Protein Structure

and Function

Dr.Koushik Biswas, M.D., D.N.B.

Assistant Professor
Department of Biochemistry
AIIMS Raebareli
 Proteins
• The term “protein” is derived from Greek word “proteios” which
means “primary”
• Of our dry body weight 3/4th is made up of protein
• Proteins contain carbon, hydrogen, oxygen, nitrogen as major
constituents, while sulphur and phosphorus are minor constituents
• The nitrogen content of ordinary protein is approximately 16% by
weight
• Abnormality in protein structure will lead to molecular diseases with
profound alteration in metabolic functions
 Proteins
• Proteins are linear polymers built of monomer units called amino
acids
• Proteins contain a wide range of functional group
(alcohol/thiol/thioether/carboxylic acids/carboxamides/etc) which
are chemically reactive
• Proteins can interact with one another and with biological
macromolecules to form complex assemblies
• Some proteins are quite rigid while others display a considerable
flexibility
 Peptides
• Proteins are built from a repertoire of 20 amino acids
• Only L-amino acids are constituents of proteins

Peptide Consist of
Dipeptide 2 amino acids
Tripeptide 3 amino acids
Tetrapeptide 4 amino acids
Oligopeptide 10 – 50 amino acids
Polypeptide > 50 amino acids
 Orders of protein structure
• Primary Structure
• Secondary structure
• Tertiary Structure
• Quaternary Structure
 Primary structure
• It is the sequence of amino acids in a polypeptide chain
• Determined from the coding sequence of mRNA
• The primary structure of a protein determines its 3D structure
(conformation)
• Chain always folds into its lowest energy conformation
• Sequence variation is the most important element of protein diversity
• The primary structure sequence is read from the N terminal to the C
terminal
Peptide bond formation
Structure of a pentapeptide (Cys-Ala-Tyr-Gly-Leu)
Peptide bond
Peptide bond
 Characteristics of a peptide bond
• Partial double bond [bond length 1.32 Å is midway
between single bond (1.49 Å) and double bond (1.27
Å)]
• C-N bond is trans in nature – there is no freedom of
rotation because of the partial double bond character
• The side chains are free to rotate
• The angles of rotation, known as Ramachandran
angles, determine the spatial orientation of the
peptide chain
Ramachandran angles: Psi (ψ) and phi (φ) angles
Primary structure of insulin
Primary structure determines biological
activity
• A protein with a specific primary structure, when put in solution will
automatically form its natural 3D structure
• So the higher levels of organisation are dependent on the primary
structure
• Even a single amino acid change (mutation) in the linear effect may
affect the protein function
• The 6th amino acid of beta chain of HbA (adult hemoglobin) is
glutamic acid
• It is changed to valine in HbS (sickle cell anemia)
Primary
structure
determines
the 3D
structure:
Anfinsen’s
experiment
 Secondary structure

• It denotes the configurational relationship between

residues which are about 3-4 amino acids apart in the
linear sequence
• Secondary structure is preserved by noncovalent
bonds (hydrogen bonds/electrostatic
bonds/hydrophobic interactions/van der Waals forces)
• Two types: alpha helix , beta pleated sheet
Alpha
helix
View down the
axis of an alpha
helix
 Characteristics of alpha helix
• Spiral structure
• 3.6 aminoacyl residues per turn of the helix
• Pitch 0.54 nm
• Twist about each alpha carbon
• Psi angle - 47°, phi angle - 57° (approx)
• Stability: mainly by hydrogen bonds
• Hydrogen bond : between oxygen of peptide bond carbonyl and
hydrogen atom of peptide bond nitrogen of the fourth residue down
the peptide chain
Hydrogen
bonds in
alpha helix
 Proline and glycine in alpha helix
• Proline can only be
stably accommodated
within the first turn of
alpha helix
• When present elsewhere
it disrupts confirmation,
producing bend
• Glycine has small R
group
• Glycine frequently
induces bend in the
alpha helix
 Amphipathic helices
• Many alpha helices have predominantly hydrophobic
R groups projecting from one side of the axis of the
helix and predominantly hydrophilic R groups
projecting from the other side
• Seen in channels and pores of membrane
Beta sheets
 Characteristics of beta sheet
• Amino acid residues form a zig zag or pleated pattern
• R groups of adjacent residues are in opposite direction
• Peptide backbone – highly extended
• Distance between adjacent amino acids: 3.5 Å
• Twist about each alpha carbon
• Adjacent strands in a sheet can run in same direction (parallel) or
opposite direction (anti parallel)
• Stability: mainly by hydrogen bonds
• Hydrogen bond : between oxygen of peptide bond carbonyl and
hydrogen atom of peptide bond nitrogen of neighbouring polypeptide
segment
Beta
sheet
Left handed
alpha helix
Right handed (uncommon)
alpha helix
(common)

Ramachandran plot
 V. Sasisekharan

Dr.G.N.Ramachandran Dr. V. Sasisekharan

Loops and bends
• Turns and bends refer to short segments of amino acids that join two
units of the secondary structure, such as two adjacent strands of an
antiparallel β sheet.
• Loops are regions that contain residues beyond the minimum number
necessary to connect adjacent regions of secondary structure.
β turn

Proline and glycine

often present in β turn
Loops

Helix loop helix motifs

present in DNA binding
proteins
Tertiary structure
• It refers to the overall folding pattern and the final 3D shape of the
polypeptide
• It refers to the spatial arrangement of amino acid residues that are far
apart in the linear sequence as well as those residues that are
adjacent
• Native conformation
• Biologically active form of the protein
• Example: Myoglobin
Factors that specify the tertiary structure
• Primary structure: Anfinsen law states that 3D shape of a polypeptide
is always determined by its primary structure
• Chaperons: These are a group of specialised proteins, required for the
native folding of many proteins
Chaperons
• Also called polypeptide chain binding (PCB) proteins
• Aid in folding during synthesis
• Not responsible for stability of final protein structure
• Bind to hydrophobic patches
• Stabilize intermediates during folding
• Prevent aggregation of incompletely folded proteins by precluding
adventitious contacts between exposed hydrophobic regions
• Defective interaction of polypeptide with chaperon may cause disease
 Chaperons

Maloy S, Hughes K, editors.

Brenner's encyclopedia of
genetics. Academic Press; 2013
Mar 22.
 Chaperons

Maloy S, Hughes K, editors.

Brenner's encyclopedia of
genetics. Academic Press; 2013
Mar 22.
Forces stabilizing tertiary structure
• Hydrophobic forces
• Hydrogen bonds
• Electrostatic forces
• Van der Waals forces
• Disulphide bonds

Tertiary structure represents a state of lowest energy

Quaternery structure
• In oligomeric proteins (consisting of two or more subunit chains),
there is a fourth level of structure, the quaternary structure
• It refers to the number, size and shape of the subunit polypeptide
chains, and their spatial relationship with one another
• Interactions among subunits may be:
• Noncovalent: hydrophobic interactions, hydrogen bonds, electrostatic
bonds
• Covalent bonds: interchain disulphide bonds
• Example: hemoglobin
Domains
• Many domains are structurally independent units
• Can fold autonomously
• Compact globular functional unit
• Length vary from 25 – 500 amino acids
• Connected by the more extended part of the polypeptide chain
• Bilobal or multilobal appearance
• Stabilised by metal ions or disulphide bridges
• Eg. Calcium binding domain of calmodulin
A domain (colored brown) is common in both these proteins
Motif / super secondary structure / folds
• Stable arrangements of several elements of secondary structure and
the connections between them
• Recognized motifs range from simple to complex, sometimes
appearing in repeating units or combinations
• A single large motif may comprise the entire protein
• Eg. coiled coil of α-keratin
Formation of secondary, tertiary and quaternary structures of protein
follows the thermodynamic principles
 Classification of proteins

Based on
shape Based on nutritional
value

Based on structural Based of

component function
Based on shape

Collagen, Elastin Myoglobin, hemoglobin

 Based on
structural
component
Based on protein function
• Catalytic proteins: enzymes
• Structural proteins: collagen
• Transport proteins: hemoglobin, albumin
• Regulatory proteins: insulin, glucagon
• Defence proteins: immunoglobulin
• Genetic proteins: histone, repressors-inducers
• Vision proteins: rhodopsin
• Contractile proteins: actin, myosin
• Others: role in respiration, storage, nutrition
 Based on nutritional value

Complete Incomplete Poor proteins

protein protein

Lack many essential

Lack one essential
Contain all amino acids (zein
amino acids (pulses
amino acids from corn lacks
lack methionine,
(egg, milk) tryptophan and
cereals lack lysine )
lysine)
Physical properties of proteins
• Diverse shape:
• Globular (hemoglobin)
• Fibrous (collagen)
• Elongated (fibrinogen)
• Oval (albumin)

Primary structure determines the overall shape, structure (and activity)

of a protein
Physical properties of proteins
• Diverse size:
• Hemoglobin 65 kD
• Myoglobin 17.2 kD
• Elastin 64 to 66 kDa
• Albumin 69 kD

Proteins contain 100 – 2000 amino acid residues

Mean molecular mass of an amino acid is 110 dalton units
Molecular weight of most protein is 11,000 to 2,20,000 D
Denaturation
• Disruption of the native conformation
• Well ordered, neatly folded 3D structure converted to an irregular
random coil
• Non covalent interactions disrupted
• Covalent bonds (including peptide bonds) left intact
• Have more exposed site for enzymatic activity

Altered Not altered

Secondary structure Primary structure
Tertiary structure
Quaternary structure
Agents causing denaturation
Agent Effect
Strong acids/bases Disrupt electrostatic forces

Detergents and organic Disrupt hydrophobic interactions

solvents
Urea and guanidine Disrupt hydrogen bonds between
hydrochloride protein and surrounding water

Heavy metal ions Disrupt disulphide bonds

Heat Disruption of noncovalent bonds

Determination of amino acid sequence of
peptide/protein
 Step 1: Determine the size of the protein
• Done by SDS PAGE electrophoresis
• All proteins are covered with negative charge of SDS
• Proteins tend to be linear in shape
• Movement of proteins in electric field is determined by their size
 Step 2: Determine the amino acid composition
of the protein
• Complete hydrolysis of the polypeptide (acid hydrolysis: 6N HCl for 24-72
hours at 100-110 °C / alkaline hydrolysis)
• Followed by
• Separation by chromatographic methods
• Followed by
• Determined quantitatively with suitable reagents

• (* This step reveals amino acid composition, but not the sequence)
• (Tryptophan is destroyed by alkaline hydrolysis but not by acid
hydrolysis)
 Step 2: Determine the N-terminal amino acid
of the protein
• N terminal is covalently bound to dansyl chloride
• Tagged protein is then hydrolysed to its constituent amino acids by
acid hydrolysis
• As only the N-terminal amino acid is dansylated, it is identified by
separating it out by column chromatography, and comparing against
dansylated standards
Step 3: Determine the C-terminal amino acid
of the protein
• Intact polypeptide is treated with hydrazine
• Hydrazine reacts hydrolytically at the carbonyl grouping (C-O) at each
peptide bond (resulting n acyl derivatives of each residue)
• But it cannot react with the C-terminal which has COOH group
• The amino acids are then analysed by column chromatography and
compared with standards
• The amino acid without hydrazine is the C-terminal amino acid
Step 4: Determine the sequence of amino
acids in the protein
• Done by Edman degradation method on automatic machines called
cyclic sequenators
• This method sequentially removes one amino acid from the N-
terminal end
• Phenylisothiocyanate reacts with the N-terminal amino group of the
peptide to form phenylthiocarbamyl derivative in alkaline solution
• Phenylthiocarbamyl derivative is released by gentle acid hydrolysis
and identified by HPLC
• The second amino acid now becomes the N-terminal amino acid and
the cycle can be repeated
See the link
• https://www.youtube.com/watch?v=GUhZoNTCCPo
Step 4: Determine the sequence of amino
acids in the protein
• Only about 20-25 amino acids can be sequenced by Edman
degradation technique
• The large polypeptides must be fragmented first by enzymes
Specific clevage of polypeptides
Step 5: Reconstructing the protein’s sequence
• After individual peptide fragments are sequenced, their order in the
original polypeptide must be elucidated
• For this a second round of protein cleavage with a different
endopeptidase is carried out
• This generates an overlapping set of peptide fragments
• The fragments are then separated and sequenced
• The sequences of the overlapping peptides are then used, as a jigsaw
puzzle, to obtain the whole protein structure
Physical properties of proteins
• Exhibit colloidal properties
• Scatter light (Tyndall effect)
• Colloidal particle size 1-200
nm
• Exert osmotic pressure
Tyndall effect
• It is the phenomenon in which the particles in a colloid scatter the
beams of light that are directed at them
• This effect is exhibited by all colloidal solutions
• Used to verify if a given solution is a colloid
• Intensity of scattered light depends on the density of the colloidal
particles as well as the frequency of the incident light
Molecular weight
• Insulin: 5700 kD
• Hemoglobin: 68000 kD
• Albumin: 69000 kD
• Immunoglobulins: 1,50,000 kD
• Rabit papilloma virus protein: 4,70,00,000 kD
Shape
• Shape of protein vary
• Insulin: globular
• Albumin: oval
• Fibrinogen: elongated

(Bigger and elongated molecules will increase the viscosity of the

solution)
Isoelectric pH
• pI of all the constituent amino acids will influence the pI of the
protein
Precipitation reaction of proteins
• Purification of enzymes / proteins usually start with precipitating
them from solution
• Stability of protein in solution mainly depend on the charge and
hydration
• Polar groups of proteins (-NH2, COOH, OH groups) tend to attract
water molecules around them to produce a shell of hydration
• Any factor which neutralises the charge or removes water of
hydration will cause precipitation of proteins
Procedures for protein precipitation
• Salting out
• Isoelectric precipitation
• Precipitation by organic solvents
• Precipitation by heavy metal ions
• Precipitation by alkaloid reagents
• Denaturation
Salting out
• Protein solution + neutral salt (sodium sulphate/ammonium sulphate)
= Protein precipitate
• Salt removes the shell of hydration
• Higher the molecular weight of a protein, salt required for
precipitation is lesser
• Globulins are precipitated with half saturation of ammonium
sulphate, but albumin is precipitated with full saturation
Isoelectric precipitation
• Proteins are lease soluble at their isoelectric pH
• Some proteins are precipitated immediately when adjusted to their
isoelectric pH
• Example: caesin forms a flocculent precipitate at pH 4.6, and
redissolves in highly acidic or alkaline solution
Casein
Precipitation by organic solvents
• When organic solvent (eg alcohol) is added
• Water molecules available for proteins are reduced
• Organic solvent also reduce the dielectric constant of the medium
• These will favour protein precipitation

(* this explains disinfectant effect of alcohol)

Precipitation by heavy metal ions
• In alkaline medium, proteins have net negative charge
• When heavy metal salts are added, positivey charged metal ions can
complex with protein molecules and metal proteinates are
precipitated
• Salts of copper, zinc, lead cadmium, mercury are toxic, because they
tend to precipitate normal proteins of the GI wall
• Based on this principle, raw egg is sometimes used as an antidote for
mercury poisoning
Precipitation by alkaloid reagents
• Tungstic acid, phosphotungstic acid, trichloroacetic acid, picric acid,
sulfosalicylic acid and tannic acid are powerful protein precipitating
agents
• These lower pH of the medium, when proteins carry net positive
charge
• These protein cations are complexed with negatively charged ions to
form protein-tungstate, protein-picrate, etc and thick flocculent
precipitate is formed
• Tanning in leather processing is based on protein precipitating effect
of tannic acid
Quantitative estimation of proteins
• Kjeldahl’s procedure
• Biuret method
• Lowry method
• Spectrophotometric method
• Radial immunodiffusion
• Nephlometry
• Turbidimetry
• ELISA
 Biuret method

Cupric ions chelates with peptide bonds of proteins in alkaline medium

to produce a pink or violet colour
Nephelometry
• Detection of light scattered by turbid particles in solution
• If albumin is to be estimated, specific antibody against albumin is
added
• The resultant antigen-antibody complex will form a turbidity of the
solution
• A beam of light (preferably laser beam) is passed through the solution
• The particles in solution will scatter light
• The light turning at 30 – 90 ° angle (generally 60 °) is collected and
passed onto detector system
Nephelometry
Turbidimetry
Turbidimetry vs
Nephelometry
Protein degradation
• ATP-independent: degradation of blood glycoproteins
• ATP and ubiquitin-dependent: degradation of regulatory proteins and
misfolded proteins
ATP-independent degradation
• Blood glycoproteins lose a sialic acid moiety from the non-reducing
ends of their oligosaccharide chain
• Asialoglycoproteins are internalised by liver-cell asialoglycoprotein
receptors
• Inside cell it is degraded by lysosomal proteases
ATP and ubiquitin-dependent degradation
• Degradation of regulatory proteins and misfolded proteins
• Ubiquitin is a small (8.5 kDa, 76 residue) polypeptide that targets
many intracellular proteins for degradation
• Ubiquitin molecules are attached by non-α-peptide bonds formed
between the carboxyl terminal of ubiquitin and the ε-amino group of
lysyl of the target protein
• Residue present at N-terminal affects ubiqutination
• Met / Ser at N-terminus retard ubiquitination
• Asp/ Arg at N-terminus accelerate ubiquitination
ATP and ubiquitin-
dependent degradation

E1: activating enzyme

E2: ligase
E3: transferase
ATP and ubiquitin-dependent degradation
• Takes place in proteasome
• Proteasome: a macromolecule ubiquitous in
eukaryotic cells
• Proteasome consists of a cylindrical complex of
proteins
• Central pore contains proteolytic enzymes
• Entry into central core is regulated by two outer
rings
• Outer rings recognise only polyubiquitinated
proteins
Noble Prize in Chemistry, 2004
Amyloid disease
• Accumumulation of insoluble spontaneously aggregating proteins,
called amyloids
• Cause Parkinson and Huntington disease
Alzheimer
disease
Prion disease