Announcement
I am Hyun-Soo Cho, in Biology Department.
This course is Biochemistry,
(1) How to get lecture slides
structure.yonsei.ac.kr/
File name: bc_ch2.ppt
(2) Exam :
 2 times (Mid, Final Exam)
 Problem types: Single or multiple choice 70% + long answer 30%
 Place: Lecture Room 과B130
 Posting of score in Exam: on the board at room SB134, 신과학
원,
Announcement (continued)
(3) Assignment (homework):
 Please read your textbook before or/and after each class
(4) Grading:
 Mid exam (40%) + Final exam (40%) + attendance, reports
(20%)
(5) Participating in this Biochemstry Course
 Thanks everyone for your interest on this class
 Have good manners : No cell phone (no message), No
chatting
 On no attendance or pointing out your bad manner, you will
get minus one per one time in your score
 I hope you would keep your honor during this course
Announcement (continued)
(6) How to study biochemstry
 Be familiar with English term
 Motivate yourself why you should study biochemistry
 Read text book carefully
 Ask any questions
(7) Interviewing with me
 You may see me during this course if you want
 My office hours: PM 3:00-4:00 on Thursday
 How: First, Contact me by E-mail or telephone
 E-mail address: hscho8@yonsei.ac.kr, 2123-5651
Chapter 2
Protein Composition and Structure
Key Properties of Proteins
1. Proteins are linear polymers built of monomer units called amino acids.
• The amino acid sequence of a protein dictates its folding process.
• The three dimensional structure of a protein determines its
biological function.
The characteristic three dimensional
structure of the beta subunit of E.coli
DNA polymerase complex allows DNA
to be copied during DNA replication
without
the
replication
dissociating from the DNA.
machinery
Key Properties of Proteins (continued)
2. Proteins contain a wide range of functional groups including alcohols, thiols,
thioethers, carboxylic acids, carboxamides, and a variety of basic
groups.Various combinations of functional groups in amino acids enable
broad spectrum of protein function.
3. Proteins can interact with one another and with other biological
macromolecules to form complex assemblies resulting in new capabilities.
A hexagonal array of
two kinds of protein filaments
in insect flight tissue
(electron micrograph)
Key Properties of Proteins (continued)
4. Some proteins are quite rigid, whereas others display limited flexibility.
•
Structural elements are rigid. Why?
(cytoskeletons, connective tissues, etc.)
•
Regulatory elements are flexible. Why?
(protein-protein interaction, signal transduction, etc.)
Conformational Changes
of Lactoferrin
upon iron binding
Proteins Are Built from a Repertoire of 20 Amino Acids
Structure and Stereoisomerism of a-Amino Acids
Ca : a-carbon (chiral)
NH3+ : amino group COO- : carboxyl group R : functional group
(side chain)
Absolute Configuration : S
Absolute Configuration : R
Left, Counter-Clockwise
Right, Clockwise
Only L-amino acids are
constituents of proteins.
Zwitterionic Character of Amino Acids (Dipolarity)
Amino Acid Nomenclature
Proteins are built from a repertoire of 20 amino acids in all species.
Twenty kinds of side chains vary in size, shape, charge, hydrogen bonding
capacity, hydrophobic character, and chemical reactivity.
Classification of Amino Acids
Based on the Characteristics of Functional Groups
Polar (Hydrophilic)
Arg, Asn, Asp, Cys,
Gln, Glu, His, Lys,
Ser, Thr, Trp, Tyr
Non Polar (Hydrophobic)
Ala, Gly, Ile,
Leu, Met, Phe,
Pro, Val
Acidic (negative charge)
Asp, Glu
Basic (positive charge)
Arg, Lys, His
Aromatic (ring)
Phe, Tyr, Trp
Aliphatic (linear chain)
Most of the rest
achiral
(non-chiral)
Most
Simple
Amino
Acids
Most
Typical
Aliphatic
Amino
Acids
Most
Typical
Non-Polar
Hydrophobic
Amino
Acids
Isoleucine
Contains
an Additional
Chiral
Carbon
The side chain of proline is bonded to
both the nitrogen and a-carbon atom.
Proline is an imino acid.
Structural flexibility is much more restricted than other amino acids.
Proline markedly influences protein architecture.
Most
Typical
Aromatic
Amino
Acids
The hydroxyl group
in tyrosine is
chemically reactive.
Tryptophan contains
an indole ring.
The aromatic rings of
Tyr and Trp contain
delocalized p electrons
absorbing UV light.
Tryptophan and Tyrosine Can Be Useful
for the Determination of Protein Concentration
Beer’s Law : A = ecl
A : Absorbance,
e : extinction coefficient (M-1cm-1),
c : concentration (M),
l : length of light pass (cm)
Maximum absorbance
at 276 nm for Tyrosine
at 280 nm for Tryptophan
Ser and Thr
Contain
Aliphatic
Hydroxyl Group.
Ser is more
hydrophilic than Ala.
Thr is more
hydrophilic than Val.
Threonine
Contains
an Additional
Chiral
Carbon
Cysteine is structurally similar to serine but contains
a sulfhydryl, thiol (-SH), group in place of the hydroxyl (-OH) group.
Pairs of sulfhydryl groups can form a disulfide bonds
which can be critical in stabilizing three dimensional structure in some proteins.
Very Polar, Highly Hydrophilic, and
Positively Charged Amino Acids
Lys : e-amino group
Arg : guanidium group
His : imidazole group
Histidine Ionization
Very Polar,
Highly Hydrophilic,
and
Negatively Charged
Amino Acids
Aspartate : b-carboxyl group
Glutamate : g-carboxyl group
Asparagine (Asn)
Uncharged Derivatives of Aspartate
b-carboxamide group
Glutamine (Gln)
Uncharged Derivatives of Glutamate
g-carboxamide group
Ka, acid dissociation constant
The equilibrium constant in acid-base reactions
HA
A- + H+
pH and pKa ?
Seven Amino Acids
Containing
Readily Ionizable
Side Chains
Aspartate
Glutamate
Histidine
Cysteine
Tyrosine
Lysine
Arginine
pKa values of
functional groups
in actual proteins
can be dramatically
changed by the
microenvironment
where
the given side chains
are located !!!
Amino Acids Are Linked by Peptide (Amide) Bonds
to Form Polypeptide Chains (Proteins)
First
Amino Acid
Second
Amino Acid
• The formation of a peptide bond requires an input of free energy
• But, the peptide bond is very stable once it is formed.
(T1/2 in aqueous solution : 1000 years, hydrolysis rate is so slow)
• The order (sequence) of amino acids in a polypeptide chain is
called the primary structure of a protein.
Formation of Polypeptide Chain
Backbone or Main Chain
(Regularly Repeating Part)
vs.
Functional Group or Side Chain
(Variable Part)
One Amino Acid in a Protein
Is Called as a Residue.
Directionality of Polypeptide Chain
N-terminus  C-terminus
(YGGFL ≠ LFGGY)
Alternative Positioning of
the Oxygen and the Hydrogen
in One Peptide Bond
Alternative Positioning of
the Oxygen and the Hydrogen
between Neighboring Peptide Bonds
Alternative Positioning of
the Functional Groups
Between Neighboring Residues
Disulfide Bonding
• In some proteins, the linear polypeptide chain can be cross-linked and the most
common cross-links are disulfide bonds between cysteine residues.
• Extracellular proteins form disulfide bonds more often than intracellular proteins.
Size of Polypeptide Chain
• Most natural polypeptide chains contain between 50 and 2000
amino acid residues and are commonly referred to as proteins.
• Less than 50 amino acids  oligopeptides or peptides
• The average molecular weight of an amino acid is about 110 Dalton.
Thus, the molecular weights of most proteins range between 5500
and 220000 dalton (i.e. 5.5 kd to 220 kd).
Amino Acid Sequences of Proteins
• Each protein has a unique and precisely defined amino acid sequence.
• Central Dogma : DNA  RNA  Protein
• Amino acid sequence of a protein determines its structure, function,
and the mechanism of biological action.
• Changes in amino acid sequences  Disease, Genetic Engineering
Chemical Properties of Peptide Bonds
?
Peptide bonds are planar
Typical Bond Lengths
within a Peptide Bond
The peptide bonds contain
• Considerable double bond character
• High H-bond forming capacity to proteins.
(Peptide Bond  Peptide Bond, or
Peptide Bonds  Functional Groups)
BUT,
still uncharged  tightly packed structure
Configurational Properties of Peptide Bonds
Trans-Configuration of a-Carbons around a Normal Peptide Bond
Balanced Configuration of a-Carbons in X-Pro linkages
Rotational Properties of Peptide Bonds
Peptide bonds are rigid…
But,
the bonds containing the a-carbon between two peptide bonds
can be rotated from -180o to +180o.
 : the angle of rotation about the bond between the nitrogen and the a-carbon
y : the angle of rotation about the a-carbon and the carbonyl carbon
Rotational Properties of Peptide Bonds (continued)
• Ramachandran Diagram Shows the Allowed Ranges of  and y Rotations.
• For Some Combinations of  and y Rotations Are Physically Impossible due to Steric
Clashes.
• Protein folding is possible by rigidity of peptide unit and restriction of  and y Rotations
Proteins’ Secondary Structures
Alpha Helix, Beta Pleated Sheet, Turns, Loops
Linus Pauling and Robert Corey’s proposal – 1951
The Double helix – 1953 James D. Watson
The a-Helix Is a Coiled Structure
Stabilized by Intra-Chain Hydrogen Bonds
• Rise of 1.5 Å per Residue
along the Helix Axis
• Rotation of 100 degree per
Residue around the Helical
Turning
• 3.6 Amino Acids per a Single
Turn of a-Helix
• Thus, amino acids spaced
three to four residues apart
are spatially quite close to
one another in an a-helix.
Most plausible H-bondings
between peptide bonds in a-helix
Coiling and Entwining of a-Helix
• Essentially all a-helices in proteins are right handed.
(Ramachandran diagram explains it why.)
• The a-helical content of proteins range from none
to almost 100%.
• Single a-helices are usually less than 45 Å long.
• Two or more helices can be entwined and form a
very stable and long coiled coil structure with a
length of 1000 Å long
Ferritin contains 75% of a-helices.
Probability of
a-Helix Coiling Direction
a-helical coiled coil; superhelix;
tropomyosin, keratin, fibrin;
bundles of fibers; filamentous structures
Beta Pleated Sheet
• Almost Fully Extended Structure
• Distance between Amino acids is 3.5 Å.
• The side chains of adjacent amino acids point
in opposite directions.
• H-bonding between different b-strands
• Why beta?
Combinations of  and y rotations
allowing the formation of b-sheet
Two Simplest b-Sheet Structures
Anti-Parallel b-Sheet
H-Bondings
between
Single Amino Acids
Parallel b-Sheet
Overlapped H-Bondings
between
Two Amino Acids
More b-Sheet Structures
Twisted b-Sheet Structure
with Multiple b-Strands
An Example of
a Protein
Rich in b-Sheet
Mixed b-Sheet Structure
with Multiple b-Strands
Fatty Acid
Binding Protein
Turns and Loops
• Omega Loop
• Reverse Turn (b-turn, hairpin bend)
enables reversals in the direction of
polypeptide chains.
• These reversals allow proteins to form
compact and globular structures.
•
•
•
•
( loop) also
enables reversals in the direction of
polypeptide chains.
No regular and periodic structures
But, loop structures could also be
rigid and well defined.
Invariably located on the surface
Protein-protein interactions
Coiled-coil protein
• Structural support for Cells and Tissues
 a-keratin: left-handed superhelix of two right-handed a helices.
from wool & hair, intermediate filaments in cytoskeleton, muscle protein
(myosin & tropomyosin)
 Heptad repeats; Every seventh residue in each helix, Leu holds two helix by
van der Waals interactions
 disulfide bond crosslinks: fewer – flexible, more – harder (horns, claws etc)
• Collagen: the most abundant protein of mammals, main fibrous
component of skin, bone, tendon, cartilage, and teeth. (피부미용)
What is Van der Waals force?
 Weak electric forces between neutral molecules by flucutating
polarization of nearby particles
 3 source; permanent dipole-permanent dipole forces, permanent dipoleinduced dipole force, Instantaneous induced dipole-induced dipole (London
dispersion forces)
 Named after Dutch physicist, Johannes Diderik van der Waals
Nobel prize winner in physics in 1910
van der waals interaction
-caused by transient dipoles, the momentary random fluctuation in the
distribution of the electrons of any atoms
- 1/r6 dependence
1-4. Bonds that Stabilize Folded Proteins
Folded proteins are stabilized mainly by weak noncovalent interactions
1 kcal = 4.2 kJ
Figure 1-10 Table of the typical chemical interactions that stabilize polypeptides
Tertiary Structure
3D Structure of Myoglobin
• Water-soluble
proteins
fold
into
compact structure with nonpolar
cores
• Three dimensional structure of a
polypeptide chain – grouping of
amino acids
• Generally, protein folding yields very
compact tertiary structures (10 fold).
1) 7 a-helices (70% of main chain) are
linked by turns and loops.
2) Heme = Protoporphyrin + Iron;
Prosthetic Group; Oxygen Binding
Key Aspects of Myoglobin 3D Structure
Surface
cross section
• The interior space consists almost entirely of non-polar residues.
(e.g. Val, Leu, Met, Phe, etc.)
• The charged residues are absent from the inside of a protein.
(e.g. Asp, Glu, Lys, Arg, etc.)
• The only polar residues inside are two His; iron and oxygen binding
• The surface outside consists of both polar and non-polar residues.
• There is very little free empty space inside.
General Rules of Protein Folding
•
In an aqueous environment, protein folding is driven by the strong tendency of
hydrophobic residues to be excluded from water.- called hydrophobic effect,
Why?
•
Contrasting distribution of polar and non-polar residues: the hydrophobic side
chains are buried inside, whereas the hydrophilic and charged functional
groups are headed to the outer surface.
•
All the NH and CO groups from the interiorly located peptide bonds holding
non-polar side chains (i.e. peptide bonds around hydrophobic environment) are
forced to form hydrogen bonds.
•
Therefore, these multiple hydrogen bonding enhance the interior structural
integrity by efficiently establishing a-helix and b-sheet structures.
•
Van
der
Waals
interactions
between
hydrophobic
contributes to the structural stability of a protein.
side
chains
also
Hydrophobic Effects?
Inside-Out Folding : Exception of Protein Folding
Membrane protein, Porin
• Proteins found in the outer membranes of
many bacteria
• The outside is covered with hydrophobic
residues interacting with neighboring alkane
chains. (cf. permeability barriers of the
biological membranes)
• The center of the protein contain a waterfilled channel lined with charged and polar
amino acids.
• Hydrophobic vs. Aqueous Environment
• Membrane vs. Cytosolic Proteins
Domain
• A compact and globular structural unit of a protein is often called as a domain
(i.e. pearls on a string)
• The size of a domain ranges from 30 to 400 amino acid residues.
• Different proteins can have a similar or the same domain.
• Domain is a structural working unit of a protein for the common function.
4 Domains in CD4 ; Each domain with approximately 100 Amino Acids
Motif
• Functional supersecondary structure
•DNA binding proteins
Four level of structural organization
•Primary structure: the amino acid sequence
•Secondary structure: spatial arrangement of a.a nearby in sequence, a helix and b strand
•Tertiary structure: spatial arrangement of a.a far apart in sequence.
Subunit
each polypeptide chain in a protein containing more than one polypeptide chain
Quaternary Structure
The spatial arrangement of subunits and the nature of their interaction
Cro
(Bacteriophage )
a dimer of
identical subunits
(homodimer)
Hemoglobin
Rhinovirus
Coat Protein
Hetero - Tetramer
(a2b2)
60 copies of each
of 4 subunits
Common cold
The Amino Acid Sequence of a protein Determines Its ThreeDimenssional Structure
Ribonuclease (124 AA; 4 Disulfide Bonds)
Denaturant
Reductant
A Lesson from Ribonuclease Observed by Anfinsen
8M urea
b-mercaptoethanol
slow dialysis &
oxidation 
slow refolding &
regaining
activity
remove b-mercaptoethanol
first and then remove urea
trace of
b-mercapto
ethanol
Random coiled ribonuclease
scrambled
The information needed to specify the catalytically active structure of ribonuclease
is contained in its amino acid sequence : Sequence specifies conformation !!!
Many sequences can adopt alternative conformations
How a.a. sequence specify protein structure?
How an unfolded polypeptide chain acquire the native tertiary structure?
How about secondary structure?
VDLLKN in a-helix
VDLLKN in b-strand
In many cases,
the context is very crucial in determining the conformational outcome.
(cf. the accuracy of predicting secondary structures using oligopeptides < 60 to 70%)
Each amino acid has its own preference
to form a-helix, b-sheet, or turns.
a-Helix could be default.
Val and Ile prefer
b-sheet due to
their branching at b-carbon.
Pro breaks
both a-helices and b-sheets
due to its ring structure.
Ser, Asp, Asn often disturb
the formation of
a-helix due to their
capability
to easily form
extra hydrogen bonds
with various side chains.
Protein Misfolding & Aggregation Can Cause Neurological Diseases
Prion Diseases
• Bovine Spongiform Encephalopathy (Mad Cow Disease), Creutzfeldt-Jakob Disease
(Human), Scrapie (Sheep); Disease transmitted purely by protein agents termed
“PRION”; Stanley Prusiner 1997 Nobel Prize
• Transmissible agents are aggregated fibrous forms of a specific protein.
• These protease resistant aggregated proteins are often referred as amyloid forms.
• These amyloid fibers are derived from a normal cellular protein, called PrP, in brain.
• Structural conversion from a-helices to b-sheets
b-amyloid plaques
Prions
Alzheimer Disease and Parkinson Disease
• amyloid plaques  Ab (b-amyloid peptide)  APP (amyloid precursor protein).
• Large aggregates not toxic, smaller aggregate damaging cell membrane.
Cooperativeness in Protein Folding : ALL or NONE Process
Cooperative
and Sharp
Transition
1:1 Mixture (Folded &
Unfolded Proteins),
no half-folded protein
Computational prediction of folding is not yet
reliable
• Ab initio method
- Equilibrium conformation is the global free-energy minimum
- potential energy parameter is accurate (H-bond, van der Waals etc)
- key intermediates?
- oligomerization can not be addressed although very many globular
proteins are oligomeric.
Protein folding funnel
STILL More to Give a Thought……
• Progressive Stabilization of Intermediates during Folding rather than random search
• Prediction of Three Dimensional Structure from Amino Acid Sequence?
- ab initio prediction
- knowledge-based methods
• Post-Translational Modification
- Phosphorylation: serine, threonine, and tyrosine, signaling switch
- Glycosylation: Asn (N) and Ser and Thr (O-GlcNAc), solubility increase and protein-protein
interaction
- Acetylation: N terminal of proteins, resistant to degradation.
- Hydroxylation: hydroxylation of proline in collagen stabilization, Vitamin C deficency
- Carboxylation: glutamate in prothrombin, Vitamin K deficency - hemorrhage
- Acylation: additon of a fatty acid to a-amino group or cysteine sulfhydryl group
-Carbamylation
Green fluorescent protein (GFP)
•composed of 238 amino acids (26.9 kDa),
originally isolated from the jellyfish Aequorea
victoria
•fluorescens green when exposed to blue light
•Used as a reporter of expression & biosensor
•The GFP gene can be introduced into
organisms (bacteria, yeast and other fungal
cells, plant, fly, and mammalian cells)
•2008 Nobel Prize in Chemistry : Martin
Chalfie, Osamu Shimomura and Roger Y.
Tsien
Fluorescence of GFP chromophore by
•A typical beta barrel structure
cyclization reaction including rearrangement
and oxidation
 Cleavage after protein synthesis
- digestive enzymes (pancreas, intestine)
- blood clotting factor (fibrinogen  firbrin)
- hormone, viral proteins
Nuclear localization of a steroid receptor
(+) corticosterone
Summary
• Proteins are built from a repertoire of 20 amino acids
• Peptide bond
• Protein structure; four levels
- primary structure
- secondary structure (a helix, b sheet, turns and loop),
- tertiary structure
- quarternary structure
• protein folding & misfolding or aggregation
• Protein modification
•How to visualize molecular structures using pymol  homeworks