components
Higher molecular weight, high speed to
settle at the bottom
Lower molecular weight needs even
higher speed to settle at the bottom
iv. Gel filtration chromatography
size exclusion or molecular sieve
Stationary phase consists of crosslinked gel particles (determines pore
size)
Porous gel beads: AGAROSE and
DEXTRAN traps smaller molecules
leaving larger molecules to be eluted
first
v. SDS-PAGE (sodium dodecylsulfatepolyacrylamide gel electrophoresis)
SDS imparts a negative charge to
proteins
Smaller proteins treated with detergent
moves faster towards positively charged
anode
Large molecules offers more resistance
to SDS
Lower molecular weight, greater
distance from origin
Higher molecular weight, smaller
distance from origin
3. CHARGE

i. Electrophoresis
Based on motion of charged particles
towards an electrode of opposite charge
Separates proteins based on attraction
towards the negative charged cathode
or positively charged anode at specific
pH
ii. Ion-exchange chromatography
Uses cation or anion exchangers (resin)
Cation exchanger
(+) slower elution
Acidic>neutral>basic
Anion exchanger
(-) slower elution
Basic>neutral<acidic
4. BINDING AFFINITY
i. Affinity chromatography
A LIGAND acts as the stationary phase
to entrap proteins
Uses BUFFER to eluate proteins
Pure but expensive results
Can be disrupted with change in pH
ii. Precipitation by antibodies
Antibodies bind to antigen
Soluble antigen binds to soluble
antibody and form lattice visible
precipitate