Protein: Structure and Function

Books:

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

 Amino acid

Properties:
1. α-carbon is bonded to four different groups except glycine
2. α-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. Leucine 6. Methionine
2. Isoleucine 7. Phenylalanine Obtained from
3. Valine 8. Threonine nutrition
4. Histidine 9. Tryptophan
5. Lysine

Non-essential amino acid

1. Alanine 8. Glycine
2. Arginine 9. Proline Synthesized by
3. Asparagine 10. Serine the body
4. Aspartic acid 11. Tyrosine
5. Cysteine
6. Glutamic acid
7. Glutamine
 Non-polar, aliphatic R groups

Gly/G Ala/A Pro/P Val/V

Leu/L Ile/I 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 Trp/W

1. Non-polar
2. Hydrophobic
3. Form H-bond
4. Absorb UV light (280 nm)
 pKa values for carboxyl and amino groups

pKa=-log10 (Ka); The smaller the pKa value, the stronger the acid
 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 =3.22 ?
2. With ionisable R group

pI = 7.59?
 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

Ψ and φ 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. Elongated
2. Dynamic, heterogeneous
3. Very rich in H-bond
4. Every protein has unique amino acid sequence
5. Sequence decide the mechanism of action
6. Sequence determine the 3-D structure
7. 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 α-helices in protein

Helical Wheel: Each residue can be plotted every

360/3.6=100° around a circle or spiral
α-helices
H bond between residues i, i+4
Rise per residue, d = 1.5 Å
# of residues per turn, n = 3.6

Pitch of helix= n x d = 5.4 Å

 The α-helix has a dipole moment

The Dipoles of
The dipole of a peptide unit. peptide units are
Numbers in boxes give the aligned along the α
approximate fractional charges helical axis
of the atoms of the peptide unit
 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 C-

terminal

7. Beta sheet formation is important in fatty acid-binding proteins and

lipid metabolism
 β-Strand

The side chain (green) are alternatively above and below the plane of the strand
Anti-parallel β-sheet

C N

N C

Adjacent β-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 β-sheet

N C

N C

Adjacent β-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 β-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 (ψ)

no steric
clashes Phi (Φ)

• Phi (Φ) and Psi (ψ) rotate,

allowing the polypeptide to assume
its various conformations

• some conformations of the

permitted polypeptide backbone result in
if atoms are steric hindrance and are disallowed
more closely
spaced
• 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 anti-

parallel 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

• α Domain structures –core is exclusively built from α

helices

• β Domain structures – core comprises of antiparallel β

sheets, usually two β sheets packed against each other

• α /β Domain structures – made from combinations of

β-α-β motifs that form a predominantly parallel β
sheets surrounded by α helices
Structure of proteins

Human plasma retinol Triosephosphate

binding protein. Retinol isomerase
molecule (vitamin A)
bound inside the barrel
Protein is us
Protein synthesis
Proteins polymerized as linear chains by the ribosome as it translates
RNA message:

ribosome

RNA
message

nascent
polypeptide
 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 Å
 Protein Structure
solved by X-ray crystallography
 PDB contains more than 2 Lakhs structures mostly
determined by X-ray crystallography and NMR. About 40
new structures per day

https://www.rcsb.org/stats/growth/growth-released-structures
 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-Lys-
Hemoglobin 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: Levinthal’s 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 (Ʈfolding) is given by N/100 µs

α-protein fold faster than the β–protein or αβ–protein

Ʈfolding = 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

Folding protein unfolded

moves over energy
surface from
unfolded to
folded state:

nativeness
Degree of
100 kBT
folded

H= bond stretching + bending of angles +

Bond rotations + van der waals interaction +
electrostatic interaction
 Folding is a complex process
Macromolecules must fold into
Structure Function
correct shape to function properly:

Misfolding (non-native structures) Disease

Many diseases with large impacts involve protein misfolding:

• Alzheimer’s Disease • Huntington’s Disease

• Aβ peptide • Huntingtin protein
• Parkinson’s Disease • Amyotrophic Lateral Sclerosis
• α-synuclein • Superoxide dismutase
• Creutzfeldt-Jakob disease • Type II Diabetes
• Prion • Amylin
 Force spectroscopy

AFM Optical Tweezers

 Misfolding and aggregation are complex
Many different species and steps involved in misfolding and aggregation:

protein partially natively

synthesis unfolded unfolded folded

ribosome
amyloid
fibrils

misfolded

degraded partially-unfolded
fragments and aggregated

disordered non-native
aggregates structured
aggregates
Chiti & Dobson, Annu. Rev. Biochem., (2006)
Aggregation of amylin protein
 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
 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, 1257–1261 (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