Chapter 1

Life characterized by
o High complexity
o Extraction, transformation, systematic use of energy to create and maintain
structures and do work
o Sense and respond to changes in surrounding
o Self-replication, enough change for evolution
o Living organisms have large # of diff compounds
o Macromolecules highly specific interactions
o Internal structures with defined functions
Hierarchy of biomolecular structure
o Supramolecular complexes are held together by noncovalent bonds
o Cell organelle supramolecular complexes macromolecules monomeric
units
o Chromatin DNA nucleotide
o Plasma membrane protein amino acid
o Cell wall cellulose sugars
Biochemistry: atomic composition, role of carbon
o Biomolecules are carbon based (>50% of cellular mass)
o Common elements
H, N, O, P, S
o Metal ions
E.g. K, Na, Ca, Mg, Zn, Fe
Play role in metabolism
o ~30 elements essential for life
o Humans ~80% water
o C (61.7%)
o N (11%)
o O (9.3%)
o H (5.7%)
E. Coli
o Water (70%)
o Protein (15%)
o DNA (1%)
o RNA (6%)
Biomolecules: Amino-Acids and proteins
o Biomolecules are hydrocarbons with Hs replaced by functional groups
o 20 common amino acids, vary by fxn group
Biomolecules contain unique combo of fxn groups
o Fxn of biomolecules depends on 3D structure
Biochemistry is stereospecific
o Stereoisomers (L and D)
o Enantiomers

Non-superimposable mirror images

o Stereoisomers have different biological properties
E.g. (R)-Carvone (spearmint) vs (S)-Carvone (caraway)
Interactions between biomolecules are stereospecific
o Macromolecules have unique binding pockets
o Only certain molecules fit/bind
Biochemistry is dynamic
o Interactions b/w biomolecules are dynamic
Biophysics: Quantitative description of biology
o Energy conversion
First law of thermodynamics
Total energy is converted but never consumed
o Chemical/light energy potential energy into:
Kinetic energy (movement, heat)
Chemical conversion (metabolism)
Macromolecular assembly loss of entropy
Cellular work (osmotic/electrical energy)
o ATP = chemical currency of cell
Thermodynamics: favorable vs unfavorable rxn
o Synthesis of complex molecules and many other metabolic reactions require
energy (ENDERGONIC)
These rxn are thermodynamically UNFAVORABLE, (G > 0)
Creating order requires work/energy
o Break down of some metabolites release significant amount of energy
(EXERGONIC)
E.g (ATP, NADH, NADHP) can be synthesized from sunlight/fuels
Cellular concentration higher than equilibrium concentration
CELL IS NEVER AT EQUILIBRIUM Dyanmic Steady State
o Most of biochemistry is endergonic

Energy Coupling: Driving Unfavorable Reactions Forward

Kinetics: How to speed up chemical rxn?

o Favorable rxn doesnt have to occur rapidly
E.g. combustion of sugar, how do we speed it up?
o Higher temperatures
Stability of macromolecules is limiting
o Higher concentration of reactants
Costly, more valuable starting materials needed
o Lower activation barrier by catalysis
Used by living organisms
Enzymes are protein catalysts: Function by increasing rate of rxn
o Catalysts DO NOT alter G
o Catalysts DO lower activation free energy of G
Stabilize transition state
o Catalysis offers:
Acceleration under mild conditions
High specificity
Possibility for regulation

o
Enzyme and pathways
o Series of related enzymatically catalyzed reactions form a pathway
o E.g pathways
Metabolic pathways produce energy or valuable materials
Signal transduction pathway transmit information

o Provides mechanism for feedback regulation

E.g. negative feedback regulation
Product of enzyme 5 inhibit enzyme 1
Genetics and evolution
o Life on earth 3.5-3.8 BYA
Form self-replicating molecules
o Replication is imperfect
Mutations occur
o Mutations give organisms advantage in given environment likely to be propagated

Chapter 2
-

Non-covalent interactions
o Dipole interactions and H-bonds
Electrostatic interactions b/w uncharged polar molecules
o Hydrogen bonds
b/w neutral groups or peptide bonds
o Ionic (Coulombic) interactions
Electrostatic interactions b/w permanent charged species or b/w ion and
permanent dipole
Attraction or repulsion
o Hydrophobic effect
Ordering of water molecules around non-polar substances
o Van der Waals interactions
Weak interactions b/w all atoms
Attractive (dispersion) and repulsive (steric) component
Any 2 atoms in close proximity
Van der Waals interactions

o
o 2 components
Attractive (London force)
Depend on polarizability
Repulsive force (steric repulsion)
Depend on size of atom
o Attraction/repulsion
Attraction dominates longer distance
0.4-0.7nm (4-7 angstroms)
o 1 ANGSTROM = 0.1 nm! Or 10-10 m

Repulsion dominates at very short distances

There is a Minimum optimal energy distance
Van der Waals contact distance
Van der Waals interactions: Biological importance
o Universal
b/w any 2 atoms near ea/o
o Weak individually
Easily broken, allow for reversible rxn
o Importance
Determines steric complementarity
E.g. macromolecular interactions shape complementarity
Stabilizes biological macromolecules (stacking in DNA)
Facilitate binding of polarizable ligands
Hydrogen bonds: biological importance
o Source of unique properties of water
o Structure and fxn of biopolymers
Proteins, nucleic acids, polysaccharides, lipids,
o Binding of a substrate to proteins
E.g. binding hormones to receptors, substrates to enzymes
o Matching of DNA, mRNA, tRNA
Hydrogen bonds: strong dipole-dipole interaction
o Typically involve 2 electronegative atoms e.g. Nitrogen and Oxygen
o Typically 4-6 kJ/mol for bonds with neutral atoms, 6-10 kJ/mol for bonds with 1
charged atom
o Strength of H-bond depend on distance and orientation
Ideally 3 atoms involved in a line
Hydrogen bonds: biological importance
o B/w hydroxyl group of an alcohol and water
o b/w carbonyl group of ketone and water
o b/w peptide groups in polypeptides
o b/w complementary bases of DNA
Water and hydrogen bonds
o Water can serve as both H-Doner and H-Acceptor
Up to 4 H-bonds per water molecule
o Gives water its anomalous properties
High boiling point
High melting point
Large surface tension
o H-bonds are weak (20 kJ/mol)
Water as a solvent
o Water is a good solvent for charged and polar substances
Amino acids and peptides
Small alcohols

Carbohydrates
o Water is poor solvent for nonpolar substances
Nonpolar gases
Aromatic moieties
Aliphatic chains
o Some polar biomolecules
Glucose, Glycine, Aspartate, Lactate, Glycerol
o Some nonpolar biomolecules
Wax, amphipatic phenylalanine, phosphatidylcholine
Water as a solvent: Ionic molecules Salts
o Highly dielectric constant reduces attraction b/w oppositely charged ions in salt
crystal
Almost no attraction at large (>40 nm) distance
o Strong electrostatic interactions b/w solvated ions and water lowers energy of
system
o Entropy increases
Ordered crystal is dissolved
Water as a solvent: Gas molecules
o Polar gases dissolve better than nonpolar gases
E.g.
Polar: Ammonia, Hydrogen sulfide
Nonpolar: N, O, CO2
o Nonpolar gases chemically converted, or bound to other molecules for biological
transport
E.g. myoglobin
Water as a solvent: Hydrophobic molecules
o Hydrophobic molecules poorly dissolved in water explained by entropy
o Bulky water has little order
High entropy
o Water near hydrophobic solute highly ordered
Low entropy
o Low entropy thermodynamically unfavorable
Hydrophobic solutes have low solubility
o Hydrophobic effect minimizes loss of water entropy
Water as solvent hydrophobic effect
o Association or folding of nonpolar molecules in aq solution
o Is one of the main factors behind
Protein folding
Protein-protein association
Formation of lipid micelles and membranes
Binding of steroid hormones to their receptors
o Does NOT arise b/c of direct attractive force b/w 2 non-polar molecules

In fact, H > 0 for transfer of many nonpolars into benzene from water;
while G < 0 (so dominated by TS)
o Origins of hydrophobic effect
Consider amphipathic lipids in water
Lipid molecules disperse in solution
Nonpolar tail is surrounded by ordered water molecules
Entropy of system decreases
System is now unfavorable
o Nonpolar chains of the amphipathic molecule aggregate
Fewer water molecules are ordered
Released water molecules will be more random, entropy increases
o Aggregation continues
Only polar head groups of ampipathic molecules are exposed
Make energetically favorable H-bonds with water molecules
Water and protein-ligand interactions: H-bonding
o Water is a ligand

Water and osmotic pressure

o Cell is surrounded by a semi-permeable membrane
Water is conducted through specialize protein called Aquaporins
Cell in Isotonic = no net water movement
Cell in hypertonic = water out, cell shrinks
Cell in hypotonic = water in, burst
Osmotic dysfunction and human disease
o Ocular cataracts, glaucoma
o Kidney hypertension, diabetes insipidis
o CNS neuromyelitis optica
o Skin eczema, wound healing
Ionization of water
o OH bonds are polar and can dissociate
Products are proton H+ and hydroxide ion OH Dissociation of water is rapid, reversible process

o Most water molecules remain unionized, thus pure water has low electrical
conductivity
o Equilibrium strongly to left
o Extent of dissociation depend on temperature
Proton hydration
o Protons do not exist free in solution
They are hydrated forming hydronium H3O+ ions
o Hydronium ions are solvated by nearby water molecules
o Covalent and hydrogen bonds are interchangeable
Allows for fast mobility of protons in water via proton hopping
Water ionization: quantitative treatment
o Concentrations of species involved in chemical equation described by the
equilibrium constant
Keq = [H+]*[OH-]/[H2O]
o Keq can be experimentally determined = 1.8*10-16 M at 25C
o [] = concentration Molarity, M, mol/Liter
o [H2O] = 55.5M determined from water density @ 25C
(1000 g/L) / 18 (g/mol) = 55.5 M

o
The pH scale
o Way to express [H+]
o In neutral solution [H+] = [OH-] and pH = 7
o pH and pOH always add to 14
o pH can be negative ([H+] > 1 M)
o What is pH?
pH = -log [H+]
log[10-7] = 7
Kw = [H+][OH-] = 1*10-14 M2
log[H+] log[OH-] = +14
pH + pOH = 14
o pH depends little on H+ from water
o pH altered by presence of other acids and bases that inc or decr [H+]
Acid release proton [H+] in water
Bases accept proton [H+]
o Acid dissociation in water

HA H+ + A Conjugate pairs

Weak acids/bases and dissociation constant

o
o
o
o
o

Biological acids/bases are often weak

Weak electrolyte dissociate only partially in water
Extent of dissociation is determined by acid dissociation constant Ka
Can calculate pH if Ka is known

o
o
o
o
o

Large Ka = greater dissociation = STRONGER ACID

Lactic acid has Ka = 1.4 x 10-4
[Lactic acid] in blood ~1 mM
Assuming no other buffers are present, what is the pH?

pKa values measure of acidity

o pKa convenient way to express Ka
pKa = -logKa (strong acid large Ka small pKa)
DONT CONFUSE WITH pH
pKa is constant and intrinsic property of the molecule
o Most biological buffers have pKa between 2-13
Buffers
o What is a buffer?

Mixture of weak acid and its Anion (conjugate base)

Buffers RESIST CHANGE in pH
When pH = pKa there is a 50:50 mixture of acid and anion forms
Buffering capacity of weak acid/base system is greatest at pH = pKa
Buffering capacity is lost when the pH differs from pKa by more than 1
pH unit
Cell is buffered by a variety of weak acids/bases
Buffers and pKa
o We can determine the pKa of a weak acid/base experimentally by producing
titration curve
o Systematic addition of strong base of known concentration while measuring the
pH
o The titration curve of acetic acid
pH is recorded at each addition of OH OH- is recorded as a fraction required to fully deprotonate the acid
equivalents
At midpoint [HA] = [A-], and pH = pKa
The blue shaded area is the pH region where the HA/A- is a good buffer

o Weak acids have unique pKas

o In the lab
Buffer systems are chosen for buffering capacity over a desired pH range
Proteins and nucleic acids are stable over a narrow pH range
Enzymes catalyze reactions at an optimal pH
Henderson-Hasselbalch Equation
o We can quantitatively describe the titration curve of a weak acid using the HHequation

o The HH equation
Quantitatively describes the shape of the titration curve
Determine pKa, given pH and molar ratio of HA/A Determine pH, given pKa and molar ratio of HA/A Determine ratio of HA/A- given pH and pKa
Proteins and other Biomolecules
o Amino acids are weak acid/bases
Useful to know their protonation state

Henderson-Hasselbalch Equation

o
o Determine the fraction of histidine group that is protonated at pH = 7.3
pK1 = 1.8 (carboxyl group)
pK2 = 6.0 (imidazole group)
pK3 = 9.2 (amino group)

Ratio vs Percentage

o
HH Equation

o
o The cell cytoplasm is maintained at pH~7.4
o Under normal conditions, lactic acid is at a concentration of 1mM
What are the concentrations of the acid and base forms?

Biological buffer systems

o Maintenance of intracellular pH is vital to all cells
Enzyme catalyzed rxn have optimal pH
Solubility of polar molecules depends on H-bond donors and acceptors
Equilibrium b/w CO2 gas and dissolved HCO3- depends on pH
o Buffer systems in vivo are mainly based on
Phosphate, concentration in millimolar range

Histidine, efficient buffer at neutral pH

Bicarbonate, important for blood plasma
o Important aspects of Drug Design/Pharmacokinetics
Drugs are exposed to various pH conditions in the body
Can change their function/recognition by substrate
Ionized molecules do not cross cell membranes easily
Water as a Reactant
o Water is not just a solvent and medium for biology it plays an active role in
biochemistry
Acid base chemistry
Another important example, conversion of CO2 to Carbonic acid

o Condensation/Hydrolysis Rxns

o
o ATP made from energy from proton gradients across membranes of chloroplasts
and mitochondria
o Other condensation reactions
Polymerization of amino acids, nucleic acids, carbohydrates
ALL THESE ARE ENDERGONIC REQUIRE HYDROLYSIS
OF ATP
o Oxidation/Reduction Rxns

Chapter 3: Amino Acids, Peptides and Proteins

-

Proteins: Main agents of biological function

o Enzymatic Catalysis
Enolase (in glycolytic pathway)
DNA polymerase (in DNA replication)
o Transport
Hemoglobin (transport O2 in blood)
Lactose permease (transports lactose across cell membrane)
o Structure
Collagen (connective tissue)
Keratin (hair, nails, feathers, horns)
o Motion
Myosin (muscle tissue)
Actin (muscle tissue, cell motility)
Amino Acids: Building blocks of proteins
o Proteins are linear heteropolymers of alpha-amino acids
o Amino acids have properties that are well-suited to carry out a variety of biological
functions
Capacity to form stable polymers
Useful acid-base properties
Varied physical properties
Varied chemical functionality

Amino Acids: Atom naming

o Organic nomeclature vs Biochemical designation
Start from one end and number down the carbons
Start from alpha-carbon and go down the R-group

Only concered with biochemical nomenclature

Amino acids: Amino acids are Chiral (EXCEPT GLYCINE)

o The -carbon has always 4 substituents and is tetrahedral
o All have an acidic carbonyl group, basic amino group, and an alpha-hydrogen
connected to the -carbon
o Each amino acid has a unique substituent R
o In glycine 4th substituent is (also) hydrogen

o L- and D- notations are in comparison to Glyceraldehyde example below

L-Levorotary
Left handed rotation of polarized light
Highest priority group on the LEFT
Proteins ONLY contain L-amino acids

Amino Acid Classification

o 20 common amino acids
o 5 basic groups depending on R-groups
Nonpolar, aliphatic (7)

Aromatic (3)
Polar, uncharged (5)
(+) charged (3)
(-) charged (2)
Nonpolar aliphatic R-groups
o G, A, P, V, L, I, M
Aromatic R-groups
o F, Y, W
These AA side chains absorb UV light at 270-280 nm
Polar, uncharged R groups
o S, T, C, N, Q
Cysteines can form disulfide bonds covalent bonds

(+) charged R-groups

o L, R, H
H, pKa ~6.0
(-) charged R-groups
o D, E
Uncommon Amino Acids
o Arise by POST-TRANSLATIONAL MODIFICATIONS of proteins
o Reversible modifications, especially phosphorylation, are important in regulation and
signaling
o Otherwise are important products/intermediates in metabolic pathways
o There are also un-natural (synthetic) AA that can be tricked into protein structures
Used to probe function of a protein, or introduce new functionalities
Uncommon/modified Amino Acids (examples)
o Phosphorylation

o Acetylation

o Methylation

o Adenylation

Ionization of Amino Acids

o At acidic pH (FULLY PROTONATED)
Carboxyl group is protonated and the amino acid is in the CATIONIC form
o At Neutral pH (ZWITTERION FORM)
Carboxyl group is DEPROTONATED but the AMINO group is
PROTONATED
Net charge is 0, these ions are called Zwitterions
o At alkaline pH (FULLY DEPROTONATED)
The amino group is neutral (-NH2) and the amino acid is in the ANIONIC
form

o Amino acids with uncharged side chains, e.g. GLYCINE, have 2 pKa values
o For glycine
pKa of -carboxyl = 2.34
pka of -amino = 9.6
It can act as a buffer in two pH regimes

o
o Zwitterions predominate at pH values b/w the pKa values of amino and carboxyl
group
for amino acid without ionizable side chains, the ISOELECTRIC POINT
(equivalence point, pI) is
pI = (pK1 + pK2) / 2
At the pI, net charge = 0
Least soluble point in water
Does not migrate in electric field
o The chemical environment affects pKa values:
-carboxy group is much more acidic than in carboxylic acids
-amino group is slightly less basic than in amines

o Some Amino acids with ionizable side chains, such as the charged amino acids, have
3 pKa values

o Ionizable side chains can be titrated

Titration curves are more complex
pKa values are discernable if two pKa values are more than 2 pH units apart

o How to calculate the pI of an amino group with an Ionizable R-group?

Identify the net zero charge species
Identify pKa value that defines the acid strength of this zwitterion: (For Glu =
pKa1)
Identify pKa value that defines the base strength for this zwitterion (For Glu =
pKR)

 Take the average of these two pKa values (For Glu = 3.22)
o Example
What is the pI of histidine?
Pk1 = 1.82
pKR = 6.0
pK2 = 9.17

(9.17 + 6)/2 = pI
pI = 7.59
MUST KNOW HOW TO DRAW TITRATION CURVE FOR GIVEN
AMINO ACID AND LABEL THE PKs and pI!

Peptide Formation: Peptide bond

o Peptide bond:
Formed by condensation reaction b/w 2 aa

Naming and convention

o Peptides are small condensation products of aas
small compared to proteins (Mw < 10kDa)
o Polypeptide chain

o Always listed in NC direction

Peptide naming/convention
o Numbering/naming starts from amino terminus
3 letter code
Written as Ser1-Gly2-Tyr3-Ala4-Leu5
One letter code
SGYAL
Should be able to recognize amino acid structure and name them using both
conventions
Peptide functions
o Hormones and pheromones
Insulin (think sugar)
Oxytocin (think childbirth)
Sex-peptide (think fruit fly mating)
o Neuropeptides
Substance P (pain mediator)
o Antibiotics
Polymyxin B (for Gram bacteria)
Bacitracin (for Gram + bacteria)
o Protection (e.g. toxins)
Apamin (bee venom)
Conotoxin (cone snails)
Chlorotoxin (scorpions)
Peptide formation: peptide bond
o Formed by condensation rxn b/w 2 aa
o Endergonic rxn
Require activation by chemical modification of N or C terminus
In cells, c terminus is activated by ATP
For chemical synthesis we use other molecules for activation
Peptide Synthesis
o Use solid-state Fmoc chemistry
o Solution state chemistry is too difficult to purify each step of rxn
o Fmoc is a protecting group! NOT AN ACTIVATING GROUP

Fmoc peptide syntehsis

o Stepwise synthesis carried out in C to N direction
o 1. Residue 1
Fmoc protected N-terminal attached to styrene bead
o 2. Remove Fmoc with mild base
o 3. Residue n+1
With DCC activated C-terminal added and peptide bond formed
o 4. Repeat
o 5. Removal of peptide from bead using TFA
Peptide formation: chemical synthesis
o Peptides over 100aa are not practical for synthesis

o
o Ribosome can synthesize 100aa in 5 seconds (error rate 1/10,000)
o Amino acid activated by ATP and ribosome catalysis
Polypeptides (covalently linked a-amino acids and possibly:
o Cofactors
general term for functional non-amino acid component
Metal ions, organic molecules
o Coenzymes
Used to designate organic cofactors of enzymes
NAD+ in lactate hydrogenase
o Prosthetic groups
Covalently attached cofactors
Heme in myoglobin
o Other modifications
Proteins size and composition
o Simple proteins contain only amino acids
o Conjugated proteins contain prosthetic groups:
Lipids
Sugars

Metals
Cofactors

o
o Peptide vs Protein (latter are typicaly ~10,000 Da)

o
o Proteins vary in their amino acid composition
o The molecular weight of a simple protein estimated by # of AA it contains
(# of AA) x 110 Da = ~Molecular weight
Note:
Average amino acid mass is 138 Da
However, small amino acids are more common
The average Mw of amino acids used by proteins is = 128 Da
In a polypeptide, H2O is removed due to peptide bond, so average mw
is reduced to 128-18 = 110 Da per amino acid
Peptides and proteins, What we trying to learn?
Sequence and composition?
o How vary b/w individuals and species?
o How related to other proteins?
What is 3d structure?
o How related to fxn?
o How did it find native fold
How achieve its biochemical role?
o How is its biochem role disrupted in disease?

Where is it localized within cell

o How does it co-localize with other proteins/organelles?
What are its physico-chemical properties?
o How do they relate to its fxn?
o How can we exploit them for analysis?
Amino acid classification: Aromatic R-groups and UV absorption
o F, Y, W
These aa side chains absorb UV light at 270-280 nm
Can be Used for detection and quantification
o Spectroscopic detection of proteins/polypeptides
o Tryptophan and Tyrosine are the strongest
o UV absorbance maxima ~275-280 nm
o Concentration ca be determined using spectrophotometry and Beers law
A = c l
A = absorbance = -log (l/lo)
= extinction coeff
C = concentration
l = path length
E.g. Purified M-E-T-S-Y-Q-N-L-L-W-T-W-S-G-G-K
Extinction coeff given
o Tyr = 1280 M-1*cm-1
o Trp = 5690 M-1*cm-1
What is the overall E for peptide?
o 1280 + (2x 5690) = 12660 M-1*cm-1
Given A = 1.23
Given 1cm cuvette
What is peptide concentration?
o (1.23)/(12660*1cm) = 9.72*10-5 M
Peptides and Proteins, Separating from a mixture (purification)
o Separation relies on differences in physical and chemical properties
Charge
Size
Affinity for ligand
Solubility
Hydrophobicity
Thermal stability
o Common techniques for preparative separation
Centrifugation
Separation based on density (i.e. differential centrifugation)
Precipitation (followed by centrifugation/filtration)
Separation based on solubility in the presence of various chemical
and/or thermal conditions (e.g. salts, pH, organic solvents, temp. etc.)
Chromatography

Separation based on interaction with solid-phase matrix

o E.g. charge, hydrophobicity, size, interaction with ligand
Most protein purification strategies utilize a combination of these approaches!
Differential centrifugation
Tissue homogenization (tissue homogenate)
Low speed centrifugation 1000 g, 10 min
o Pellet contains whole cells, nuclei, cytoskeletons, plasma
membranes
Supernatant subjected to Medium speed centrifugation 20,000 g 20min
o Pellet contains mitochondria, lysosomes, peroxisomes
Supernatant subjected to high speed centriguation 80,000 g, 1h
o Pellet contains microsomes(fragments of ER), small vesicles
Subject to very high speed centrifugation (150,000 g 3h)
o Supernatant contains soluble proteins
o Pellet contains ribosomes, large macromolecules
Peptides and proteins, separating from a mixture (purification)
o Column Chromatography
Mobile phase protein solution
Stationary phase porous matrix
Proteins in mobile phase interact with the stationary phase matrix based on
unique physico-chemical properties
o Ion-exchange chromatography
Separation based on the surface charge characteristics of the
protein/biomolecule
Cation exchange negatively charged resin
Choose if buffer pH < pI (positively charged)
Anion exchange positively charged resin
Choose if buffer pH > pI (negatively charged)
Strongly bound proteins are eluted with high salt buffer
Method is selected based on the proteins isoelectric point pI
How it works:
Proteins move through the column at rates determined by their net
charge at the pH being used. With cation exchangers, proteins with a
more negative net charge move faster and elute earlier
Example, protein of interest has pI = 8.9, buffer pH = 7
What ion exchange method would you choose for purifaction?
First decide is the protein + or charged at pH = 7.4?
Buffer pH is below pI, so positively charged
Select a cation-exchange column
o Size-exclusion chromatography
AKA Gel-filtration
Separation based on size of protein/biomolecule

 Larger proteins flow through faster than smaller ones

Many different pore sizes available
Selected based on expected MW of the protein
May also be used to determine the MW of your protein by comparing a
set of calibrated standards
How it works:
protein mixture is added to column containing cross-linked polymers
protein molecules separate by size; larger molecules pass more freely
appearing in the earlier fractions
Example:
You have protein of 376 aa
You have choice b/w S75 or S200 SEC column
o Note: S75 is for proteins (10-100 kDa)
o S200 is for proteins (100-1000 kDa)
Which column would you use?
(376 AA) x (110 Da) = 41,360 Da
o Use the S75 column
o Affinity chromatography
Separation based on affinity to a ligand
Variety of ligands
Small molecule
Metal
Antibody/protein
Affinity tags can be genetically engineered into a protein target
These methods can be very specific
How it works:
Solution of ligand is added to column
Protein mixture is added to column containing polymer bound ligand
specific for protein of interest
Unwanted proteins are washed through column
Proteins of interest are eluted by ligand solution
Peptides and proteins, assessing the quality of purification
o Enzymatic analysis
Total activity vs specific activity
Enzymatic activity
o 1 Unit = transforms 1 umol of product per min at 25C
Specific activity
o = total activity/total protein
During purification total activity should not change much; specific activity
should get higher


For non-enzymes require a different assay
o General assay to assess protein purity
Gel Electrophoresis
Electric field pulls proteins according to their charge
Gel matrix hinders mobility of proteins according to their size and
shape
o Smaller proteins migrate faster than larger ones

o
red one is cathode (+)
o Polyacrylamide Gel Electrophoresis (PAGE)
Separates biological macromolecules, usually proteins or nucleic acids,
according to their electrophoretic mobility.

o Denaturing Electrophoresis (SDS-PAGE)

SDS sodium dodecyl sulfate a detergent

SDS micelles bind and unfolds denatures the proteins

Gives all proteins a uniformly negative charge
Gives all proteins a uniform shape linear polypeptide
Rate of movement will only depend on linear size: small proteins will
move faster
Typically incorporates a reducing agent
o E.g. B-mercaptoethanol (BME) or Dithiothreitol (DTT)
These are used to reduce disulfide bonds

Assessing quality of purification

o Denaturing electrophoresis SDS-PAGE
o Typically incorporates reducing agent
E.g. B-Mercaptoethanol (BME) or Dithiothreitol (DTT)
These reagents used to reduce disulfide bonds

Analyzing physico-chemical properties

o
Protein sequencing
o Primary AA sequence provides
Protein ID
Insight into physico-chemical properties (MW + pI)
Insight into 2, 3 structure (sometimes 4 structure)
Insight into biological function, disease and evolutionary history

Actual sequence generally determined from DNA sequence

Edman Degradation (Classical method)
Successive rounds of N-terminal modification, cleavage, and
identification
Can be used to identify protein with known sequence
Mass Spectroscopy (modern method)
MALDI MS and ESI MS can precisely identify the mass of a peptide,
and thus the aa sequence
Can e used to determine PTM
Protein Sequencing (EDMAN DEGRADATION)

o
o Benefits
Only require 10-100 pico-mole of protein
o Drawbacks
Can only sequence ~30 AA
o How to overcome drawback?
Cleave larger proteins into smaller pieces first
E.g. using proteases, or chemical cleavage
Protein Sequencing Protease Enzymes

Protein Sequencing Mass Spectroscopy

o Modern methods of protein mass and sequence analysis done by Mass-Spec

o
o Electrospray Ionization Mass Spectroscopy (ESI-MS)

o
DNA Sequence analysis

Primary Sequence Analysis

Chapter 4

3D structure of proteins
o Major themes
3D structure determined by 1 sequence
Protein fxn depend on structure Form & Function
Most proteins exist in one or a few # of stable structures
Protein structure is primarily stabilized by non-covalent interactions
Proteins may share common structural features
Protein structures are not static Dynamics is important to function, and some
are completely unstructured
Overview of Protein Structure
o Unlike most organic polymers, protein molecules adopt a specific 3D conformation
o This structure is able to fulfill a specific biological function

o This structure is called NATIVE FOLD or NATIVE CONFORMATION

4 levels of protein structure

o
Overview of Protein Structure
o Native fold is stabilized by a large # of favorable interactions w/I protein
Typically, this is the THERMODYNAMICALLY MOST STABLE
There is a COST IN CONFORMATIONAL ENTROPY of folding the protein
into one specific native fold
Proteins MAY HAVE MORE THAN ONE STABLE CONFORMATION
Important to catalysis (e.g. binding and release of substrate)
Non-covalent interactions are maximized
o Hydrophobic effect (Major component)
Release of water molecules from the structured solvation layer around the
molecule as protein folds increase the net ENTROPY
o Hydrogen bonds
Interactions of N-H and C=O of the peptide bond leads to local regular
structures such as a-helices and B-sheets
o London dispersion
Medium-range weak attraction b/w all atoms contribute significantly to the
stability in interior of protein
o Electrostatic interactions
Long-range strong interactions b/w permanently charged groups
Salt-bridges, esp. buried in hydrophobic environment strongly stabilize the
protein
Structure of the peptide
o Protein structure partially dictated by properties of the peptide bond
o Peptide bond is a resonance hybrid of 2 canonical structures
o Resonance causes peptide bonds
To be less reactive (compared to esters for example)
To be rigid and nearly planar
To exhibit large dipole moment in favored trans configuration

Phi/Psi Dihedral Angles

o Rotation around the peptide bond is not permitted
o Rotation of bonds connected to the a-cabon is permitted

Protein Secondary Structure

o Secondary structure refers to local spatial arrangement of polypeptide backbone
o 2 regular arrangements are common
a-helix
stabilized by H-bonds b/w residues nearby in sequence
B-sheet
Stabilized by H-bonds b/w adjacent segments that may not be nearby
in sequence
o Irregular arrangement of polypeptide chains is called RANDOM COIL
Right handed a-helix
o 3.6 residues (5.4) per turn
o Stabilized by H-bonds b/w nearby backbone amides

 (n and n+3 residues)

o Polypeptide bonds are aligned roughly parallel with helical axis
o Side chains point out roughly perpendicular w/ helical axis

The Alpha Helix

o Inner diameter of the helix (no side chains) is about 4-5

Too small for anything to fit inside
o The outer diameter of the helix (with side chains) is 10-12
o Residue 1 and 8 align nicely on top of each other
o Not all polypeptide sequences adopt a-helix structures
o Small hydrophobic residues Ala and Leu are strong helix formers
o Pro acts as a helix breaker because the rotation around N-Ca bond is impossible
o Gly acts as helical breaker because the tiny R-group supports other conformations
o Attractive or repulsive interactions b/w side chains 3-4 amino acids apart will affect
formation

o
The Helix Dipole
o Recall that the peptide bond has a strong dipole moment
Carbonyl O = negative
Amide H = positive
o All peptide bonds in the a-helix have a similar orientation
o Result in a large macroscopic dipole moment
Negatively charged residues often occur near the positive end of the helix
dipole

Beta-sheet
o The backbone is in a more extended conformation

o The pleated sheet-like arrangement of backbone is held together by hydrogen bonds

b/w the backbone amides in different strands
o Side chains protrude from the sheet alternating in up and down direction

o
o Parallel or Antiparallel orientation of two chains w/I a sheet are possible
o Parallel B-sheets
H-bonded strands run in the SAME DIRECTION
Resulting in bent H-bonds (weaker)
o Antiparallel B-sheets
H-bonded strands run in the OPPOSITE DIRECTIONS
Resulting in linear H-bonds (stronger)

Beta-turns
o B-turns occur frequently whenever strands in B-sheets change the direction
The 180 turn is accomplished over 4 amino acids
o The turn is stabilized by H-bond from carbonyl oxygen to amide proton at the n+3
position
o Proline in position 2 or Glycine in position 3 are common in B-turns

o
Proline Isomers (Cis and Trans)
o Most peptide bonds not involving proline are in the trans configuration (<99.95(
o For peptide bonds involving proline, about 6% are in the cis configuration. Most of
this 6% involves B-turns
o Proline isomerization is catalyzed by proline isomerases

o
Analysis of Secondary Structure Circular Dichroism (CD)
o Spectroscopic technique to quantitatively assess secondary structure content

o CD measures the molar absorption differences or left- and right-circularly

polarized light = L R
Chromophores in a chiral environment produce characteristic signals
CD signals from peptide bonds depend on the chain conformation

o
Protein Tertiary Structure
o Tertiary structure
Overall spatial arrangement of atoms in a polypeptide chain or in a protein
o 3 major classes
Globular proteins
Water-soluble proteins
Lipid-soluble membranous proteins
Fibrous proteins
Typically insoluble; made from a single secondary structure
Intrinsically disordered proteins
Lack stabile/ordered structure
o Found with a huge variety of shapes and sizes but some general features are shared
Compact fold
Hydrophobics concentrated at the interior of hydrophilics at surface
Stabilized by many H-bonds and ion-pairs
o While overall structures are often very complex, most proteins are assembled by
linking together common structural features motifs or folds
Protein Motifs (Folds)
o Motif/Fold recognizable folding pattern of 2 or more connected 2ndary structural
elements
o All alpha, All beta, or Mixed alpa/beta
o Motifs can be found as reoccurring structures in numerous proteins
o Most proteins are made of different motifs folded together

o
o Specific connectivity b/w sequential 2ndary elements are favored

o
o Larger and more complex folds may be constructed from smaller simpler motifs

o
o Hierarchy
Class: all , all , /, and +
Each class has many folds
Some are very common, some are unique to only 1 known protein

o Structural classification of proteins (SCOP) database

o

Protein Domains
o Domain part of a protein that is independently stable
Multiple domains may have independent functions within the entire protein
May be separated by a linker (independent), or closely packed with eachother

Protein Quaternary Structure

o Quaternary Structure formed by spontaneous assembly of individual polypeptides
into a larger functional oligomer
o e.g. Hemoglobin Functional Unit is a Tetramer
Protein Structure Database (PDB)
o There are 20-25,000 protein coding regions in the human genome
o Only ~1,400 unique folds in nature
Globular Protein Structure
o Myoglobin
First atomic model of a protein
o Features
Hydrophobic core
Hydrophilic surface
Shape is adapted to function
o Group of enzymes involved in glycolysis
Even w/i the small group of related function, you can observe a variety of
size, shape, symmetry, oligomeric state, etc.
Globular Protein Structure and Function
o Globular Proteins Most common in biology
o Perform variety of biological functions
Enzymes
Transporters
Regulatory proteins
Antibodies

o Signal transduction nearly half

Fibrous Protein Structure and Function
o Adapted for Structural Roles
o Simple repeating element of secondary structure
o Insoluble in water
o Hydrophobic surfaces of similar peptides pack together = fibrils
o

Fibrous Protein Quaternary Structure

o
Fibrous Protein Structure
o Collagen
Gly and Pro-rich left handed helix
Three chains form a right handed triple helix
Higher tensile strength than steel wire
Gly residues facilitate fibril packing
Many triple helices assemble into a collagen fibril
Stabilized by inter-strand H-bonding and crosslinking b/w modified lysines
(unusual)


o 4-Hydroxyproline in Collagen
Forces the proline into a favorable pucker conformation
Offer more hydrogen bonds b/w 3 strands of collagen
The post-translational processing is catalyzed by prolyl-hydroxylase and
requires: a-ketoglutarate, oxygen and ascorbate (vitamin C)
Lack of vitamin C can cause scurvy (degeneration of connective tissue)

o
o Example of B-sheet fibrous protein: Silk Fibroin
Main protein in silk from moths and spiders
Anti-parallel B-sheet structure
Small side chains (Ala- and Gly) allow the close packing
Structure is stabilized by H-bonding w/i sheets
London dispersion interactions b/w sheets

Extremely strong material

Strong than steel
Can stretch a lot before breaking
Intrinsically Disordered Protein Structure
o Proteins, or protein segments that lack definable structure
o Composed of amino acids whose higher concentration forces less-defined structure
Lys, Arg, Glu and Pro
o Functions include
Protein/protein interactions
Can adopt many different conformations, facilitating highly specific
interactions with several partner proteins
Cell signaling
Common sites of post-translational modification (e.g. phosphorylation)
involved in signaling pathways
Domain Tethering
Linking together functional domains into a single molecule or
oligomeric complex
o Phosphorylated Kinase Inducible Domain (pKID)
Protein interaction induces secondary structure
Allows for intimate binding interactions
o P53 protein
Several different conformations possible
Allows for multiple binding partners

o Protein Kinase A
Tethering of Functionally Coupled Proteins/Domains
o

Methods of determining protein structure

o 3 major methods
X-ray crystallography
NMR spectroscopy
Electro microscopy

Determining Protein Structure (X-ray Crystallography)

o X-ray crystallography is by far the most common and successful technique
Diffraction technique: uses coherent X-ray light source
o Required steps:
Purify the protein (mg quantities)
Crystalize the protein
Collect diffraction data
Calculate electron density map
Build model into density

o Pros

o Cons

No size limits
Well-established
High resolution

Crystallization can be difficult/impossible

Especially for membrane proteins
Determining Protein Structure (Biomolecular NMR Spectroscopy)
o NMR Spectroscopy
NMR active nuclei (e.g. 1H , 13C) absorb radio frequencies when placed in a
strong magnetic field

o Required steps
Isotope labeling
Purify protein (mg quantities)
Collect NMR data
Assign NMR spectra
Calculate structure
o Pros
Does not require crystals
Information on structure and dynamics
o Cons
Difficult for insoluble membrane proteins
Difficult for larger proteins (>50 kDa)
Determining Protein Structure (Biomolecular CryoEM)
o Electron Cryo-Microscopy (cryoEM)
Imaging technique using electron beam as light source
o Required steps
Purifying the protein (ug quantities)
Collect image data
Calculate 3d reconstruction
Build model into density map
o Pros
Does not require crystals
No size limit (except for small proteins)
o Cons
Technically difficult
Best for large proteins (>200 kDa)