2. 2
Primary Structure - Amino Acids
• It is the amino acid
sequence (1940) that
“exclusively”
determines the 3D
structure of a protein
• 20 amino acids –
modifications do occur
post translationally
3. 3
Amino Acids Continued…
• Chirality – amino acids are
enatiomorphs, that is mirror
images exist – only the L(S)
form is found in naturally
forming proteins. Some
enzymes can produce D(R)
amino acids
• Think about a data structure for
this information – annotation
and a validation procedure
should be included
• Think about systematic versus
common nomenclature
Primary Structure
5. Amino acids
• Polar, uncharged amino acids
– Contain R-groups that can form hydrogen bonds with water
– Includes amino acids with alcohols in R-groups (Ser, Thr, Tyr)
– Amide groups: Asn and Gln
– Usually more soluble in water
• Exception is Tyr (most insoluble at 0.453 g/L at 25 °C)
– Sulfhydryl group: Cys
• Cys can form a disulfide bond (2 cysteines can make one cystine)
6. Amino acids
• Acidic amino acids
– Amino acids in which R-group contains a carboxyl group
– Asp and Glu
– Have a net negative charge at pH 7 (negatively charged pH
> 3)
– Negative charges play important roles
• Metal-binding sites
• Carboxyl groups may act as nucleophiles in
enzymatic interactions
• Electrostatic bonding interactions
7. Amino acids
• Basic amino acids
– Amino acids in which R-group have net positive charges at pH 7
– His, Lys, and Arg
– Lys and Arg are fully protonated at pH 7
• Participate in electrostatic interactions
– His has a side chain pKa of 6.0 and is only 10% protonated at pH 7
– Because His has a pKa near neutral, it plays important roles as a proton
donor or acceptor in many enzymes.
– His containing peptides are important biological buffers
8. Nonstandard amino acids
• 20 common amino acids programmed by genetic code
• Nature often needs more variation
• Nonstandard amino acids play a variety of roles: structural, antibiotics,
signals, hormones, neurotransmitters, intermediates in metabolic cycles,
etc.
• Nonstandard amino acids are usually the result of modification of a
standard amino acid after a polypeptide has been synthesized.
• If you see the structure, could you tell where these nonstandard amino
acids were derived from?
11. Peptide bonds
• Proteins are sometimes called polypeptides since they contain many peptide bonds
H
C
R1
H3N
+
C
O
OH NH
H
C
R2
O-
C
OH
+
H
C N
R1
H3N
+
C
O
H H
C
R2
O-
C
O
+ H2O
12. Structural character of amide groups
• Understanding the chemical character of the amide is important since the peptide bond is an amide bond.
• These characteristics are true for the amide containing amino acids as well (Asn, Gln)
• Amides will not ionize:
R C
O
NH2 R C
O
NH2
13. Acid-base properties of amino acids
K1=
Gly+
+ H2O Gly0
+ H3O+
[Gly0
][H3O+
]
[Gly+
]
Gly0
+ H2O Gly-
+ H3O+
K2=
[Gly-
][H3O+
]
[Gly0
]
The dissociation of first proton
from the α-carboxyl group is
The dissociation of the second
proton from the α-amino group
The pKa’s of these two groups are far enough apart that they can be
approximated by Henderson-Hasselbalch
pK1 + logpH =
[Gly0
]
[Gly+
]
pK2 + logpH =
[Gly-
]
[Gly0
]
15. Titration of Gly
H
C H
COO-
H3N
+
H
C H
COO-
H2N
H
C H
COOH
H3N
+
pK1 pK2
Gly0Gly+
Gly-
pH 2.3 pH 9.6
From the pK values we can calculate the pI (isoelectric point) where the
amino acid is neutral.
pI ≈ average of (pK below neutral+ pKabove neutral)
So, for Gly,
pI = (pK1 + pK2)/2
= (2.3 + 9.6)/2 ≈ 6
16. General rules for amino acid ionization
• Alpha carboxylic acids ionize at acidic pH and have pKs less than 6;
So in titrating a fully protonated amino acid, alpha carboxylic acids
lose the proton first.
• Alpha amino groups ionize at basic pH and have pKs greater than 8;
So after acids lose their protons, amino groups lose their proton.
• Most of the 20 amino acids are similar to Gly in their ionization
properties because their side chains do not ionize at biological pHs.
• However, there are 5 exceptions worth noting (the amino acids with
polar charged side chains)
• Glu, Asp, Lys, Arg, His
• Each has 3 ionizible groups and thus, 3 pKs.
17. Titration curve of arginine
The neutral form of Asp is close to pH 10.8
Take the pKs for +1 and -1 from this point and average to get approximate
pI,
pI = (pK2 + pK3)/2 = (9.0 + 13.0)/2 = 11.0
18. Acid-base properties of amino acids
Amino acid α-COOH pKa α-NH3
+
pKa
R-group pKa
Gly 2.3 9.6 -
Ala 2.4 9.7 -
Val 2.3 9.6 -
Leu 2.4 9.6 -
Iso 2.4 9.7 -
Met 2.4 9.2 -
Pro 2.1 10.6 -
Phe 1.8 9.1 -
Trp 2.4 9.4 -
Ser 2.2 9.2 13
Thr 2.6 10.4 13
Tyr 2.2 9.1 10.1
Cys 1.7 10.8 8.3
Asn 2.0 8.8 -
Gln 2.2 9.1 -
Asp 2.1 9.8 3.9
Glu 2.2 9.7 4.3
Lys 2.2 9.0 10.5
Arg 2.2 9.0 12.5
His 2.4 9.2 6.0
19. More rules for amino acid ionization
• Carboxylic acid groups near an amino group in a molecule have a more acidic
pK than isolated carboxylic groups.
• Amino groups near a carboxylic acid group also have a more acidic pK than
isolated amines.
• Aromatic amines like His have a pK about pH 6.
• When titrating an amino acid that is fully protonated (ie starting at pH = 1),
the alpha carboxylic acids lose their proton first (all free amino acids have this
group), then side chain carboxylic acids, then aromatic amine side chains
(His), then alpha amino groups, then side chain amino groups.
• These rules apply to small peptides too.
20. Amino acids are optically active
• All amino acids are optically active (exception Gly).
• Optically active molecules have asymmetry; not superimposable (mirror images)
• Central atoms are chiral centers or asymmetric centers.
• Enantiomers -molecules that are nonsuperimposable mirror images
21. Asymmetry
• Molecules are classified as Dextrorotatory (right handed), D or
Levrotatory (left handed) L depending on whether they rotate the plane
of plane-polarized light clockwise or counterclockwise determined by a
polarimeter
24. Diastereomers
• Stereoisomers or optical isomers are molecules with different
configurations about at least one of their chiral centers but are otherwise
identical
• Since each asymmetric center in a chiral molecule can have two possible
configurations, a molecule with n chiral centers has 2n
different possible
stereoisomers and 2n-1
enantiomeric pairs
• Ex. Threonine and Isoleucine both have two chiral centers, and thus 4
possible stereoisomers.
26. Diastereomers
• Special case: 2 asymmetric centers are chemically identical (2
asymmetric centers are mirror images of one another)
• A molecule that is superimposable on its mirror image is optically
inactive (meso form)
27. Nomenclature
• Glx can be Glu or Gln
• Asx can be Asp or Asn
• Polypeptide chains are always described from the N-terminus to the C-
terminus
29. 29
Peptide Bond Formation
• Individual amino acids form a polypeptide chain
• Such a chain is a component of a hierarchy for describing
macromolecular structure
• The chain has its own set of attributes
• The peptide linkage is planar and rigid
Primary Structure
30. 30
Geometry of the Chain
• A dihedral angle is the angle
between two planes defined by 4
atoms – 123 make one plane; 234
the other
• Omega is the rotation around the
peptide bond Cn – Nn+1– it is
planar and is 180 under ideal
conditions
• Phi is the angle around N –
Calpha
• Psi is the angle around Calpha C’
• The values of phi and psi are
constrained to certain values
based on steric clashes of the R
group. Thus these values show
characteristic patterns as defined
by the Ramachandran plot
From Brandon and ToozeSecondary Structure
32. Properties of alpha helix
• 3.6 residues per turn, 13 atoms between H-bond donor and acceptor
∀ φapprox. -60º; ψ approx. -40º
• H- bond between C=O of ith
residue & -NH of (i+4)th
residue
• First -NH and last C=O groups at the ends of helices do not participate in H-
bond
• Ends of helices are polar, and almost always at surfaces of proteins
• Always right- handed
• Macro- dipole
33. 33
Alpha Helix Continued
• There are 3.6 residues
per turn
• A helical wheel will
outline the surface
properties of the helix
Secondary Structure
35. Introduction to Molecular Biophysics
Association of α helices: coiled coils
These coiled coils have a heptad repeat abcdefg with nonpolar residues at
position a and d and an electrostatic interaction between residues e and g.
Isolated alpha helices are
unstable in solution but are
very stable in coiled coil
structures because of the
interactions between them
The chains in a coiled-coil have
the polypeptide chains aligned
parallel and in exact axial
register. This maximizes
coil formation between chains.
The coiled coil is a protein motif that is often used to control oligomerization.
They involve a number of alpha-helices wound around each other in a highly
organised manner, similar to the strands of a rope.
36. Introduction to Molecular Biophysics
The Leucine Zipper Coiled Coil
Initially identified as a structural motif in proteins involved in eukaryotic
transcription. (Landschultz et al., Science 240: 1759-1763 (1988). Important
part of Eugenetics.
Originally identified in the liver transcription factor C/EBP which has a Leu
at every seventh position in a 28 residue segment.
37. Association of α helices: coiled coils
The helices do not have to run in the same direction for this type of
interaction to occur, although parallel conformation is more common.
Antiparallel conformation is very rare in trimers and unknown in
pentamers, but more common in intramolecular dimers, where the two
helices are often connected by a short loop.
Chan et al., Cell 89, Pages 263-273.
38. Since the dipole moment of a peptide bond is 3.5 Debye units, the alpha
helix has a net macrodipole of:
n X 3.5 Debye units (where n= number of residues)
This is equivalent to 0.5 – 0.7 unit charge at the end of the helix.
Basis for the helical dipole
In an alpha helix all of the peptide
dipoles are oriented along the
same direction.
Consequently, the alpha helix has
a net dipole moment.
The amino terminus of an alpha helix is positive and the
carboxy terminus is negative.
41. 41
Beta Sheets Continued
• Between adjacent polypeptide chains
• Phi and psi are rotated approximately 180 degrees from
each other
• Mixed sheets are less common
• Viewed end on the sheet has a right handed twist that may
fold back upon itself leading to a barrel shape (a beta
barrel)
• Beta bulge is a variant; residue on one strand forms two
hydrogen bonds with residue on other – causes one strand
to bulge – occurs most frequently in parallel sheets
Secondary Structure
43. Secondary Structure:
Phi & Psi Angles Defined
• Rotational constraints emerge from interactions with bulky
groups (ie. side chains).
• Phi & Psi angles define the secondary structure adopted by
a protein.
44. 44
Other Secondary Structures – Loop
or Coil
• Often functionally significant
• Different types
– Hairpin loops (aka reverse turns) – often
between anti-parallel beta strands
– Omega loops – beginning and end close (6-16
residues)
– Extended loops – more than 16 residues
Secondary Structure
1AKK
45. The dihedral angles at Cα
atom of every residue
provide polypeptides requisite conformational
diversity, whereby the polypeptide chain can fold
into a globular shape
48. Beyond Secondary StructureBeyond Secondary Structure
Supersecondary structure (motifs): small, discrete, commonly
observed aggregates of secondary structures
β sheet
helix-loop-helix
βαβ
Domains: independent units of structure
β barrel
four-helix bundle
*Domains and motifs sometimes interchanged*
49. 49
Secondary Structure
• The chemical nature of the carboxyl and amino groups of
all amino acids permit hydrogen bond formation (stability)
and hence defines secondary structures within the protein.
• The R group has an impact on the likelihood of secondary
structure formation (proline is an extreme case)
• This leads to a propensity for amino acids to exist in a
particular secondary structure conformation
• Helices and sheets are the regular secondary structures, but
irregular secondary structures exist and can be critical for
biological function
Secondary Structure
50. 50
Other (Rarer) Helix Types - 310
• Less favorable
geometry
• 3 residues per turn
with i+3 not i+4
• Hence narrower and
more elongated
• Usually seen at the
end of an alpha helix
Secondary Structure 4HHB
51. 51
Other (Very Rare) Helix Types - Π
• Less favorable geometry
• 4 residues per turn with i+5 not i+4
• Squat and constrained
Secondary Structure
53. • Consists of two perpendicular 10 to 12 residue alpha helices
with a 12-residue loop region between
• Form a single calcium-binding site (helix-loop-helix).
• Calcium ions interact with residues contained within the loop
region.
• Each of the 12 residues in the loop region is important for
calcium coordination.
• In most EF-hand proteins the residue at position 12 is a
glutamate. The glutamate contributes both its side-chain oxygens
for calcium coordination.
EF Hand
Calmodulin, recoverin : Regulatory proteins
Calbindin, parvalbumin: Structural proteins
55. •Consists of two α helices and a short extended amino acid chain between them.
•Carboxyl-terminal helix fits into the major groove of DNA.
•This motif is found in DNA-binding proteins, including λ repressor, tryptophan
repressor, catabolite activator protein (CAP)
Helix Turn Helix Motif
58. What is a Protein Fold?
Compact, globular folding arrangement of the polypeptide chain
Chain folds to optimise packing of the hydrophobic residues in the interior
core of the protein
60. Tertiary structure examples: All-α
Alamethicin
The lone helix
Rop
helix-turn-helix
Cytochrome C
four-helix bundle
61. 61
Tertiary Structure
• Myoglobin (Kendrew 1958) and hemoglobin
(Perutz 1960) gave us the proven experimental
insights into tertiary structure as secondary
structures interacting by a variety of mechanisms
• While backbone interactions define most of the
secondary structure interactions, it is the side
chains that define the tertiary interactions
Tertiary Structure
62. 62
Components of Tertiary Structure
• Fold – used differently in different contexts – most
broadly a reproducible and recognizable 3 dimensional
arrangement
• Domain – a compact and self folding component of the
protein that usually represents a discreet structural and
functional unit
• Motif (aka supersecondary structure) a recognizable
subcomponent of the fold – several motifs usually
comprise a domain
Like all fields these terms are not used strictly making
capturing data that conforms to these terms all the more
difficult
Tertiary Structure
63. Domains
• A domaindomain is a basic structural unit of a
protein structure – distinct from those that
make up the conformations
• Part of protein that can fold into a stable
structure independently
• Different domains can impart different
functions to proteins
• Proteins can have one to many domains
depending on protein size
65. 65
Tertiary Structure as Dictated by the
Environment
• Proteins exist in an aqueous environment where hydrophilic residues
tend to group at the surface and hydrophobic residues form the core –
but the backbone of all residues is somewhat hydrophilic – therefore it
is important to have this neutralized by satisfying all hydrogen bonds as
is achieved in the formation of secondary structures
• Polar residues must be satisfied in the same way – on occasion pockets
of water (discreet from the solvent) exist as an intrinsic part of the
protein to satisfy this need
• Ion pairs (aka salt bridge) form important interactions
• Disulphide linkages between cysteines form the strongest (ie covalent
tertiary linkages); the majority of cysteines do not form such linkages
Tertiary Structure
66. 66
Tertiary Structure as Dictated by
Protein Modification
• To the amino acid itself eg
hydroxyproline needed for
collagen formation
• Addition of carbohydrates
(intracellular localization)
• Addition of lipids (binding
to the membrane)
• Association with small
molecules – notably
metals eg hemoglobin
Tertiary Structure
67. 67
There are Different Forms of
Classification apart from Structural
• Biochemical
– Globular
– Membrane
– Fibrous
myoglobin
Collagen
Bacteriorhodopsin
70. Four helix bundle
•24 amino acid peptide with a hydrophobic surface
•Assembles into 4 helix bundle through hydrophobic regions
•Maintains solubility of membrane proteins
71. TIM Barrel
•The eight-stranded α /β barrel (TIM barrel)
•The most common tertiary fold observed in
high resolution protein crystal structures
•10% of all known enzymes have this domain
73. Domains are independently folding structural units.
Often, but not necessarily, they are contiguous on the peptide chain.
Often domain boundaries are also intron boundaries.
74. Domain swapping:
Parts of a peptide chain can reach into neighboring
structural elements: helices/strands in other domains or
whole domains in other subunits.
Domain swapped diphteria toxin:
75. • Helix bundles
Long stretches of apolar amino acids
Fold into transmembrane alpha-helices
“Positive-inside rule”
Cell surface receptors
Ion channels
Active and passive transporters
• Beta-barrel
Anti-parallel sheets rolled into cylinder
Outer membrane of Gram-negative bacteria
Porins (passive, selective diffusion)
Transmembrane Motifs
76. Quaternary Structure
• Refers to the organization of subunits in a protein with multiple subunits
• Subunits may be identical or different
• Subunits have a defined stoichiometry and arrangement
• Subunits held together by weak, noncovalent interactions (hydrophobic,
electrostatic)
• Associate to form dimers, trimers, tetramers etc. (oligomer)
• Typical Kd for two subunits: 10-8
to 10-16
M (tight association)
–Entropy loss due to association - unfavorable
–Entropy gain due to burying of hydrophobic groups - very favourable
77. 77
Quaternary Structure
• The biological function of some molecules
is determined by multiple polypeptide
chains – multimeric proteins
• Chains can be identical eg homeodimer or
different eg heterodimer
• The interactions within multimers is the
same as that found in tertiary and secondary
structures
78. • Stability: reduction of surface to volume ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity (allostery)
Structural and functional advantages
of quaternary structure
81. Useful Proteins
• There are thousands and thousands of different
combinations of amino acids that can make up
proteins and that would increase if each one had
multiple shapes
• Proteins usually have only one useful
conformation because otherwise it would not be
efficient use of the energy available to the system
• Natural selection has eliminated proteins that do
not perform a specific function in the cell
82. Protein
Families
• Have similarities in amino acid sequence and 3-D
structure
• Have similar functions such as breakdown
proteins but do it differently
83. Proteins – Multiple Peptides
• Non-covalent bonds can form interactions
between individual polypeptide chains
– Binding site – where proteins interact with one
another
– Subunit – each polypeptide chain of large
protein
– Dimer – protein made of 2 subunits
• Can be same subunit or different subunits
86. Protein Assemblies
• Proteins can form very
large assemblies
• Can form long chains if
the protein has 2 binding
sites – link together as a
helix or a ring
• Actin fibers in muscles
and cytoskeleton – is
made from thousands of
actin molecules as a
helical fiber
87. Types of Proteins
• Globular ProteinsGlobular Proteins – most of what we have
dealt with so far
– Compact shape like a ball with irregular
surfaces
– Enzymes are globular
• Fibrous ProteinsFibrous Proteins – usually span a long
distance in the cell
– 3-D structure is usually long and rod shaped
88. Important Fibrous Proteins
• Intermediate filaments of the cytoskeleton
– Structural scaffold inside the cell
• Keratin in hair, horns and nails
• Extracellular matrix
– Bind cells together to make tissues
– Secreted from cells and assemble in long fibers
• Collagen – fiber with a glycine every third amino
acid in the protein
• Elastin – unstructured fibers that gives tissue an
elastic characteristic
90. Stabilizing Cross-Links
• Cross linkages can be between 2 parts of a protein or
between 2 subunits
• Disulfide bonds (S-S) form between adjacent -SH
groups on the amino acid cysteine
91. Proteins at Work
• The conformation of a protein gives it a unique
function
• To work proteins must interact with other
molecules, usually 1 or a few molecules from the
thousands to 1 protein
• Ligand – the molecule that a protein can bind
• Binding site – part of the protein that interacts
with the ligand
– Consists of a cavity formed by a specific arrangement
of amino acids
93. Formation of Binding Site
• The binding site forms when amino acids from within
the protein come together in the folding
• The remaining sequences may play a role in regulating
the protein’s activity
94. Antibody Family
• A family of proteins that can be created to
bind to almost any molecule
• AntibodiesAntibodies (immunoglobulins) are made in
response to a foreign molecule ie. bacteria,
virus, pollen… called the antigenantigen
• Bind together tightly and therefore
inactivates the antigen or marks it for
destruction
95. Antibodies
• Y-shaped molecules with 2 binding sites at
the upper ends of the Y
• The loops of polypeptides on the end of the
binding site are what imparts the
recognition of the antigen
• Changes in the sequence of the loops make
the antibody recognize different antigens -
specificity
97. Binding Strength
• Can be measured directly
• Antibodies and antigens are mixing around in a
solution, eventually they will bump into each
other in a way that the antigen sticks to the
antibody, eventually they will separate due to the
motion in the molecules
• This process continues until the equilibriumequilibrium is
reached – number sticking is constant and number
leaving is constant
• This can be determined for any protein and its
ligandligand
98. Equilibrium
Constant
• Concentration of antigen, antibody and antigen/antibody
complex at equilibrium can be measured – equilibriumequilibrium
constant (K)constant (K)
• Larger the K the tighter the binding or the more non-
covalent bonds that hold the 2 together
99. Enzymes as Catalysts
• Enzymes are proteins that bind to their ligand as the
1st
step in a process
• An enzyme’s ligand is called a substratesubstrate
– May be 1 or more molecules
• Output of the reaction is called the product
• Enzymes can repeat these steps many times and
rapidly, called catalysts
• Many different kinds – see table 5-2, p 168
100. Enzymes at Work
• Lysozyme is an important enzyme that protects us
from bacteria by making holes in the bacterial cell
wall and causing it to break
• Lysozyme adds H2O to the glycosidic bond in the
cell wall
• Lysozyme holds the polysaccharide in a position
that allows the H2O to break the bond – this is the
transition statetransition state – state between substrate and
product
• Active siteActive site is a special binding site in enzymes
where the chemical reaction takes place
103. Prosthetic Groups
• Occasionally the sequence of the protein is not
enough for the function of the protein
• Some proteins require a non-protein molecule to
enhance the performance of the protein
– Hemoglobin requires heme (iron containing compound)
to carry the O2
• When a prosthetic groupprosthetic group is required by an enzyme
it is called a co-enzymeco-enzyme
– Usually a metal or vitamin
• These groups may be covalently or non-covalently
linked to the protein
104. Feedback Regulation
• Negative feedbackNegative feedback –
pathway is inhibited by
accumulation of final
product
• Positive feedbackPositive feedback – a
regulatory molecule
stimulates the activity of
the enzyme, usually
between 2 pathways
↑ ADP levels cause the
activation of the glycolysis
pathway to make more ATP
105. Allostery
• Conformational coupling of 2 widely separated
binding sites must be responsible for regulation
– active site recognizes substrate and 2nd
site
recognizes the regulatory molecule
• Protein regulated this way undergoes allosteric
transition or a conformational change
• Protein regulated in this manner is an allosteric
protein
106. Phosphorylation
• Some proteins are regulated by the addition
of a PO4 group that allows for the attraction
of + charged side chains causing a
conformation change
• Reversible protein phosphorylations
regulate many eukaryotic cell functions
turning things on and off
• Protein kinaseskinases add the PO4 and protein
phosphatasephosphatase remove them
107. Phosphorylation/Dephosphorylation
• Kinases capable of
putting the PO4 on 3
different amino acid
residues
– Have a –OH group on R
group
• Serine
• Threonine
• Tyrosine
• Phosphatases that
remove the PO4 may be
specific for 1 or 2
reactions or many be
non-specific
108. GTP-Binding Proteins (GTPases)
• GTP does not release its PO4
group but rather the guanine
part binds tightly to the protein
and the protein is active
• Hydrolysis of the GTP to GDP
(by the protein itself) and now
the protein is inactive
• Also a family of proteins
usually involved in cell
signaling switching proteins on
and off
110. Motor Proteins
• Proteins can move in the cell,
say up and down a DNA strand
but with very little uniformity
– Adding ligands to change the
conformation is not enough to
regulate this process
• The hydrolysis of ATP can
direct the the movement as
well as make it unidirectional
– The motor proteins that move
things along the actin
filaments or myosin
111. Protein Machines
• Complexes of 10 or more
proteins that work together
such as DNA replication,
RNA or protein synthesis,
trans-membrane signaling
etc.
• Usually driven by ATP or
GTP hydrolysis
• See video clip on CD in
book
112. Functions of Globular Proteins
• Storage of ions and molecules
– myoglobin, ferritin
• Transport of ions and molecules
– hemoglobin, serotonin transporter
• Defense against pathogens
– antibodies, cytokines
• Muscle contraction
– actin, myosin
• Biological catalysis
– chymotrypsin, lysozyme
113. Protein Interaction with Other Molecules
• Reversible, transient process of chemical equilibrium:
A + B AB
• A molecule that binds to a protein is called a ligand
– Typically a small molecule
• A region in the protein where the ligand binds is called
the binding site
• Ligand binds via same noncovalent forces that dictate
protein structure (see Chapter 4)
– Allows the interactions to be transient
114. Oxygen Binding Curves
EOC Problem 6
gets you further
into cooperativity in
oxygen binding.
Knowing this will
help in Class.
116. Bohr Effect
• Hemoglobin's affinity for oxygen is decreased in the
presence of carbon dioxide and at lower pH.
• Carbon dioxide reacts with water to give bicarbonate,
carbonic acid free protons via the reaction:
CO2 + H2O ---> H2CO3 ---> H+
+ HCO3
-
• Protons bind at various places along the protein and carbon
dioxide binds at the alpha-amino group forming
carbamate.
• This causes a conformational change in the protein and
facilitates the release of oxygen.
117. Bohr Effect
• Blood with high carbon dioxide levels is also lower in pH
(more acidic). (recall the equilibrium)
• Conversely, when the carbon dioxide levels in the blood
decrease (i.e. around the lungs), carbon dioxide is released,
increasing the oxygen affinity of the protein.
118. Bohr Effect Summary
• High CO2 in tissues
• Higher H+
• Lower pH
• Affinity for O2
decreases
• O2 released to tissues
• T state favored
• Low CO2 in lungs
• Lower H+
• Higher pH
• Affinity for O2
increases
• O2 binds hemoglobin
• R state favored
120. Amyloid diseases
Disease Protein/peptide Aggregate
Alzheimer’s disease Aβ Senile plaq
Primary systemic amyloidosis Ig light chain
Senile systemic amyloidosis Transthyretin
Diabetes type II Amylin
Hemodialysis-associated amyloidosis β2
-microglobulin
Familial systemic amyloidosis Lysozyme mutant
Huntingon’s disease Huntingtin Huntingtin inclusion
Parkinson’s disease α-synuclein Lewy body
CJD, other prion diseases PrPSc
Prion aggregate
Taupathies, Pick disease, FTDP-17 Tau protein PHF, Pick-body
121. 1) Protein (AL, ATTR, ALys)
2) Cause (spontaneous, mutation, induced)
3) Mechanism (loss or gain of function)
Amyloid diseases: modern classification
Amyloids are insoluble fibrous protein aggregates sharing specific
structural traits. They are insoluble and arise from at least 18
inappropriately folded versions of proteins
and polypeptides present naturally in the body
→ protein misfolding diseases
124. • Stanley B. Prusiner coined the term proin from Proteinaceous
infective particle
and changed to prion to sound it rhythmic.
• Prion diseases were caused by misfolded proteins.
• Elucidated the gene and mechanism by which wild type protein
bring about the
clinical disease.
PRION DISEASES
126. Classification of prion diseasesClassification of prion diseases
• Infectious/ExogenousInfectious/Exogenous
– e.g., Kuru, BSE (mad cow disease), Scrapie
– Spread by
• Consumption of infected material.
• Transfusion.
• SporadicSporadic
• Familial/HereditaryFamilial/Hereditary
– Due to autosomal dominant mutation of PrP.
127. Differences between cellular and scrapie proteinsDifferences between cellular and scrapie proteins
PrPPrPCC
PrPPrPSCSC
Solubility
Soluble Non soluble
Structure
Alpha-helical Beta-sheeted
Multimerisation state
Monomeric Multimeric
Infectivity
Non infectious Infectious
Susceptibility to Proteinase K
Susceptible Resistant
Hinweis der Redaktion
2,3,4 completely encoded by primary structure
Linear polymer theory
Set of 20 amino acids not determined until 1940
The formation of Cystine can take place between 2 polypeptide chains to make a cross link between them. Extracellular proteins often contain Cys-Cys bonds, while cellular proteins rarely have them in the m since the environment in the cell is reducing. In the presence of oxygen or oxidizing conditions, the 2 thils react to form a disulfide bond between them. Since this is a redox reaction, the hydride ion released by each thil is usually coupled to an electonacceptor reaction or in simple oxidation with oxygen, hydrogen peroxide is usually formed with further reduction to water.
These are amino acids derivatives found in proteins
4-hydroxyproline and 5-hydroxylysine are structural components of collagen a structural protein
N-formyl methionine is the N-terminal residue of all prokaryotic proteins but is usually removed as the protein matures
Gamma-carbocyglutamate is part of proteins involved in blood clotting
Methylated and acetylated amino acids are important parts of ribosomal proteins and chromosomal proteins called histones (important for chromatin formation in eukaryotes)
Amino acids with specialized roles
GABA is a glutamate decarboxylation product, dopamine and glycine are neurotransmitters
Histamine is a mediator of allergic reactions
Thyroxine is an iodine-containing hormone that stimulates metabolism
Some amino acids are important intermediates in metabolic processes
Citrulline and ornithine are important in urea biosynthesis
Homocysteine is an intermediate in amino acid metabolism
S-adnosylmethionine is methylating agent (adds methyl groups to other compounds)
Azaserine is an antibiotic
Beta-cyanoalanine is an intermediate in cyanide production in plants.
At low pH values both acid-base groups of glycine are fully protonated.
The pK values of glycine’s two ionizable groups are different enough that the Henderson-Hasselbalch equation closely approximates each phase of the titration.
pI = 1/2(pKi +pKj) Where Ki and Kj are the dissociation constants of the two ionizations involving the neutral species.
The pH at which the molecule carries no net electric charge is the isoelectric point.
At low pH values both acid-base groups of glycine are fully protonated.
The pK values of glycine’s two ionizable groups are different enough that the Henderson-Hasselbalch equation closely approximates each phase of the titration.
pI = 1/2(pKi +pKj) Where Ki and Kj are the dissociation constants of the two iionizations involving the neutral species.
The pH at which the molecule carries no net electric charge is the isoelectric point.
Enantiomers of fluorochlorobormomethane
In Fisxher projections all horizontal bonds point above the page and all vertical bonds below the page.
The designation of the relative configuration of chirl centers is the same as the absolute configuration. We can use the CORN crib mnemonic.
We look at the alpha carbon from its H atom the other substituents should read CO-R-N in the clockwise direction.
The mirror images on the top of this figure are the L and D forms.
The two other optical isomers are the Diastereomers or allo forms of the L and D forms.
The D-allo and L-allo forms are mirror images of one another just like the D and L forms
Neither allo form is symmetrically related to either of the D or L forms
Diastereomers are physically and chemically distinct. We see differences in melting points, spectra, and chemical reactivity. VERY Different from one another.
D and L isomers are mirror images of one another but the meso has internal mirror symmetry and therefore lacks optical activity.
Amino acid residues in polypeptides are named by dropping of the suffix -ine and replacying it by -yl
The C- terminus is given the name of the parent amino acid.
So this compound is called alanyltyrosylaspartylglycine. We can replace this by Ala-Tyr-Asp-Gly or AYDG
Greek letter used to identify the atoms in the glutamyl and lysyl R groups
Take Home Message – Beta sheet is a secondary structural element that is often observed in proteins, description of beta sheet structure – antiparallel strands, hydrogen bonding across strands
Take Home Message: Phi & Psi determine secondary structure
In order to compute secondary structure elements in proteins, you need to understand the spacial constraints that protein sequences are under due to the rotation around peptide bond.
Take Home Message: Ramachradan was a really smart interesting scientist 1922 – 2001
Defined - A graphical representation in which the dihedral angle of rotation about the alpha-carbon to carbonyl-carbon bond in polypeptides is plotted against the dihedral angle of rotation about the alpha-carbon to nitrogen bond.
Ramachandran – calculations that elegantly accounted for the structure and amino acid composition of collagen in 1954, training in physics and electrical engineering – really significant contribution early on to the field of structural biology – developed the above Rhamachandran plot in response to stereochemical critisicisms of the collagen structure – notable critic Crick.
G N Ramachandran used computer models of small polypeptides to systematically vary phi and psi with the objective of finding stable conformations. For each conformation, the structure was examined for close contacts between atoms. Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii. Therefore, phi and psi angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone.
In the diagram above the white areas correspond to conformations where atoms in the polypeptide come closer than the sum of their van der Waals radi. These regions are sterically disallowed for all amino acids except glycine which is unique in that it lacks a side chain. The red regions correspond to conformations where there are no steric clashes, ie these are the allowed regions namely the alpha-helical and beta-sheet conformations. The yellow areas show the allowed regions if slightly shorter van der Waals radi are used in the calculation, ie the atoms are allowed to come a little closer together. This brings out an additional region which corresponds to the left-handed alpha-helix.
Today, for beginners in biochemistry protein structures are introduced with a discussion of the Ramachandran map, which also forms the cornerstone for many discussions of protein folding.
Alpha and beta predicted by Pauling
Stabiliz
There are a number of advantages in forming oligomers:
size without loss of stability
modular construction – one gene – big protein
complex catalytic sites in enzymes
regulation – will return to this briefly later
The high affinity state is R and the low affinity state is T. Real functioning Hb is the middle sigmoid response. This shows that Hb goes through a conformational change based on oxygen concentration.