Protein structure

Protein Conformation
• Spatial arrangement of atoms in a protein is called
conformation
• It include structural state that can be achieved
without breaking covalent bond
• Protein in any of the functional, folded conformation
are called native proteins
 Levels of protein structure
• There are four distinct levels of protein structure.
• To be able to perform their biological function,
proteins fold into one or more specific spatial
conformations driven by a number of non covalent
interactions.
• Primary
• Secondary
• Tertiary
• Quaternary
Levels of protein structure
• To determine the structure of proteins, the scientist
employs the following techniques: X-ray
crystallography, NMR spectroscopy, cryo-electron
microscopy and dual polarisation interferometry
 Primary structure
• Protein primary structure is the linear sequence of
amino acids in a peptide or protein.

• By convention the primary structure of protein is

reported starting from the amino terminal (N) end to
the carboxyl terminal (c) end.
 Secondary structure
• This refers to highly regular local sub-structures on the
actual polypeptide backbone chain.
• They are defined by patterns of hydrogen bonds hydrogen
bonds between the main-chain peptide groups.
• They have a regular geometry, some parts of the protein
are ordered but do not form any regular structures. They
should not be confused with random coil, an unfolded
polypeptide chain lacking any fixed three-dimensional
structure.
• Common secondary structures have characteristics amino
acid content
 Alpha helix
• In this structure, the polypeptide backbone is tightly
wound around an imaginary axis drawn longitudinally
through the middle of the helix, and the R groups of
the amino acid residues protrude outward from the
helical backbone.
• Amino Acid Sequence Affects stability of the α Helix
• Interactions between amino acid side chains can
stabilize or destabilize the α-helical structure.
• Asn, Ser, Thr, and Cys residues can also destabilize an
α helix , adjacent Glu, Lys Arg are also repel each
other
• Pro, Gly are helix breaker, because of size and shape
 Β pleated sheet
• In the β conformation, the backbone of the
polypeptide chain is extended into a zigzag rather
than helical structure
• Hydrogen bond are formed between adjacent
segment of polypeptide chain
• A single protein segment in the β conformation is
often called a β strand.
• The arrangement of several strands side by side, all in
the β conformation, is called a β sheet.
 turns
• In globular proteins nearly 1/3 of the amino acid
residues are in turns or loops, where the polypeptide
reverse direction
• These are connecting elements that link successive
runs of alpha or beta conformation
• Most common is beta turns (connect the ends of
two adjacent segment of antiparallel beta sheets)
• Gly and Pro residues occurs in beta turn (amenable
to tight turn
 Tertiary structure
• This refers to the three-dimensional structure created by a
single protein molecule (a single polypeptide chain).
• It may include one or several domain.
• The α-helices and β-pleated-sheets are folded into a compact
globular structure.
• The folding is driven by the non-specific hydrophobic
interactions the burial of hydrophobic residues from water.
• the structure is stable only when the parts of a protein
domain protein are locked into place by specific tertiary
interactions, such as salt bridge, hydrogen bonds, and the
tight packing of side chains and disulfide bonds.
• The disulfide bonds are extremely rare in cytosolic proteins,
since the cytosol (intracellular fluid) is generally a reducing
environment.
 Quaternary structure
• This is the three-dimensional structure consisting of
the aggregation of two or more individual
polypeptide chains (subunits) that operate as a single
functional unit ( multimer).
• The resulting multimer is stabilized by the non
convalent interactions and disulfide bond.
• There are many possible quaternary structure
organisations.
• Complexes of two or more polypeptides (i.e. multiple
subunits) are called multimers
• Dimer contains two subunits,
• a trimer contains three subunits,
• a tetramer contains four subunits, and
• a pentamer contains five subunits, and so forth.
• The subunits are frequently related to one another by
symmetry operations , such as a 2-fold axis in a dimer.
• Multimers made up of identical subunits are referred to with
a prefix of "homo-“
• and those made up of different subunits are referred to with
a prefix of "hetero-", for example, a heterotetramer, such as
the two alpha and two beta chains of hemoglobin.
 Examples of multimers
• Chymotrypsin (bovine pancreas) – 3 subunit
• Hemoglobin (human) - 4
• Hexokinase (yeast) - 2
• RNA polymarse (E coli) – 5
• Glutamine synthetase - 12
 Fibrous proteins
• These are adapted for structural functions
• Share properties that give strength or flexibility to
the structure
• All are insoluble in water, due to high concentration
of hydrophobic amino acid residues, both interior
and on the surface
 Alpha Keratin
• Found in hairs, nails, claws, quills, horns, hooves,
skin
• Rich in hydrophobic residues Ala, Val, Leu, Ile, Met,
and Phe
• The secondary structure is a right handed alpha helix
• Two strands of alpha keratin in parallel are wrapped
about each other to form super twisted coiled coil
• An individual polypeptide in alpha keratin has a
tertiary structure
• Two alpha helix polypeptide is an example of
quaternary structure (crosslink between polypetide
stabilizes the structure)
 Collagen
• Found in connective tissues: tendon, cartilage,
organic matrix bone, cornea of the eyes
• Comprises of 3 polypeptide called alpha chain (super
twisted about each other which is right handed)
• The amino acid is a repeat of tripeptide Gly-X-Y,
where X- Proline, Y- 5, hydroxproline
• Hyp allow super twist and very close packing of the
three polypeptide
• The secondary structure is a left handed alpha helix,
with 3 amino acid per turns
• It is a coiled coil with distinct secondary and tertiary
structure
 Collagen

• Typically collagen contain 35% Gly, 11% Ala, and 21%

Pro and 4 Hyp
• Mammals has more than 30 structural variant of
collagen
• Crosslink of Lys Hyl makes collagen to be rigid and
brittle (accumulation at old age) of
Dehydrohdroxylysinorleucine.
• Genetic defect in collagen : Osteogenesis imperfecta,
Ehler-Danlos syndrome result in loose joint in infant.
This is as a result of subtitution of Gly by Cys & Ser
• In scruvy there is connective tissue issues due to
deficiency of vit C, which cannot hydroxylate Pro at
the Y position
 Globular proteins
• Polypetide chain folded into a spherical or globular
shapes
• This contain several types of secondary structures
• In globular proteins different segments of
polypeptide chain fold back on each other
• The folding provide structural diversity for protein to
carry out the biological functions
• They includes enzymes, transport protein, motor
proteins, regulatory proteins, immunoglobulins
 Super-secondary structure
• This is also called motifs or folds
• They are particular stable arrangements of
several elements of secondary structure and
connection between them.
 Domains
• is a part of a polypeptide chain that is independently
stable or could undergo movements as a single entity
with respect to the entire protein.
• a domain from a large protein will retain its native
three-dimensional structure even when separated
(for example, by proteolytic cleavage) from the
remainder of the polypeptide chain
• Different domains often have distinct functions, such
as the binding of small molecules or interaction
with other proteins.
 Hemoglobin
• It is the 1st Oligomeric protein with 3 dimensional
structure elucidated
• It consist of 4 polypeptide chains and 4 heme
prosthetic group in which iron is in ferrous state
• The protein portion (globin) consist of 2 alpha chain
(141 residues) and 2 beta chains (146 residues)
• Hemoglobin is a multimeric protein consisting of 4
poly peptide chain, therefore it is known as tetramer
or dimer of alpha-beta protomer
 Sickle cell Anaemia
• About 500 genetic variant of haemoglobin in humans
• Occur in individual who inherit alleles for sickle
haemoglobin from both parents
• The erythrocytes are fewer and abnormal large immature
cells, the blood contain many long thin sickle shaped
erythrocytes
• Altered portion of HbS is as a result of single amino acid
substitution: Val, instead of Glu residue at position 6 of
beta chain
• R group of Val has no electric charge, whereas Glu has at
pH 7
• Haemoglobin is use for oxygen transport.
• In sickle cell deoxygenated HbS becomes insoluble
and form polymers.

• The point of replacement on the HbS from Glu to Val,

produce a sticky surface on the beta chain, which
cause abnormal association of deoxyHbS, forming
long fibrous aggregate
 Protein separation and purification
• Crude extraction: open cells to release proteins
• Differential centrifugation: Isolation of specific
organelles
• Fractionation: this depends on properties of protein
(solubility, pH, size and charge)
• Salting out: Use of salt like ammonium sulfate,
solubility of protein is lowered at high salt
concentration
• Dialysis: separating protein from solvent using semi-
permeable membrane
• Column chromatography (Affinity, HPLC) separate
with respect to charge, size, binding affinity and
other
• Electrophoresis: separation based on charge
 Determination of short poly peptide
sequence
• Automated procedure
• FDNB – 1 flouro dinitrobenzene
• Dansyl chloride
• Dabsyl choride
• These reagent labels the amino terminal end of the
polypeptide
 Sequencing entire peptide
• Edman degradation procedure in a sequenator
(PITC –Phenyl isothiocyanate, residue of amino group
Phenylthiohydratoin)
• Mass spectrometry
• NMR
• Amino acid analyser
• From the sequence of nucleotide