Principle of protein structure and function

1
Principles of Protein Structure
By: Asheesh Kumar Pandey

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
Amino Acids Continued…
• Chirality – amino acids are enatiomorphs, that is mirror images exist – only the
L(S) form is found in naturally forming proteins.
Primary Structure

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)
Ca : a-carbon (chiral) NH3
+
: amino group COO-
: carboxyl group

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)

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

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

Amino Acids
Hydrophilic Hydrophobic

The amino-acid side chains have different
tendencies to participate in interactions
• Hydrophobic residues: van der Waals interactions – tendency to
avoid contact with water and pack against each other 
hydrophobic effect
- Ala & Leu are strong helix-favoring residues, Proline are not
because its backbone nitrogen isn’t available for H-bond
- Aromatic side chain of Phe participates in weakly polar
interactions
• Hydrophilic residues : Hydrogen bonds to one another, to peptide
backbone, to polar organic molecules, and to water.
- pKa shift: Asp & Glu (57 in hydrophobic interior or nearby (-)
charge), Lys (106 in ?)
- His: most versatile, most often found in enzyme active sites, pKa is
6, neutral, proton donator and acceptor

- Arg: completely protonated at neural, compared to Lys?
- Cys: common in enzyme active site, most powerful nucleophile.
Compared to Ser?
• Amphipatic residues : both polar and nonpolar character
- Lys: hydrophic charged region, long hydrophobic region
(methylene) involved in van der Waals interactions with
hydrophobic side chains
- Tyr: pKa is 9, in some enzyme active site, hydroxyl group can be
donor and acceptor of H-bond. Aromatic ring can form weakly polar
interactions
- Trp: similar to Tyr, indole amide hydrogen don’t ionize.
- Met: least polar among amphipatic residues, thioether sulfur is
excellent ligand for metal ions.

Genes and Proteins
The genetic code
The genetic code is degenerate

The flow of genetic information in prokaryotes (left) and eukaryotes (right)
Genes and Proteins
Splicing
And ?

• Alternative splicing can lead to truncated
proteins, proteins with different stretches in
the middle, and frameshifts.
• Coding sequences can also be modified by
RNA editing; some nucleotides can be
changed and additional nucleotides inserted
into the mRNA sequence before translation.

Genetic code organization
• Single-base changes (single-nucleotide
polymorphism) in the third position in a codon
produce the same amino acid. Changes
elsewhere in the codon produce a different
amino acid, but with the same physical-chemical
propherties. The second base specifies if the
amino acid is polar or hydrophobic. 
Conservative substitutions.

Genes and Proteins
Table of the frequency with which one amino acid is replaced by others
in amino-acid sequences of the same protein from different organisms

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.

• 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.

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

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

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
]

Titration curve of glycine
H
C H
COO-
H3N
+ Neutral
form

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

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.

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

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

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.

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

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

Asymmetry
• Fischer projections are a shorthand way to write molecules with chiral
centers

Asymmetry
• All α-amino acids from proteins have the L-stereochemical
configuration

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.

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)

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

Nomenclature
• Nonhydrogen atoms of the amino acid side chain are named in sequence
with the Greek alphabet

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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

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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

Rotational Properties of Peptide Bonds
φ : 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
Peptide bonds are rigid…
But,
the bonds containing the a-carbon between two peptide
bonds
can be rotated from -180o
to +180o
.

Extended polypeptide chain showing the typical backbone bond lengths and angles
The Peptide Bond
Ramachandran plot

Typical beta turn
Importance and Determinants
of Secondary Structure
Prediction of secondary structure elements from a. a sequence is accurate
to only about 70%.  convenient way of fold classification
N
N+3
beta turn, reverse turn, hairpin turn

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

Figure 1-13. The alpha helix
1-6. Properties of the Alpha Helix
N
N+4

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Alpha Helix Continued
• There are 3.6 residues
per turn
• A helical wheel will
outline the surface
properties of the helix
Secondary Structure

Table of helical parameters
Properties of the Alpha Helix
 Lipid bilayer thickness? 30A. To span the cell membrane, how long
helix?
at least 20 residues long helix All helices in real protein structures are right-handed. Why?
because of steric hindrance caused by L-configuration
 Helix dipole increase with increasing length of the helix
At the N-terminal ends of helices  negative side chain

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.

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.

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.

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.

Common Secondary Structure Elements
• The Beta Sheet

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Beta Sheets
Secondary Structure

55
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

Figure 1-11. Ramachandran plot
1-5. Importance and Determinants
of Secondary Structure
Peptide back bone C=O and N-H tend to hydrogen bond with one another,
which result in the secondary structure. Especially in the interior of proteins.

Secondary structure: reverse turns

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.

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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

The dihedral angles at Cα
atom of every residue
provide polypeptides requisite conformational
diversity, whereby the polypeptide chain can fold
into a globular shape

Structure Phi (Φ) Psi(Ψ)
Antiparallel β-sheet -139 +135
Parallel β-Sheet -119 +113
Right-handed α-helix +64 +40
310 helix -49 -26
π helix -57 -70
Polyproline I -83 +158
Polyproline II -78 +149
Polyglycine II -80 +150
Phi & Psi angles for Regular Secondary
Structure Conformations
Table 10
Secondary Structure

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*

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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

65
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

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Other (Very Rare) Helix Types - Π
• Less favorable geometry
• 4 residues per turn with i+5 not i+4
• Squat and constrained
Secondary Structure

Supersecondary structure:
Crossovers in β-α-β-motifs
Right handed
Left handed

• 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

EF Fold
Found in Calcium binding proteins such as Calmodulin

•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

•The beta-alpha-beta-alpha-beta subunit
•Often present in nucleotide-binding proteins
Rossman Fold

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

Tertiary structure examples: All-α
Alamethicin
The lone helix
Rop
helix-turn-helix
Cytochrome C
four-helix bundle

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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

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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

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

80
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

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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

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There are Different Forms of
Classification apart from Structural
• Biochemical
– Globular
– Membrane
– Fibrous
myoglobin
Collagen
Bacteriorhodopsin

Tertiary structure examples: All-β
β sandwich β barrel

Tertiary structure examples: α/β
placental ribonuclease
inhibitor
α/β horseshoe
triose phosphate
isomerase
α/β barrel

Four helix bundle
•24 amino acid peptide with a hydrophobic surface
•Assembles into 4 helix bundle through hydrophobic regions
•Maintains solubility of membrane proteins

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

Domains are independently folding structural units.
Often, but not necessarily, they are contiguous on the peptide chain.
Often domain boundaries are also intron boundaries.

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:

• 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

Figure 1-33. Three-dimensional structure of the bacterial potassium channel
1-11. Membrane Protein Structure
•View looking down the channel
•The pore-forming loop, 2 K+
ions
• Nobel prize in chemistyr 2003
- Roderick Mackinnon (kcsA K+ channel)
- Peter Agre (water channel)

"for structural and mechanistic studies of ion channels"
Roderick Mackinnon succeeded in
determining the first high-resolution
structure of an ion channel, the kcsA
K+ channel from streptomyces lividans.
The Nobel Prize in Chemistry 2003The Nobel Prize in Chemistry 2003

13.6 Specific channels increase the permeability of
some membranes to water
-Some tissues need to transport water.
-kidney, secretion of saliva and tears.
※ Aquaporin (Peter Agre in red-blood-cell membrane)
-Water channel. 24kda membrane protein
-6 membrane spanning helices.
-Positive residues in the center prevent the transport of protons through
aquaporin; maintain proton gradients

Potassium and sodium ion
• The atomic radius K+ is 1.33 Å and that of Na+ is 0.95 Å .
For the potassium ions the
distance to the oxygen atoms
in the ion filter is the same as
in water.
The sodium ions, which are
smaller, do not fit in between
the oxygen atoms in the filter.
This prevents them from
entering the channel.

The different kinds of K+ channels gating (opening)
• ligand gated
- dependent on the intracellular Ca2+ concentration
- the level of certain G-protein subunits in the cell
- cytoplasmic or extracellular domains for binding ligands.
• voltage gated
- the membrane voltage-dependent.
- integral membrane domains for sensing voltage differences.

The Structure of the Potassium Channel
four usually identical subunits that encircle with four-fold symmetry
→ inner, outer, pore α -helix

Molecular surface of KcsA and contour of the pore
the entire internal pore

a pore helix (red) and a selectivity filter (gold). Blue mesh shows
electron density for K+ ions and water along the pore.
The ion conduction pore of K+ channelsThe ion conduction pore of K+ channels

The ion conduction pore of K+ channelsThe ion conduction pore of K+ channels

K+ channels conduct K+ ions specifically because the
selectivity filter contains multiple binding sites that mimic
a hydrated K+ ion’s hydration shell. Potassium channels
achieve high conduction rates by exploiting electrostatic
repulsion between closely spaced ions and by coupling the
conformation of the selectivity filter to ion binding within the
filter.
Selective ion conduction

Figure 1-34. Illustration of the ordered arrays of water molecules surrounding exposed
1-12. Protein Stability
: Weak Interactions and Flexibility
Water’s role in weak interactions
1.Small enthalpy difference.
2. Hydrophobic effect – entrophy
(nonpolar groups in water tend to be
Surrounded by a cluster of water mols
•Folding decrease the entropy of the proteins
; but increase of water entropy is much bigger
(hydrophobic effect)
The folded protein is a thermodynamic compromise
•Stability is a net loss of free energy (entropy + enthalpy)
Free energy difference between the folded and unfolded states;
~21-42kJ/mole, marginally stable.

Figure 1-35. Computed circular dichroism spectra for the evaluation of protein conformation
• CD shows protein conformation.
• Denaturants (urea, guanidinium
hydrochloride, SDS) competes for
H-bonds with polar groups of the
back bone and side chains.
• Features of themophilic proteins
- more salt bridges
- more hydrophobic interactions &
shorter loops
Protein structure can be disrupted by a variety of agents

Figure 1-36. Results of a molecular dynamics simulation of two interacting alpha helices
All chemical bonds are flexible because of
Vibration and rotation
Proteins are much more flexible because of
weak interactions to break and reform frequently
– Thermal fluctuation is essential for protein
function (up to a few angstroms)
- Adjust to the binding of ligand or substrate
- Water penetration into the interior of the
protein
The marginal stability of protein tertiary structure allows
proteins to be flexible

Figure 1-37. The structure of the small protein bovine pancreatic trypsin inhibitor, BPTI
: Post-Translational Modifications
1.Disulfide bond between cysteine side chains
-Oxidation of two sulfhydryl groups in ER
- not found in most intracellular proteins,
but common in secreted proteins
Covalent bonds can add stability to tertiary structure

Figure 1-38. Stabilization by coordinate covalent bonds
2. Coordinate covalent bonds
-Coordination of a metal ion to side chains or water molecules
- Ca2+
, Zn2+
most common
-removal of the metal ions can leads to denaturation (EDTA)

Figure 1-39. Examples of stabilization by cofactor binding
3.Organic or organometallic cofactor at the active site
Pyridoxal phosphate in D-amino
acid aminotransferase Heme iron in myoglobin
Methionine S and heme iron in Cytochrome C
Porphyrin cofactor - covalent bonds
PQQ in polyamine oxidases

Figure 1-40. Table of post-translation modifications affecting protein stability
Glycosylation at serine, threonine or asparagine residues
-N-glycosylation site : NxS/T motif
-Most important for protein stability, folding, protein-protein recognition
(blood cell surface proteins, prevent cells from sticking to one another, cell walls)
-Deglycosylation can lead to unfolding or to aggregation. Stability change!
-Generally not alter the tertiary structure of a protein, Crystal Structure?
Phosphorylation and N-acetylation are
reversible and conformational switches
Histones or DNA methylases &
Demethylases (JMJD2A, LSD1)
Histone acetylation & deacetylation
(CBP, HDACs)

Each histone is organized
in two domains, a characteristic ‘histone fold’ and an unstructured
N-terminal ‘tail’. The histone-fold domains constrain the
DNA in a central core particle and, thereby, restrict access of
DNA-binding proteins.
This histone tail is a flexible amino terminus of 11-37 residues.
Several positively charged lysine side chains in the histone tail may
Interact with linker DNA, and the tails of one nucleosome likely interact with
Neighboring nucleosomes  higher-order coiling.

The histone tail lysine, especially those in H3 and H4, undergo reversible
acetylation and deacetylation by enzymes such as CBP (P300) and HDACs
In the acetylated form, the positve charge of the lysine e-amino group is
neuralized. This eliminate its interaction with a DNA phosphate group.
So the greater the acetylation of histone N-terminus, the less likely chromatin
is to form condensed 30-nm fibers and possibly higher-order folded structures.

Lac repressor tetramer binding to DNA
1-14. The Protein Domain
Globular proteins are composed of structural domains
-Domain is a structural and functional unit composed of generally continuous amino
acids (50~200 a.a.)
-Domains have hydrophobic cores
Tetramerization and DNA-binding domain
Alanine racemase
Interuption!

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

114
• 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

• Stability: reduction of surface to volume ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity (allostery)
Structural and functional advantages of
quaternary structure

Quaternary structure of
multidomain proteins

117
Cooperativity
Co-location of
Function
Combination
Structural
Assembly
Hemoglobin:
Enhanced binding
capability of oxygen
Glutamine sythetase:
Controlled use of
Nitrogen from
Multiple active sites
Immunoglobulin:
Multiple receptor
responses
Actin:
Giving the cell shape
and form

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

Protein
Families
• Have similarities in amino acid sequence and 3-D
structure
• Have similar functions such as breakdown
proteins but do it differently

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

Different Subunit Proteins
• Hemoglobin
–2 α globin
subunits
–2 β globin
subunits

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

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

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

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

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

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

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

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

Principle of protein structure and function

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

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

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

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

Lysozyme
• Non-covalent bonds hold the polysaccharide in the
active site until the reaction occurs

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

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

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

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

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

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

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

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

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

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

Oxygen Binding Curves
EOC Problem 6
gets you further
into cooperativity in
oxygen binding.
Knowing this will
help in Class.

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.

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.

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

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

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

AD plaque Neurofibrillary tangle (PHF)
Alzheimer’s disease

Amyloid precursor protein (APP)
(TACE, ADAM10)
(PSEN)

• 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

• Kuru
• Fatal Familial Insomnia
(FFI)
• Creutzfeldt-Jakob disease
(CJD)
• Scrapie
• Bovine Spongiform
Encephalopathy (BSE)
• Chronic Wasting Disease
(CWD)
Prion DiseasesPrion Diseases
HumanHuman AnimalAnimal

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.

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

Principle of protein structure and function

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