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Protein: Structure and Function

Books:
1. Lehninger Principle of Biochemistry ( by Nelson and Cox)

Amino acid

Properties:
1. -carbon is bonded to four different groups except glycine
2. -carbon is a chiral center
3. Two possible stereoisomers (called enantiomers)
4. Optically active (except glycine): rotate plan polarized light
5. Can act as acids and bases

Amino acids in the human body


Essential amino acid
1.
2.
3.
4.
5.

Leucine
Isoleucine
Valine
Histidine
Lysine

6.
7.
8.
9.

Methionine
Phenylalanine
Threonine
Tryptophan

Obtained from
nutrition

Non-essential amino acid


1.
2.
3.
4.
5.
6.
7.

Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamic acid
Glutamine

8.
9.
10.
11.

Glycine
Proline
Serine
Tyrosine

Synthesized by
the body

Non-polar, aliphatic R groups

Gly/G

Ala/A

Leu/L

Pro/P

Ile/I

Val/V

Met/M

Polar, uncharged R groups


1. Cysteine forms dimer
called cystine
2. Disulfide residues are
strongly hydrophobic
Ser/S

Thr/T

Cys/C

Asn/N

Gln/Q

Positively charged R groups

Lys/K

Arg/R

His/H

Negatively charged R groups

Asp/D

Glu/E

Aromatic R groups

Phe/F

Tyr/Y

1. Non-polar
2. Hydrophobic
3. Form H-bond
4. Absorb UV light (280 nm)

Trp/W

pKa values for carboxyl and amino groups

Amino acid have characteristic titration curve


1. Without ionisable R group

pK1 = for acid


pK2 = for base
pI = (pK1 + pK2 )/2

2. With ionisable R group

pI = ?

2. With ionisable R group

pI = ?

Protein
1. All protein polymers are constructed from the same set
of 20 amino acids.
2. Polymers of proteins are called polypeptides.
3. A protein consists of one or more polypeptides folded
and coiled into a specific conformation
4. The physical and chemical characteristics of the R group
determine the unique characteristics of a particular
amino acid.

Peptide Bond

Properties of Peptide bond


1. Planar
2. Double bond character (which prevent rotation
about this bond)
3. Uncharged (helps to form tightly packed globular
structure
4. Two conformations possible (cis and trans)
5. All peptide bonds are in proteins are trans
6. Other than peptide bonds in protein helps to take
many conformational structure
7. Average half-life of peptide bond is 7 years
(intracellular condition)

Fully extended polypeptide chain

Both bond can rotate

and are zero

trans-Peptide group

cis-Peptide group

Peptide and Protein


Peptides:
1. Short polymers formed from the linking of (usually less
than or equal to 100) amino acids and comprise
2. Some of the basic components of human biological
processes, including enzymes, hormones, and antibodies.

Protein:
1. A functional, polypeptide chain composed of at
least around fifty amino acids put together.
2. They play a critical role in biochemical reactions
within cells.

Types of Protein
1. Simple protein: contain only amino acid residues
2. Conjugated proteins: contain permanently associated chemical
components in addition to amino acids
(a) Lipoproteins: contain lipid
(b) glycoproteins: contain sugar
(c) metalloprotein: contain specific metal

Structure of Protein
Primary Structure
I.

The primary structure determines the folding of the


polypeptide to give a functional protein

II. Polar amino acids (acidic, basic and neutral) are


hydrophilic and tend to be placed on the outside of the
protein.
III. Non-polar (hydrophobic) amino acids tend to be placed
on the inside of the protein
IV. The possible conformations are very large
V. Most are useless, natural selection picks out the best

Primary structure of protein


1. Starting of the polypeptide chain :
N-terminal (amino group)
2. End terminal of the polypeptide chain:
C-terminal (carboxylic group)

1.
2.
3.
4.
5.
6.
7.

Elongated
Dynamic, heterogeneous
Very rich in H-bond
Every protein has unique amino acid sequence
Sequence decide the mechanism of action
Sequence determine the 3-D structure
Sequence reveals about its evolutionary history

Human insulin

Molecular interactions
1. Strong interactions
(a) Covalent bonding
(b) Ionic bonding
(c) Resonance bonding
2. Weak Interaction
(a) Van der Waals interaction
(i) Polar-polar
(ii) Polar-non polar
(iii) Non polar- non polar
(b) Hydrogen bonding
3. Effect of medium
(a) Screening of field
(b) Surface interaction
(c) Hydrophobic environment
(d) Hydrophilic environment

Secondary structure of protein


The folding of the N-C terminals of the chain
using many different interactions
The polypeptide chain can fold into regular structures like:
1. Alpha helix
2. Beta-sheet
3. Turns and loops
These are called secondary structure which helps to form final 3-D structure

Alpha-helix
1. It is coiled structure stabilized by intra-chain H-bonds
between NH and CO groups (situated 4 residue ahead in
the sequence) except end terminal groups
2. Pitch of alpha-helix 5.4 A
3. Both right handed and left handed helix are allowed ,
however right handed alpha-helices are energetically
more favourable because there is less steric clash
between the side chain and backbone
4. Content in the protein: alpha-helices may be 100%
5. Glycine, serine and threonine make amino terminal
residue (N-cap) in alpha-helix
6. Glycine and asparagine makes carboxyl terminal (C-cap)
of alpha-helix

Representation of -helices in protein


Helical Wheel: Each residue can be plotted every
360/3.6=100 around a circle or spiral

-helices
H bond between residues i, i+4

Rise per residue, d = 1.5


# of residues per turn, n = 3.6

Pitch of helix= n x d = 5.4

The -helix has a dipole moment

The dipole of a peptide unit.


Numbers in boxes give the
approximate fractional charges
of the atoms of the peptide unit

The Dipoles of
peptide units are
aligned along the
helical axis

Beta- sheet/strand
1. It is stabilized by H-bonding between chains
2. This secondary structure may associated through side chain interactions
and form super-secondary structure called motif
3. Beta-sheet is almost fully extended
4. The distance between adjacent amino acids along a beta-strand is
roughly 3.5 A (in contrast to 1.5 A in alpha-helix)
5. A beta- sheet is formed by linking two pr more beta strands by H-bonds
6. Beta strand represented by broad arrows pointing in the direction of Cterminal
7. Beta sheet formation is important in fatty acid-binding proteins and
lipid metabolism

-Strand

The side chain (green) are alternatively above and below the plane of the strand

Anti-parallel -sheet
C

C
Adjacent -strand run in opposite direction. H-bond between
NH and CO groups connect each amino acid on an
adjacent strand and stabilize the structure

Parallel -sheet

C
Adjacent -strand run in same direction. H-bond between NH
and CO groups connect each amino acid on one strand with
two different amino acids on the adjacent strand

Mixed -sheet

Turns and Loops


1. Polypeptide chain can change direction with the help of turn and loops
2. CO group of residue i is H-bonded with NH group of residue i+3
3. This particular H-bond interaction stabilizes abrupt changes in the
direction of polypeptide chain
4. Turn and loops connect alpha-helices and beta strand and allow a
peptide chain to fold back on itself to make a compact structure
5. Loops often contain hydrophilic residues and are found on the protein
surface
6. Turn or loops contain 5 residues or less
7. Beta turn connects different anti-parallel beta strands

Ramachandran plot
Psi ()
no steric
clashes

Phi ()

Phi () and Psi () rotate,


allowing the polypeptide to assume
its various conformations

permitted
if atoms are
more closely
spaced

some conformations of the


polypeptide backbone result in
steric hindrance and are disallowed
glycine has no side chain and is
therefore conformationally highly
flexible (it is often found in turns)

Tertiary structure of protein


Spatial arrangement of amino acid residues that are far
apart in the sequence
1. This folding is sometimes held together by strong covalent bonds
(e.g. cysteine-cysteine disulphide bridge)
2. Bending of the chain takes place at certain amino acids
(e.g. proline)
3. Hydrophobic amino acids tend to arrange themselves inside
the molecule
4. Hydrophilic amino acids arrange themselves on the outside

Quaternary structure of protein


1. Polypeptide chains can assemble into multi sub-units
2. Sub units are spatially arranged
3. Helix-loop-helix: two helices connected by a turn
4. Coiled-coil: two alpha helices interact in parallel through
their hydrophobic edge
5. Helix-bundle: several alpha-helices that associate in an antiparallel manner
6. Beta-alpha-beta unit: two parallel beta strand linked to an
intervening alpha helix by two loops

Levels of structure in proteins

Protein structure: overview


Structural element

Description

primary structure

amino acid sequence of protein

secondary structure

helices, sheets, turns/loops

super-secondary structure (motif)

association of secondary structures

domain

self-contained structural unit

tertiary structure

folded structure of whole protein


includes disulfide bonds

quaternary structure

assembled complex (oligomer)


homo-oligomeric (1 protein type)
hetero-oligomeric (>1 type)

Physical parameters of protein


Size of the protein roughly between 1nm
to 10nm
Persistence length of protein ranges
between 0.3 nm to 0.8 nm
Elastic modulus of protein ranges between
1200 to 2000 pN/nm2

The protein structure must obey

1. The bond lengths and bond angles should be


distorted as little as possible
2. No two atoms should approach one another more
closely than is allowed by there van der Waals radii
3. The amide group must remain planar and in the
trans configuration. This allows only rotation about
the two bonds adjacent to the alpha-carbon
4. Some kind of non-covalent binding is necessary to
stabilized a regular folding

Structure of proteins
Domain structures core is exclusively built from
helices
Domain structures core comprises of antiparallel
sheets, usually two sheets packed against each other
/ Domain structures made from combinations of
-- motifs that form a predominantly parallel
sheets surrounded by helices

Structure of proteins

Human plasma retinol


binding protein. Retinol
molecule (vitamin A)
bound inside the barrel

Triosephosphate
isomerase

Solving Protein Structures


Atomic resolution pictures of macromolecules
X-ray Crystallography (first applied in 1961 - Kendrew & Perutz)
NMR Spectroscopy (first applied in 1983 - Ernst & Wuthrich)
Structure

Function

Structure

Mechanism

Structure

Origins/Evolution

Structure-based Drug Design


Solving the Protein Folding Problem

QHTAWCLTSEQHTAAVIWDCETPGKQNGAYQEDCA
HHHHHHCCEEEEEEEEEEECCHHHHHHHCCCCCCC

Crystallographic structure of Myoglobin

(1958, Sir John Kendrew)

10

Protein Structure
solved by X-ray crystallography

PDB contains 75000 structures mostly determined by X-ray


crystallography and NMR. About 3-5 new structures per day

Total
Yearly

Importance of Protein Structure


Using electrophoresis, Pauling showed that individuals with
sickle cell disease had a modified form of Hb
Hemoglobin A: Val-His-Leu-Thr-Pro-Glu-Glu-LysHemoglobin S: Val-His-Leu-Thr-Pro-Val-Glu-Lys-

sticky patch causes hemoglobin S to agglutinate (stick together) and form


fibers which deform the red blood cell

Protein folding: Levinthals paradox


101 residues.
each residue can assume three different conformations
the total number of structures would be 3100,
which is equal to 5 1047
If it takes 10-13 s to convert one structure into another
the total search time would be 5 1047 10-13 s
which is equal to 5 1034 s, or 1.6 1027 years.
The enormous difference between calculated and actual
folding times is called Levinthal's paradox.

There should be some pathways for folding

Factors affecting protein folding


1. Space packing
proteins are like liquid and gases instead of crystalline solid
it helps in forming structure but space packing is not enough
2. Internal residue: Folding is directed mainly by internal residues
not by surface residues. (Hydrophobic force-driven folding)
3. Protein structures are hierarchically organized
4. Protein structures are highly adaptable
5. Secondary structure can be context dependent and can be
predicted by algorithms
6. Changing the fold of a protein

Speed limit of protein folding

For a single domain protein


The approximate folding time (folding) is given by N/100 s
-protein fold faster than the protein or protein
folding = k exp(G/kBT)

Protein folding
Hydrophobic effect
Conformational entropy
Electrostatics
Hydrogen bonding
van der Waals interaction

The main driving force for folding water soluble globular


protein molecules is to pack hydrophobic side chains into
the interior of the molecule , thus creating a
HYDROPHOBIC CORE &
HYDROPHILLIC SURFACE.
Problem- How to create such a hydrophobic core from
a protein chain ???

Protein folding
Insulin

?
Compact (in general)
Defined structure

Molten Globule
1. Secondary structure that is present in a native
protein forms within a few microsecond
2. This is because of hydrophobic collapse
3. It is larger by (5-15%) in size of native conformations
4. Side chains are not ordered/packed
5. Structure fluctuation is much larger
6. Not thermodynamically stable

Energy landscape governs folding


unfolded

100 kBT

Degree of
nativeness

Folding protein
moves over energy
surface from
unfolded to
folded state:

folded

H= bond stretching + bending of angles +


Bond rotations + van der waals interaction +
electrostatic interaction

Folding is a complex process


Macromolecules must fold into
correct shape to function properly:

Structure

Misfolding (non-native structures)

Function

Disease

Many diseases with large impacts involve protein misfolding:


Alzheimers Disease
A peptide
Parkinsons Disease
-synuclein
Creutzfeldt-Jakob disease
Prion

Huntingtons Disease
Huntingtin protein
Amyotrophic Lateral Sclerosis
Superoxide dismutase
Type II Diabetes
Amylin

Misfolding and aggregation are complex


Many different species and steps involved in misfolding and aggregation:
protein
synthesis

unfolded

partially
unfolded

natively
folded

ribosome

amyloid
fibrils

misfolded
partially-unfolded
and aggregated

degraded
fragments

disordered
aggregates

non-native
structured
aggregates
Chiti & Dobson, Annu. Rev. Biochem., (2006)

Force-extension curves
Move traps apart at constant rate to stretch
handles and apply force to a single-molecule:

unfolding
molecule unfolds

Apparently
two-state
folding

refolding

stretching
handles
molecule unfolds

50 mM MOPS, 200 mM KCl, pH 7.0

Reconstructing landscapes from FECs


Based on Jarzysnki equality for relating equilibrium free energy to
non-equilibrium work:

Force

But energy
is dissipated!

Work

Geqm W

Distance

Probability

Recover equilibrium energy from


fluctuations in work done:

Geqm
W
Work

Method to calculate free energy


profile G(x)

Jarzynski equality:

Hummer & Szabo, PNAS (2001)

Geqm
W

= exp noneqm
exp
k BT
k BT

Many applications, never


validated experimentally

Jarzynski, Phys. Rev. Lett. (1997)

Reconstructing energy landscape profiles


Using equilibrium probability distributions:

G ( x ) =
k BT log P ( x )

Time (s)

Probability density

Extension (nm)

Invert

Free energy (kJ/mol)

G ( x )
P ( x ) exp

k
T
B

Thousands of
transitions!

Extension (nm)
Woodside et al., Science (2006)

Deconvolution to remove effects of compliant handles:


P(x)

G(x)

raw data
Extension (nm)

parameterfree model
for folding

Free energy (kJ/mol)

Residual

Probability

deconvolution

Slow and time consuming


experiments

Woodside et al.,
PNAS (2006)
Extension (nm)

Computational approach for protein folding


1. Energy minimization
(a) Steepest
(b) Conjugated gradient
2. Monte Carlo Simulation
(a) Random Sampling
(b) Stimulated annealing
3. Molecular dynamics
(a) Compute conformational change
(b) Calculate trajectories at thermal condition and fond
the ensemble averaged physical quantity

Protein folding/unfolding

Denaturants
high temperatures
- cause protein unfolding, aggregation
low temperatures
- some proteins are sensitive to cold denaturation
heavy metals (e.g., lead, cadmium, etc.)
- highly toxic; efficiently induce the stress response
proteotoxic agents (e.g., alcohols, cross-linking agents, etc.)
oxygen radicals, ionizing radiation
- cause permanent protein damage
chaotropes (urea, guanidine hydrochloride, etc.)
- highly potent at denaturing proteins;
often used in protein folding studies

Force spectroscopy

AFM

Optical Tweezers

Protein-Protein Interaction Networks


Yeast ~6000 proteins, ~3 interactions per protein, i.e. ~>20,000 interactions. Humans ~100,000
interactions

Which two proteins will interact?


AND, which will not?
The ANSWER lies in the nature of the
interacting surfaces
Nat. Biotechnol. 18, 12571261 (2000)

A-B, A-C forms poorly matched surfaces, few


weak bonds are formed, broken apart by
thermal motion
A-D offers well matched surfaces, enough
noncovalent bonds are formed to create a
stable interface

Forces driving protein-protein interaction


Long-range attractive interactions
electrostatic steering
Short-range non-covalent forces:
Hydrophobic interactions
van der Waals attraction
Hydrogen bonds
Ion pairs
Other factors:
Shape and charge complementarity
Secondary structure
Amino acid composition

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