Module 2-Lesson 2

FOUR LEVELS OF PROTEIN STRUCTURE

Protein Hierarchy

TERMINOLOGY
 Supersecondary structure is the next level up from secondary structure and involves
the association of secondary structures. Also known as structural motifs.
 Domains are larger associations of two or more secondary structures, two or more
supersecondary elements, or mixtures of two or more secondary and supersecondary
structures.
 They can also be known as 'folds', and 'modules'.

 In Protein Tertiary Structure domains are independently folding units of tertiary

structure and contain between 35 and 200 amino acids.

 Motif - can be either a structural or a sequence motif:

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 A structural motif is the arrangement of atoms in 3-D space to produce a particular
structural pattern eg. alpha helix, beta sheet.
 A sequence motif is a particular pattern in the sequence of amino acids or
nucleotides.

Primary structure
The primary structure of a protein is its linear sequence of amino acids and the location
of any disulfide (-S-S-) bridges.
METHODS OF DETERMINING THE SEQUENCE OF A POLYPEPTIDE
 SANGER’S REACTION
 Frederick Sanger was the first to determine peptide sequence
 Sanger reduced the disulfide bonds to separate the A chain from the B chain into
smaller peptides using trypsin, chemotrypsin, and pepsin
 THE EDMAN’S REACTION
 The use of phenylisothiocyanate (Edman’s reagent) to label the amino-terminal
residue of a peptide
 BY DNA SEQUENCING

SECONDARY STRUCTURE OF PROTEINS

The Alpha Helix
 A common secondary structure encountered in proteins of the globular class
 Stabilized by H-bonding between amide N’s and carbonyl C’s of peptide bonds
spaced four residues apart
 This orientation of H-bonding produces a helical coiling of the peptide backbone such
that the R-groups lie on the exterior of the helix and perpendicular to its axis
The Alpha helix
 Amino acids such as A, D, E, I, L and M favor the formation of -helices
 G and P favor disruption of the helix (producing a bend)
 The disruption of the helix is important as it introduces additional folding of the
polypeptide backbone to allow the formation of globular proteins

3.6 amino-acid residues per turn (0.54 nm per turn) of the helix

Proteins in -helix
 KERATIN – fibrous protein whose structure is nearly entirely -helical
 HEMOGLOBIN – a globular, flexible molecule whose structure is approximately 80%
-helical;
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The Beta () sheet
 Composed of 2 or more different regions of stretches of at least 5-10 amino acids
 Stabilized by H-bonding between amide N’s and carbonyl C’s
 H-bonding residues are present in adjacently opposed stretches of the polypeptide
backbone
Pleated -Sheets
 This is due to positioning of the -carbons of the peptide bond which alternates
above and below the plane of the sheet

Parallel and antiparallel sheets

• When the H-bonds are formed between the polypeptide backbones of separate
polypeptide chain, they are termed INTERCHAIN BONDS
• The H-bonds of a -sheet formed by a single polypeptide chain folding back on
itself are termed INTRACHAIN BONDS
-sheet PROTEIN
• Found in both fibrous and globular proteins
• AMYLOID PROTEIN – composed of twisted - pleated sheet fibrils whose
3D structure is identical to that of silk fibrils

LOOPS, TURNS and BENDS

 TURNS and BENDS refer to short segments of amino acids that join two units of
secondary structure (e.g., 2 adjacent strands of antiparallel -sheet)
  turn – involves 4 aminoacyl residues, in which the 1
st
residue is H-bonded to the
th
4 , resulting in a 180° turn
 Proline and Glycine are often present in  turns
 Loops are regions that contain residues beyond the minimum number necessary to
connect adjacent regions of secondary structure

Supersecondary Structures or Folds

 Also known as Structural motifs

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Zinc finger Helix-loop-helix

Leucine zipper Helix-turn-helix

Four Most common motifs

Tertiary Structure
 Refers to the complete three-dimensional conformation of a polypeptide
 It indicates in a 3-D space, how secondary structural features—helices, sheets,
bends, turns, and loops---assemble to form domains and how these domains relate
spatially to one another.
 The interaction of different domains is governed by several forces: These include
hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der
Waals forces.

FUNCTIONS OF DOMAIN
 Binding of a substrate or other ligands
 Anchor a protein to a membrane

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 Module 2-Lesson 2
 Interact with a regulatory molecule that modulates its function

Three domains of pyruvate kinase

FORCES CONTROLLING PROTEIN STRUCTURES

I. Hydrogen Bonding
 R-group containing O- and N-bound hydrogen (-OH of S and T) can form H-bonds
with electron rich atom (such as O of carboxyl or carbonyl groups of peptide bond)
 H-bonds between polar groups on the surface of a protein and the aqueous solvent
enhances the solubility of the protein
II. Hydrophobic Forces
 The hydrophobicity of certain amino acid R-groups tends to drive them away from the
exterior of proteins and into the interior, shielding them from water.
 This driving force restricts the available conformations into which a protein may fold
III. Electrostatic Forces
Three types
1. Charge-charge
 Favor protein folding
 Between oppositely charged R-groups such as K or R and D or E
2. Charge-dipole
 Interaction of ionized R-groups of amino acids with the dipole of the water molecule
3. Dipole-dipole
 The slight dipole moment that exist in the polar R-groups of amino acid also
influences their interaction with water.
 Majority of the amino acids found on the exterior surfaces of globular proteins
contain charged or polar R-groups.
IV. van der Waals Forces:
 There are both attractive and repulsive van der Waals forces that control protein
folding.
 Attractive van der Waals forces involve the interactions among induced dipoles that
arise from fluctuations in the charge densities that occur between adjacent uncharged
non-bonded atoms.
 Repulsive van der Waals forces involve the interactions that occur when uncharged
non-bonded atoms come very close together but do not induce dipoles.
 Although van der Waals forces are extremely weak, relative to other forces governing
conformation, it is the huge number of such interactions that occur in large protein
molecules that make them significant to the folding of proteins.

Disulfide (S-S) bonds

 Bond that links the sulfhydryl groups of cysteinyl residues
 Involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen

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 Intrapolypeptide disulfide bonds – enhances the stability of the folded conformation of
a peptide
 Interpolypeptide disulfide bonds – stabilize the quaternary structure of certain
oligomeric proteins
Quaternary Structure
 2 or more different polypeptide chains that are held in association by the same non-
covalent forces that stabilize the tertiary structures of proteins
 Oligomeric proteins - proteins with multiple polypeptide chains
 Oligomeric proteins can be composed of multiple identical polypeptide chains or
multiple distinct polypeptide chains.
 Homooligomers - proteins with identical subunits
 Heterooligomers - proteins containing several distinct polypeptide chains
HEMOGLOBIN
 The oxygen carrying protein of the blood
 Contains two  and two  subunits arranged with a quaternary structure in the form,
22
 A hetero-oligomeric protein

PROTEIN FOLDING
 Protein fold and unfold in milliseconds
 Unfolding and refolding occur 100 or 1000 times during their lifetime
 Ribosomes participate the first time the protein is folded, but not in subsequent
folding
 Increase concentration of proteins affect the kinetics of protein folding
 Information needed for correct protein folding is contained in the primary structures

Factors that facilitate folding and refolding

I. Native conformation of a protein is thermodynamically favored
II. Folding is modular
Stage 1
Newly synthesized polypeptide folds into 2ndary structure
- 2ndary structures facilitates proper folding
Stage 2
Hydrophobic regions are driven into the interior of the protein and away from the solvent
Final stage - Native conformation
III. Auxilliary proteins assist folding
 Chaperone proteins
 Protein disulfide isomerase
 Proline - cis, trans - isomerase
CHAPERONES
 Specialized group of protein required for the proper folding of many species of
proteins
 PCB (Polypeptide chain-binding) protein
 Acts as catalysts by increasing the rates of the final stage in the folding process

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Types:
 hsp 70 – bind short sequences of hydrophobic amino acids thus, shielding them
from solvent
 hsp 60 (chaperonins) – provides a sheltered environment in which a polypeptide
can fold until all hydrophobic regions are buried in its interior, thus eliminating
aggregation.
PROTEIN DISULFIDE ISOMERASE
 Facilitates formation of disulfide bonds that stabilize a protein’s native conformation
PROLINE – cis, trans – ISOMERASE
 Catalyzed isomerization from trans to cis
 Cis configuration is common in -turns
IV. Folding is a dynamic process
 Unfold  refold
 Chaperone “rescue” unfold proteins
 Glutathione reduces inappropriate disulfide bond
PROTEIN DENATURATION
 Caused by heat, organic solvents, mechanical mixing, strong acids and bases,
detergents, and ions of heavy metals such as lead and mercury
 EFFECT: unfolding and disorganization of protein structure (not accompanied by
hydrolysis of peptide bond)
PROTEIN DENATURATION

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REVERSIBLE OR PERMANENT?
REVERSIBLE
 In rare cases, upon removal of denaturing agents
 Protein refolds into original native structure
PERMANENT
 Most proteins remain permanently disordered

Pathologic Consequences of Protein Conformation Perturbation

PRION DISEASES
 Transmissible spongiform encephalopathies
 Fatal neurodegenarative diseases characterized by spongiform changes,astrocytic
gliomas, and neuronal loss resulting from the deposition of insoluble protein
aggregates and neural cell
 Include:
a. Creutzfeldt-Jacob disease (Human)
b. Scrapie (sheep)
c. Bovine spongiform encephalopathy (mad Cow Disease) in cattle
PRION
PrPc (for cellular)
 The normal protein
 has its secondary structure dominated by alpha helices (probably 3 of them)
 is easily soluble
 is easily digested by proteases
 is encoded by a gene designated (in humans) PRNP located on our chromosome 20.

PrPSc (for scrapie)

 The abnormal, disease-producing protein
 Primary structures are identical but its secondary structure is dominated by beta
conformation
 is insoluble in all but the strongest solvents
 is highly resistant to digestion by proteases
 When PrPSc comes in contact with PrPC, it converts the PrPC into more of itself
 These molecules bind to each other forming aggregates.

ALZHEIMER’S DISEASE
 Refolding or misfolding of  - amyloid in human brain tissue
 Elevated levels of  - amyloid undergoes conformational transformation
BETA - THALASSEMIA
 Genetic defects that impair the synthesis of one polypeptide sub-units of hemoglobin

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