Proteins

 Proteins are biopolymers of amino acid residues linked by peptide bonds.

 Two amino acids can be covalently linked through a bond called peptide bond

to form a dipeptide.

 When a peptide bond is formed, a water molecule is released.

 A peptide formation requires condensation of the α-carboxyl group of one

amino acid with a α-amino group of the other amino acid.

 Similarly, many amino acids can be linked together by multiple peptide bonds

to form large peptides or polypeptides or proteins.

 Polypetide: Is a polymer containing many amino acid residues joined by

peptide bonds.
 Oligopeptide: Is a peptide containing up to 20 amino acid residues.

 Proteins may contain hundreds to thousands of amino acid residues.

 Many proteins are composed of multiple polypeptides.

Levels of protein organization

 Proteins are structurally and functionally highly complex biological

macromolecules.

 The functions carried out by proteins are highly diverse which is inextricably

linked to its native structure or conformation in vivo.

 Structurally, four levels of protein organization are observed, namely

1). Primary structure

2). Secondary structure

3). Tertiary structure, and

4). Quaternary structure

 We shall next discuss these levels of organization _ _

Protein primary structure

 Primary structure refers to the amino acid sequence present in a polypeptide

or protein.

 Amino acid sequence means the order of arrangement of different amino acids

in a polypeptide chain.
 Different proteins have different amino acid sequences and hence different

structures and functions.

 It is the amino acid sequence of a protein that determines its three

dimensional structure and function.

 In fact protein amino acid sequence and function is closely linked.

 In the 1950s, Christian Anfinsen conducted a series of experiments on an

enzyme called ribonuclease’s activity.

 He determined that all the information needed to form the three-dimensional

structure of the polypeptide is stored in the specific sequence of amino acids in

that polypeptide.

 Later experiments confirmed this fact - that primary structure determines the

conformation of proteins.
 In 1953, British biochemist Fredrick Sanger deduced for the first time the

amino acid sequence of the polypeptide, the bovine insulin.

 He was awarded with the Nobel prize in chemistry in 1958 for his work on

amino acid sequencing of insulin.

 In 1980 again he received the Nobel prize in chemistry for nucleic acid

sequencing.
 Short proteins and peptides (up to 100 residues) can be synthesized chemically

in labs.

 Proteins can be fragmented or lysed in labs using proteases (such as trypsin,

chymotrypsin, pepsin etc.) or other chemicals (cyanogen bromide) that breaks

the peptide bonds to release free peptides and amino acids.

 Protein size/mass is indicated in:

Protein Molecular mass: Dalton (Da) or kDa,

1 kDa = 1000 D

or

Relative molecular mass/ molecular weight: has no units

The peptide bond & the peptide group

  A peptide bond in polypeptide is rigid and planar.

 The C–N peptide bond in peptides/polypeptides is somewhat shorter than

the non-peptide C–N bond in a simple amine.

 Moreover, the C–N peptide bond is not truly a single bond but exhibit a

partial double-bond character.

 This is due to resonance in the peptide bond because of localization of 2

pairs of electrons between the carbonyl oxygen and amide nitrogen.

 The resonance in the peptide bond gives it a partial double-bond nature and

hence rotation at the peptide bond is restricted/not permitted.

 Disallowed rotation of the peptide bond has significant impact on polypeptide

secondary and tertiary structure.

The peptide group

 A peptide group comprises of six atoms around the peptide bond in a

polypeptide.

 The 6 atoms lay in a single plane with the oxygen of the carbonyl trans in

position to the hydrogen of the amide nitrogen.

 Rotation of covalent bonds other than the peptide bonds is possible in the

peptide group of a polypeptide.

 Rotation is permitted about the N – Cα and the Cα – C bonds.

 The rigid peptide bonds limit the range of conformations possible for a

polypeptide.

 Peptide conformation is defined by three dihedral or torsion angles called phi

(φ), psi (ψ) and omega (ω) which reflects rotation about each of the three

repeating bonds in the peptide backbone.

  Dihedral angle between the N – Cα bond is the φ angle which can have any value

between +180° to -180°.

 Similarly, dihedral angle between the Cα – C bond is the ψ angle which can have

any value between +180° to -180°.

 A graphical representation of φ and ψ angle for a given protein can be shown by

Ramachandran plot introduced by G N Ramachandran.

 Ramachandran plot exhibit allowed values for φ and ψ when plotted against

each other.

 Ramachandran plot is useful to test the quality of 3D protein structures available

in protein databases.

Protein Secondary structure

 The secondary structure arises from the hydrogen bonds formed between

atoms of the polypeptide backbone.

 The hydrogen bonds can form between the partially negative oxygen atom and

the partially positive hydrogen atom.

 Most proteins have segments of their polypeptide chains that are either coiled or

folded in patterns that contribute to the protein’s shape.

 Many of these coils and folds repeat so often that they have been given names.

 Two folds that are extremely common in biochemistry are the alpha-helix and

the beta-pleated sheet.

 The alpha-helix is a right-handed helical coil that is held together by hydrogen

bonding between every fourth amino acid.

 Linus Pauling and Robert Corey in 1948 put forward the alpha helix structure in

proteins.

 Each helical turn accommodates 3.6 amino acid residues.

 Many globular proteins have multiple alpha-helical portions separated by long

stretches of non-helical regions.

 Not all polypeptides can form a stable α helix.

 Some amino acids like ala, arg, leu, lys etc. favours α helix whereas, gly, pro,

cys discourage α helix structure in proteins.

 Some fibrous proteins, including alpha-keratin, are almost completely

comprised of alpha-helices.

 Fingernails and toenails are also made up of alpha-helices.

 The other common secondary structure is the β-confromation.

 Pauling and Corey, 1951 predicted the β-conformation in proteins.

 In this structure, two different regions of a polypeptide chain lie side by side

and are bound by hydrogen bonds to form β sheets.

 β sheets make up the core of many globular proteins.

 In β sheets, the polypeptide backbone is organized into a zigzag structure rather

than helical strcture.

 The two types of beta-pleated sheets are parallel beta-pleated sheets and

antiparallel beta-pleated sheets.

 If two beta-strands run in the same direction ( ) then it is a parallel beta-

pleated sheet, and if they run in opposing directions ( ) then it is an

antiparallel beta-pleated sheet.

 A third type of secondary structure encountered in globular proteins is

the β turns.

 In globular proteins, the compact folded structure is comprised of turns or

loops where the polypeptide chain reverses direction.

 At the turns or loops the secondary structure β turns are observed.

 β turns are commonly observed in the turn of two adjacent segments of an

antiparallel β sheet.
  β turn structure is a 180° turn involving 4 amino acid residues, with the

carbonyl oxygen of the first forming a hydrogen bond with the amino

group hydrogen of the fourth.

 Gly and pro residues most often encountered in β turns, as gly is small and

flexible and pro forms a favorable cis peptide bond.

 Two common types of β turns are the type I and type II.

 β turns are often found at the surface of a protein where it can form H-bond

with water molecules.

Protein tertiary structures

 Tertiary structures refer to the three-dimensional arrangement of all atoms in

a polypeptide chain.
 Whereas secondary structure refers to the spatial arrangement of amino acid

residues that are adjacent in a segment of a polypeptide.

 Tertiary structure involves longer-range aspects of amino acid sequence.

 In tertiary structure, the polypeptide chain undergoes folding, as a result amino

acids that are far apart may interact with each other due to weak forces and

sometimes disulfide bond(s) within the folded structure.

  Tertiary structure generally comprises of bends (including β turns) whose

direction and angle is determined by amino acid resides present at the bends

such as gly, pro, thr and ser.

 Tertiary structure of proteins can be classified into two major groups where

many proteins belong, namely:

Fibrous proteins and globular proteins

Fibrous proteins

 Fibrous proteins assume the shape and structure of fibers due to arrangement of

the polypeptide chain as long strands or sheets.

 The fundamental structural unit of fibrous proteins is a simple repeating element

of secondary structure.

 Fibrous proteins usually consist of one type of secondary structure and hence

their tertiary structure is relatively simple.

 All fibrous proteins are water insoluble due to high concentration of

hydrophobic amino acid residues in its surface and interior.

 Fibrous proteins are best examples of relationship between protein structure and

function.

 Fibrous proteins have properties that give them strength and flexibility to its

structures.

 Examples of fibrous proteins include fibroin (in silk), collagen (in bone,

cartilage etc) and α-keratin (in hair, nail etc)

 α-keratin is found only in mammals and have a right handed alpha helix and

arranged as coiled coil.

 Collagen on the other hand has a unique structure as the alpha helix is

left-handed and has 3 amino acid residues per turn of helix.

 In silk fibroin, the polypeptide chain is predominantly in anti-parallel

β sheet conformation.
Globular proteins

 In globular proteins, different segments of a polypeptide chain fold back on

each other, generating a compact and roughly spherical structure.

 The folding provides the structural diversity necessary for the proteins to carry

out different biological functions.

 Examples include enzymes, receptors, ion channels, antibodies, transport

proteins, gene regulatory proteins etc.

 Muscle myoglobin was the first protein whose 3D globular structure was

determined by John Kendrew using x-ray diffraction studies in the 1950s.

 In myoglobin the single polypeptide chain almost completely assumes alpha

helix conformation.
Protein super-secondary structures

 Globular proteins exhibit complex tertiary structures with common structural

patterns.

 The common structural pattern recurs in different and unrelated proteins and is

an assemblage of α helix and β sheet conformations.

 The α helix and β sheet conformations are linked by connecting

segments which in turn stack on one another and undergo precise

arrangement.

 Motifs or folds are super-secondary structures found in numerous

globular proteins.

 Super-secondary structures are a recognizable folding pattern

involving two or more elements of secondary structure and the

connecting segments between them.

 A motif can be very simple such as two elements of secondary

structure and the connection between them and represent a small part

of a protein.

 An example is β- α- β loop.

 Also, a motif can be a very elaborate structure involving many protein

segments folded together, such as the β barrel.

 A single large motif may comprise the entire protein.

 The coiled coil of alpha keratin is a well-studied motif found also in some

other proteins.

 Motif or super-secondary structure is simply a folding pattern and does not fall

between secondary and tertiary structure.

  The term ‘super-secondary structure’ is hence somewhat misleading as it

suggests hierarchy.

Protein quaternary structure

 Quaternary structure is the ultimate level of organization that many proteins

attain.

 However, it is to be noted that not all proteins exists as quaternary structure.

 Quaternary structure exists in proteins consisting of two or more identical or

different polypeptide chains (subunits).

 These proteins are called oligomers or multimers because they have two or

more subunits.

 The repeating structural unit in oligomers are known as protomers.

 Proteins with identical subunits are referred as homo-oligomers and with non-

identical subunits as hetero-oligomers.

 The quaternary structure describes the manner in which subunits are arranged in

native proteins.

 Many proteins have multiple subunits ranging from two to hundreds.

 The association of polypeptide chains in proteins can serve a variety of

functions.

 Subunits are held together by noncovalent forces; as a result, oligomeric

proteins can undergo rapid conformational changes that affect biological

activity.

 Oligomeric proteins include the hemoglobins, allosteric enzymes responsible

for the regulation of metabolism, contractile proteins such as actin and tubulin,

etc.
 The first oligomeric protein whose 3D structure was determined was

hemoglobin (molecular mass 64.50 kDa).

 Hemoglobin is a tetramer of 2 dissimilar subunits α and the β chains.

 So two chains of α and β forms the tetramer hemoglobin (α2β2).

 Max Perutz and John Kendrew and their colleagues revealed the three

dimensional structure of hemoglobin using x-ray analysis in 1959.

 The quartenary structure of hemoglobin is intrinsically linked to its

function that facilitates efficient oxygen binding and dissociation in living

tissue.