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2 times (Mid, Final Exam) Problem types: Single or multiple choice 70% + long answer 30% Place: Lecture Room 과 B131 Posting of score in Exam: on the board at room SB134, 과학원 ,
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AnnouncementAnnouncement
I am Hyun-Soo Cho, in Biology Department.This course is Molecular Biochemistry,
(4) Grading: (4) Grading: Mid exam (40%) + Final exam (40%) + attendance, reports (20%)
Mid exam (40%) + Final exam (40%) + attendance, reports (20%)
(3) Assignment (homework): (3) Assignment (homework): Please read your textbook before or/and after each class Please read your textbook before or/and after each class
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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
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(6) How to study biochemstry (6) How to study biochemstry Be familiar with English term Motivate yourself why you should study Molecular biochemistry Read text book carefully Ask any questions
Be familiar with English term Motivate yourself why you should study Molecular biochemistry Read text book carefully Ask any questions
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You may see me during this course if you want My office hours: PM 5:00-6:00 on Thursday How: First, Contact me by E-mail or telephone E-mail address: [email protected], 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
machinery dissociating from the DNA.
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.)
Key Properties of Proteins (continued)
Conformational Changes
of Lactoferrin
upon iron binding
Absolute Configuration : S
Left, Counter-Clockwise
Absolute Configuration : R
Right, Clockwise
Structure and Stereoisomerism of a-Amino Acids
Only L-amino acids are constituents of proteins.
R : functional group (side chain)
C : -carbon (chiral) NH3+ : amino group COO- : carboxyl group
Proteins Are Built from a Repertoire of 20 Amino Acids
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 AcidsBased on the Characteristics of Functional Groups
Ala, Gly, Ile,
Leu, Met, Phe,
Pro, Val
Arg, Asn, Asp, Cys,
Gln, Glu, His, Lys,
Ser, Thr, Trp, Tyr
Non Polar (Hydrophobic)Polar (Hydrophilic)
Arg, Lys, HisAsp, Glu
Basic (positive charge)Acidic (negative charge)
Most of the restPhe, Tyr, Trp
Aliphatic (linear chain)Aromatic (ring)
achiral(non-chiral)
Most
Simple
Amino
Acids
MostTypical
AliphaticAminoAcids
MostTypical
Non-PolarHydrophobic
AminoAcids
IsoleucineContains
an AdditionalChiral
Carbon
The side chain of proline is bonded toboth the nitrogen and -carbon atom.
Proline is an imino acid.
Structural flexibility is much more restricted than other amino acids.
Proline markedly influences protein architecture.
MostTypical
AromaticAminoAcids
The hydroxyl groupin tyrosine is
chemically reactive.
Tryptophan containsan indole ring.
The aromatic rings ofTyr and Trp contain
delocalized electronsabsorbing UV light.
Tryptophan and Tyrosine Can Be Usefulfor the Determination of Protein Concentration
Beer’s Law : A = clA : Absorbance,
: 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 ThrContainAliphatic
Hydroxyl Group.
Ser is morehydrophilic than Ala.
Thr is morehydrophilic than Val.
ThreonineContains
an AdditionalChiral
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, andPositively Charged Amino Acids
Histidine Ionization
Lys : -amino group
Arg : guanidium group
His : imidazole group
Very Polar,
Highly Hydrophilic,
and
Negatively Charged
Amino Acids
Aspartate : -carboxyl group
Glutamate : -carboxyl group
Asparagine (Asn)
Uncharged Derivatives of Aspartate
-carboxamide group
Glutamine (Gln)
Uncharged Derivatives of Glutamate
-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)
• 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.
FirstAmino Acid
SecondAmino Acid
Directionality of Polypeptide ChainN-terminus C-terminus
(YGGFL ≠ LFGGY)
Backbone or Main Chain(Regularly Repeating Part)
vs.Functional Group or Side Chain
(Variable Part)
One Amino Acid in a ProteinIs Called as a Residue.
Alternative Positioning ofthe Oxygen and the Hydrogen
in One Peptide Bond
Alternative Positioning ofthe Oxygen and the Hydrogen
between Neighboring Peptide Bonds
Alternative Positioning ofthe Functional Groups
Between Neighboring Residues
Formation of Polypeptide Chain
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. Why?
• 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).
Size of Polypeptide Chain
• 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
Amino Acid Sequences of Proteins
Chemical Properties of Peptide Bonds
Peptide bonds are planar
Typical Bond Lengthswithin 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 -Carbons around a Normal Peptide Bond
Balanced Configuration of -Carbons in X-Pro linkages
Rotational Properties of Peptide Bonds
: the angle of rotation about the bond between the nitrogen and the -carbon
: the angle of rotation about the -carbon and the carbonyl carbon
Peptide bonds are rigid…But,
the bonds containing the -carbon between two peptide bonds
can be rotated from -180o to +180o.
Rotational Properties of Peptide Bonds (continued)
• Ramachandran Diagram Shows the Allowed Ranges of and Rotations.
• For Some Combinations of and Rotations Are Physically Impossible due to Steric
Clashes.
• Protein folding is possible by rigidity of peptide unit and restriction of and Rotations
Proteins’ Secondary Structures
Alpha Helix, Beta Pleated Sheet, Turns, Loops
Linus Pauling and Robert Corey’s proposal – 1951The Double helix – 1953 James D. Watson, 책소개
The -Helix Is a Coiled StructureStabilized 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 -Helix
• Thus, amino acids spaced
three to four residues apart
are spatially quite close to
one another in an -helix.
Most plausible H-bondingsbetween peptide bonds in -helix
(n, n+4)
• Essentially all -helices in proteins are right
handed.
(Ramachandran diagram explains it why.)
• The -helical content of proteins range from none
to almost 100%.
• Single -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 -helices.
-helical coiled coil; superhelix;tropomyosin, keratin, fibrin;
bundles of fibers; filamentous structures
Probability of-Helix Coiling Direction
Coiling and Entwining of -Helix
Beta Pleated Sheet
Combinations of and rotationsallowing the formation of -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 -strands
• Why beta?
Anti-Parallel -Sheet
H-Bondings
between
Single Amino Acids
Parallel -Sheet
Overlapped H-Bondings
between
Two Amino Acids
Two Simplest -Sheet Structures
More -Sheet Structures
Mixed -Sheet Structurewith Multiple -Strands
Twisted -Sheet Structurewith Multiple -Strands
An Example ofa Protein
Rich in -Sheet
Fatty AcidBinding Protein
Turns and Loops
• Reverse Turn (-turn, hairpin bend)
enables reversals in the direction of
polypeptide chains.• These reversals allow proteins to form
compact and globular structures.
• Omega Loop ( 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
-keratin: left-handed superhelix of two right-handed 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 Why 7?
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 dipole-induced 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
Figure 1-10 Table of the typical chemical interactions that stabilize polypeptides
1-4. Bonds that Stabilize Folded Proteins
1 kcal = 4.2 kJ
Folded proteins are stabilized mainly by weak noncovalent interactions
Tertiary Structure
• 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).
3D Structure of Myoglobin
1) 7 -helices (70% of main chain) are linked by turns and loops.
2) Heme = Protoporphyrin + Iron; Prosthetic Group; Oxygen Binding
• 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.
Key Aspects of Myoglobin 3D Structure
Surface cross section
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 -helix and -sheet
structures.
• Van der Waals interactions between hydrophobic side chains also
contributes to the structural stability of a protein.
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 water-
filled channel lined with charged and polar
amino acids.
• Hydrophobic vs. Aqueous Environment
• Membrane vs. Cytosolic Proteins
4 Domains in CD4 ; Each domain with approximately 100 Amino Acids
• 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.
Domain
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, helix and strand
•Tertiary structure: spatial arrangement of a.a far apart in sequence.
Quaternary Structure
The spatial arrangement of subunits and the nature of their interaction
Subunit
each polypeptide chain in a protein containing more than one polypeptide chain
Cro(Bacteriophage )
a dimer ofidentical subunits
(homodimer)
HemoglobinHetero - Tetramer
(22)
RhinovirusCoat Protein
60 copies of eachof 4 subunits
Common cold
Ribonuclease (124 AA; 4 Disulfide Bonds) Denaturant
Reductant
The Amino Acid Sequence of a protein Determines Its Three-Dimenssional Structure
A Lesson from Ribonuclease Observed by Anfinsen
The information needed to specify the catalytically active structure of ribonuclease
is contained in its amino acid sequence : Sequence specifies conformation !!!
8M urea
-mercaptoethanol
trace of-mercapto
ethanol
slow dialysis &oxidation
slow refolding & regaining activity
remove -mercaptoethanol first and then remove urea
Random coiled ribonucleasescrambled
VDLLKN in -helix VDLLKN in -strand
Many sequences can adopt alternative conformations
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%)
How a.a. sequence specify protein structure?How an unfolded polypeptide chain acquire the native tertiary structure?How about secondary structure?
-Helix could be default.
Val and Ile prefer
-sheet due to
their branching at -carbon.
Pro breaks
both -helices and -sheets
due to its ring structure.
Ser, Asp, Asn often disturb
the formation of
-helix due to their
capability
to easily form
extra hydrogen bonds
with various side chains.
Each amino acid has its own preference
to form -helix, -sheet, or turns.
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 -helices to -sheets
Alzheimer Disease and Parkinson Disease• amyloid plaques A (-amyloid peptide) APP (amyloid precursor protein).• Large aggregates not toxic, smaller aggregate damaging cell membrane.
Prions
-amyloid plaques
Cooperativeness in Protein Folding : ALL or NONE Process
1:1 Mixture (Folded & Unfolded Proteins), no half-folded protein
Cooperative and SharpTransition
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
Nuclear localization of a steroid receptor
(+) corticosterone
Fluorescence of GFP chromophore by cyclization reaction including rearrangement and oxidation
•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
•A typical beta barrel structure
Green fluorescent protein (GFP)
Cleavage after protein synthesis - digestive enzymes (pancreas, intestine) - blood clotting factor (fibrinogen firbrin) - hormone, viral proteins
Summary
• Proteins are built from a repertoire of 20 amino acids
• Peptide bond
• Protein structure; four levels - primary structure - secondary structure ( helix, sheet, turns and loop), - tertiary structure - quarternary structure
• protein folding & misfolding or aggregation
• Protein modification
•How to visualize molecular structures using pymol homeworks