1. CHAPTER 3 : PROTEINS

2. PROTEINS ARE BUILT FROM REPERTOIRE OF AMINO ACIDS

3. 3.1 LEVELS OF PROTEIN STRUCTURE
Individual protein molecules can be described by…
“ up to four levels of structure”
: primary structure : : secondary structure, tertiary structure and quatenary structure :
 linear sequence of amino acid  The 3D structures
residues in protein  Forces responsible for maintaning OR stabilizing these 3 levels are primarily “noncovalent”
PROTEOMICS  the study of large sets of proteins
PROTEINS (structurally)
Globular (e.g. enzymes)
Fibrous/ structural proteins (e.g. α-keratin)

4. FOUR LEVELS OF PROTEIN STRUCTURE

5. PROTEIN MODELS
STICK
FLAT RIBBON
SPACE FILLING
3D RIBBON
CARTOON

9. 1) PRIMARY STRUCTURE
Defined as the linear arrangement/ specific sequence of amino acids in a polypeptide chain linked through covalent peptide bonds.
-In order to function properly, peptides and proteins must have the correct sequence of amino acids
- Sometimes called "covalent structure of proteins” = all of the covalent bonding within proteins defines the primary structure.
-In contrast, the higher orders of proteins structure (i.e. secondary, tertiary and quaternary) involve mainly non-covalent interactions.

10. e.g. INSULIN
- In the protein hormone insulin, 51 amino acids are found
- Using 51 amino acids there are 1.55 x 1066 different possible sequences
- Many other proteins contain many more amino acids then insulin, but only the correct precise sequence is produced by the body
-The procedure used to synthesize the correct sequence of amino acids in proteins is guided by the genetics of DNA and RNA.

12. 2) SECONDARY STRUCTURE DEFINITION
1. Long chains of amino acids will commonly fold or curl into a regular repeating structure
2. Repeating conformations of peptide chains which are stabilised by hydrogen bonds between H of amino groups and O of carbonyl group of the peptide backbone
Protein conformation = local conformation of the backbone
= 3D arrangement of various atoms of the molecule
  This structure will add new properties to a protein like strength, flexibility and etc…

13. Carbonyl: carbonyl functional group which is a divalent group consisting of a carbon atom with a double-bond to oxygen
Amide: a functional group containing a carbonyl group linked to a nitrogen atom.

14. common secondary structure right-handed screw a) α helices
left-handed screw
a) Resembles a ribbon wrapped around a tube
(similar to a circular staircase)
b) Hydrogen bonds are found parallel to the helix axis
c) Very stable but flexible
(therefore it is often seen in parts of a protein that need to bend or move)
parallel
b) β structure (β strands and β sheets)
anti-parallel
a) Two or more ribbons of amino acids are involved
b) Hydrogen bonds are formed between the two (or more) polypeptide strands
c) These line up to form a pleated like structure (similar to folds in fabric)
d) Tends to be rigid but less flexible than α helices.
c) β turn
A structure in which the polypeptide backbone folds back on itself
Often responsible for;-
-sharp bends and twists (in α helices)
- hair-pins (in β sheets)
c) Useful for connecting helices and sheets

15. THE α HELIX
A spiral formed by coiling of the polypeptides chain around the fibre axis
α helix present in more complex globular proteins
All carbonyl group (in α helix) point toward C-terminus
Average content of α helix in the proteins = 26%
Bottom : N-terminus
Top : C-terminus
The α helices found in proteins are almost right-handed
“right handed α helix”
 the backbone turns in a clockwise direction (when viewed along the axis from its N-terminus)
If you imagine that the right handed helix is spiral staircase, you will be turning to the right as you walk down the staircase
-blue ribbon  indicates the shape of the polypeptide backbone
- All the side chains (R groups)  project outward from the helix axis

16. HYDROGEN BOND IN A HELIX

17. HYDROGEN BOND IN A HELIX
H bond

18. β SHEETS
Hydrogen bonding occurs between neighbouring polypeptide chains rather than within one as in α helix
Fibrous and insoluble in aqueous solvents
This class includes
i) β STRANDS
ii) β SHEETS
β STRANDS
Portions of the polypeptide chain that are most fully extended
Proteins rarely contain isolated β strands because the structure by itself is not significantly more stable than other conformations
When multiple β strands are arranged side-by-side
-Are stabilised by H bonds between carbonyl oxygen and amide hydrogens on adjacent β strands
-β sheets sometimes called as β pleated sheet
-A typical β sheet contains  from 2 to as many as 15 individual β strands
β SHEETS

19. β SHEETS a) parallel b) anti-parallel
The hydrogen bonded chains extended in the
same directions
Neighbouring hydrogen bonded polypeptide chains run in
opposite directions
-Running in the same N- to C- terminal direction
-the H bonds  not perpendicular to the β strands
-The H bonds are evenly spaced but slanted
-Are less stable than anti-parallel sheets
-running in opposite N- to C- terminal directions
-the H bonds  essentially perpendicular to the β strands
-the space between H-bonded pairs alternatively wide and narrow

20. TURNS and LOOPS In α helix or β strand BUT turns and loops
Consecutive residues have a similar conformation that is repeated throughout the structure
In α helix or β strand
BUT
Regions that found in proteins which contain stretches of non-repeating 3D structure
These regions can cause directional changes in the polypeptide backbone
Connect α helices and β strands
Allow the polypeptide chain to fold back on itself, producing 3-dimensional shape seen in native structure
turns and loops

21. LOOPS TURNS Usually found on the surfaces of proteins
(often contains hydrophilic residues)
-Loops containing only a few (up to five) residues will be referred as turns if they cause an abrupt change in the direction of a polypeptide chain
-β turns (or reverse turns);-
a) the most common types of tight turns
b) usually connect different antiparallel β strands
c) common types of β turns are;-
“TYPE I” and “TYPE II”
(both types produce an rapid/ abrupt change in the direction of the polypeptide chain)
-All turns have internal hydrogen bonds that stabilize the structure

22. LOOPS

23. β TURNS “Note that the hydrogen bonds between
Occur more than twice as frequently as type II
Always have Gly as the third residue
“Note that the hydrogen bonds
between
the peptide groups of the 1st and 4th residues of the bends”

24. 3.3 TERTIARY STRUCTURE OF PROTEINS
During and after protein synthesis, a protein folds into α helices and β sheets
These areas of secondary structure bind together and fold on each other in specific ways
Once the process of protein synthesis is completed, the protein takes its final shape.
This stable form of the protein is known as the mature form, also known as the tertiary structure
by DEFINITION….
1) The shape of fully folded polypeptide chains
2) Results from the folding of its secondary structural elements
(which may already posses some regions of α helix and β structure)
into a closely packed 3D structure

25. 2 types of tertiary structures:
…important feature of tertiary structure…
Amino acid residues that are far apart in the primary structure are brought together, permitting interactions among their side chains
2 types of tertiary structures:
Supersecondary structures (MOTIFS)
Domains

26. Native protein = the naturally occurring form of protein
TERTIARY STRUCTURE
Most native proteins occurs at this state.
Native protein = the naturally occurring form of protein

27. TERTIARY STRUCTURE STABILIZED BY..
so that distant regions of the chain are brought closer

28. TERTIARY STRUCTURE
Disulfide bridge  though covalent, are also element of tertiary structure (they are not part of the primary structure since they form only after the protein folds)

29. EXAMPLES OF TERTIARY STRUCTURE
Triose phosphate isomerase = catalyzes the reversible interconversion of the triose phosphate isomers dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP), an essential process in the glycolytic pathway.

30. COMMON TYPES OF TERTIARY STRUCTURES
(a) SUPERSECONDARY STRUCTURES (a.k.a motifs)
-Are recognizable combinations of α helices + β strands + loops (that appear in a number of different proteins)
-Sometimes  motifs are associated with a particular function (although structurally similar motifs may have different functions in different proteins)
…common motifs…
One of the simplest motifs
-Occurs in a number of calcium-binding proteins
(since Glutamate and Aspartate residues in the loop of calcium-binding proteins form part of the calcium-binding site)
-Also called as helix-turn-helix in certain DNA-binding proteins
(since the residues that connect the helices form a reverse turn. The α helices residues in DNA-binding proteins bind DNA)

31. The individual α helices have opposite orientations
Consists of two amphipathic α helices that interact through their hydrophobic edges
- e.g. ;- leucine zipper
The individual α helices have opposite orientations
-Parallel in the coiled-coil motif
Consists of two parallel β strands linked to an intervening
α helix by 2 loops
- The helix connects the C-terminal end of one β strand to the N-terminal end of the next
Often runs parallel to the two strands

32. - Consists of 2 adjacent antiparallel β strands connected by a β turn
Is an antiparallel β sheet composed of sequential β strands connected by loops or turns
-May contain one or more hairpins but, more typically the strands are joined by larger loops
- This is a β sheet motif linking four antiparallel β strands such that;-
strands 3 and 4 (form the outer edges of the sheet) strand 1 and 2 (are in the middle of the sheet)

33. Is formed when β strands or sheets stack on the top of one another
Figure shows an example of a β sandwich where the β strands are connected by short loops and turns..
BUT
β sandwich can also be formed by the interaction of 2 β sheets in different regions of the polypeptide chain

34. COMMON TYPES OF TERTIARY STRUCTURES
(b) DOMAINS
Are compact units which are discrete and independently folded
May consist of combinations of motifs
Are usually connected by loops,
BUT
they are also bound to each other THROUGH weak interactions
(formed by the amino acid side chains on the surface of each domain)
Some domain structures occur in many different proteins while others are unique
In general, proteins can be grouped into families according to…
“ similarities in domain structure and amino acid sequence”

35. CLASSIFICATION OF DOMAINS
- Commonly used classification scheme groups are;-
α category domains
consist almost entirely of α helices and loops
consist only β sheets and nonrepetitive structures that link β strands
β category domains
have supersecondary structures such as the βαβ motif and others (in which regions of α helix and β strand alternate in the polypeptide chain)
α/β category domains
consist of local clusters of α helices and β sheet (where each type of secondary structure arises from separate contiguous regions in the polypeptide chain)
α+β category domains
- Within each of the 4 main structural categories, protein domains can be further classified by the presence of characteristic folds;-
Is a combination of secondary structures that form the core of a domain
“ some domain have easily recognizable folds such as the β meander. However, other folds are more complex”
folds

36. COMMON DOMAIN FOLDS

37. 3.4 QUATERNARY STRUCTURE
-Many protein exhibit an additional level of organization called as quaternary structure

38. QUATERNARY STRUCTURES (quaternary structure)
by DEFINITION…
(1) Quaternary structure refers to the organization and arrangement of subunits
… IN …
a protein with multiple subunits
multisubunit protein = an oligomer (proteins with only one polypeptide chain are monomers)
(2) Quartenary structure is the stable association …OF... multiple polypeptide chains resulting in an active unit
Not all proteins exhibit quartenary structure
Usually, each polypeptide within a multisubunit protein folds more-or-less independently into a stable tertiary structure
the folded subunits then associate with each other to form the final structure
(quaternary structure)

39. common shorthand method for describing oligomeric proteins
SUBUNITS
each subunit = a separate polypeptide chain
the subunits within an oligomeric protein always have a defined stoichiometry
the arrangement of the subunits give rise to a stable structure
-the subunits of a multisubunit protein may be…
identical
different
Dimers and tetramers predominate
Each type often has a different function
common shorthand method for describing oligomeric proteins
Uses ;-
Greek Letters  to identify types of subunits
Subscript numerals  to indicate number of subunits
Example ;-
α2βγ protein  contains 2 subunits designated α
 EACH 1 of subunits designated β and γ

40. proper alignment of the subunits
-Subunits are held together by many weak, noncovalent interactions
hydrophobic interactions
electrostatic forces
-principle forces involved
-contribute to the
proper alignment of the subunits
Hydrogen bonding
ionic bonding
Van der Walls interaction
involved in the interactions between subunits
in rare instances
between cysteine residues in different polypeptide chains
disulfide bonds
- The subunits of an oligomeric protein can be often separated in the laboratory because…intersubunit forces are usually rather weak…

41. EXAMPLE OF QUATERNARY STRUCTURE
This protein has 2 identical subunits with α/β barrel folds
-The identical subunits associate through weak interactions between the side chains found mainly in loop regions

42. EXAMPLE OF QUATERNARY STRUCTURE
This protein has identical all-β subunits that bind symmetrically
-The identical subunits associate through weak interactions between the side chains found mainly in loop regions

43. Determination of the subunit composition of an oligomeric protein
SUBUNITS
Determination of the subunit composition of an oligomeric protein
-essential step in the physical description of a protein
Molecular weight of native oligomer  estimated by gel-filtration chromatography
Molecular weight of each chain  SDS-polyacrylamide gel electrophoresis

44. 3.5 PROTEIN DENATURATION AND RENATURATION
Disruption of the protein native conformation, commonly caused by
HEATING (but only with small range of temperature)
as a result
Most of the denatured proteins ;-
Retain considerable internal structure
Some of the denatured proteins ;-
May unfold completely to form a random coil
-Small denatured proteins can spontaneously renature, or refold
Can cause loss of biological activity since the protein will malfunction
Amount of energy needed for the process is often small
(perhaps equivalent to the energy needed for the disruption of 4 hydrogen bonds)
Is a cooperative process  the destabilization (of just a few weak interactions) leads to
almost complete loss (of native conformation)

45. How DENATURATION can occur ??
Modest increase in temperature will result in unfolding and
loss of secondary and tertiary structure 1
Thermal
Most proteins are stable at temperatures up to 50oC to 60oC
Requires a reducing agent that disrupts disulfide bridge, which will finally allowing protein to unfold
e.g. urea and guanidinium salts 2
Chaotropic Agents
By allowing molecules to solvate nonpolar groups in the interior of proteins
e.g. hydrophobic tails of detergents  sodium dodecyl sulfate
The water molecules disrupt the hydrophobic interactions that normally stabilize the native conformations 3
Detergents
Denature proteins by penetrating the protein interior and disrupting hydrophobic interactions

46. Thermal denaturation of horse apomyoglobin and ribonuclease A
PROTEIN DENATURATION
Thermal denaturation of horse apomyoglobin and ribonuclease A
(the midpoint of T range over which denaturation occurs = Tm)

47. Regain its native conformation
RENATURATION
Occur at Tm ;-
Most proteins have a characteristics “melting” T (Tm) that corresponds to the T at the midpoint of the transition between the native and denature forms
 Tm depends on pH and the ionic strength of the solution
Regain its native conformation
Regain correct set of disulfide bonds
For this case, renaturation of Ribonuclease A can occur if :-
Urea is removed
Small amount of 2-mercaptoethanol is added
Regain full enzymatic activity

48. 3.7 STRUCTURES MYOGLOBIN ND HEMOGLOBIN
3.6 COLLAGEN
3.7 STRUCTURES MYOGLOBIN ND HEMOGLOBIN
3.8 ANTIBODIES AND BIND AND SPECIFIC ANTIGENS
2 groups (a group of 5) for each sub topic
Reference : Text Book Only
Presentation Date : Week 10