Structure of ProteinsDr. Kiran Meena

13/09/2019

9:00-10:00 AM

Specific learning objectives

Structure organization of the Proteins includes:

1) Primary structure

2) Secondary Structure

3) Tertiary Structure

4) Quaternary Structure

Enumerate and describe pathophysiological conditions arising due to defective protein

structure

Describe structure of collagen and its structure-function relationship with clinical correlation

Describe the structure of Myoglobin and Hemoglobin and its structure-function relationship

Structural organization of Proteins

Fig.3.16. Lehninger Principles of Biochemistry

Protein’s conformation stabilized by mainly weak interactions

In protein structure, stability defined as tendency to maintain a native

conformation

Protein conformation with lowest free energy (most stable) is the one with

maximum number of weak interactions

Native proteins are marginally stable; ΔG separating the folded and

unfolded states in proteins under physiological conditions is in range of

20-65kJ/mol

Cont--

• For all proteins, weak interactions imp in folding of polypeptide chain into

2° and 3° structure, association of multiple polypeptides to form

4°structure

• Net stability contributed by hydrogen bond, or the difference in free

energies of the folded and unfolded states, may be close to zero

Protein stability depends on hydrophobic interactions: Hydrophobic aa

side chains tend to cluster in protein’s interior, away from water.

• AA seq of most proteins contains hydrophobic aa side chains, positioned

so that form clusters when protein fold, forming a hydrophobic protein

core.

Primary Structure

Amino acids (aa) are linked by peptide bonds to form polypeptide

chains.

Ordered sequence of aa in a polypeptide chains is primary structure

of protein.

It is unique primary structure that enables a polypeptide chain to fold

into a specific 3-D structure that gives protein its chemical and

physiological properties.

Amino Acids are Polymerized into Peptides and Proteins

Condensation reaction: α-carboxyl group of

one aa with side chain R1 forms a covalent

peptide bond with α-amino group of other aa

with side chain R2 by elimination of a

molecule of H2O

Exergonic reaction: Ex. Hydrolysis of a

peptide bond, it occurs slowly because it has

a high activation energy, so peptide bond

become quit stable, with average half life

7yrs under intracellular conditions

Fig3.13: Lehninger Principles of Biochemistry by David L Nelson, 6th Ed

Cont--

Dipeptide (contain two aa bonded to each

other via a single peptide bond)

Tripeptides (contains three aa) form a

second peptide bond through its terminal

carboxylic acid group and amino of a third aa

(R3).

Repetition of this process form a polypeptide

or protein of specific aa sequence

(R1R,2R,3R4∙∙ ∙Rn).Fig. 2.8. Peptide bond formation:

Textbook of Biochemistry with Clinical Correlations

4th edition by Thomas M Devlin

Components of a Polypeptide Chain

It consists of a repeating backbone part,

and a variable part consists distinctive

side chains.

Polypeptide backbone is rich in hydrogen-

bonding. Each residue contains a

carbonyl group (exception of proline, an

NH group)

Mass of a protein expressed in units of

daltons; one Dalton (Da) is equal to one

atomic mass unit.

Fig.2.15: Components of a polypeptide chain: Biochemistry 7th

edition by Berg, Tymoczko and Stryer

Peptide Bond has Partial Double-Bond Character

Single bond linked α-carboxyl and α-nitrogen

atoms, this peptide bond exhibits partial

double-bond character.

Bond that connects a carbonyl carbon to an α-

nitrogen cannot rotate and thus conformation

of peptide backbone is constrained.

Almost all peptide bonds in proteins are trans.

Therefore, O, C, N, and H atoms of a peptide

bond are coplanar.

Fig.2.18: Peptide bonds are planar: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Rotation about Bonds in a Polypeptide

Peptide bond conformation defined by values

of Phi (φ) and psi (ψ) angle or dihedral angles

φ is angle of rotation about bond between N-Cα

bond

ψ is angle of rotation about bond between Cα-C

bond

Both φ and ψ defined as ±180° when

polypeptide fully extended and all peptide

groups in same plane

Fig.2.22: Biochemistry 7th edition by Berg, Tymoczko and Stryer

α

Cont--

Dihedral angles increases as the distal (4th) atomis rotated clockwise

From ±180° position, dihedral angle increasesfrom -180° to 0°, at which point 1st and 4th atomseclipsed

Rotation continue from 0° to +180° to bringstructure back to starting point

φ and ψ have any value bet -180° to +180°, butmany values prohibited by steric hindrance betatoms in polypeptide backbone and aa sidechains

Fig.4.2 (d). Lehninger Principles of Biochemistry, 6th Ed.

Ramachandran Plot

Ramachandran plot introduced by G.N.Ramachandran in 1963

Give visual description of combinations of φ and ψ angles that permitted

in peptide backbone or not permitted due to steric hindrances

Allowed values for φ and ψ become evident when ψ is plotted versus φ

in a Ramachandran plot

This plot are useful tools used to test the quality of 3-D protein

structures deposited in international databases

In case of Gly residue, less sterically hindrance, has much broader

ranges of allowed confirmations

Cont--

φ and ψ values bet. -180° and +180°, but

many values are prohibited by steric

interference between atoms in polypeptide

backbone and aa side chains

Allowed/favorable regions with no steric

overlap are shown in dark green; borderline

regions are shown in light green

In peptides, for aa other than glycine, most

combinations of φ and ψ angles are

disallowed due to steric hindrance

Fig.2.23: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Amino Acid Sequences have Direction

A polypeptide chain has polarity because

its ends are different: an α-amino group is

present at one end and an α-carboxyl

group at the other.

Amino end is taken to be beginning of a

polypeptide chain, and so sequence of aa

in a polypeptide chain is written starting

with amino-terminal residue.

Fig.2.14: Amino acid sequences have direction: Biochemistry 7th

edition by Berg, Tymoczko and Stryer

Secondary Structure of Proteins

Regular arrangements of protein chain are stabilized by hydrogen

bonding

Polypeptide chains fold into regular structures such as alpha (α) helix,

beta (β) sheet, and turns and loops.

α-helices, β-strands, and turns are formed by a regular pattern of

hydrogen bonds between peptide N-H and C=O groups of aa that are

near one another in linear sequence. Such folded segments are called

secondary structure.

Alpha Helix Is a Coiled Structure Stabilized by Intrachain Hydrogen Bonds

α-helix is stabilized by intrachain hydrogen

bonding between CO and NH groups along

parallel to helical turn.

R groups of each aminoacyl residue in an α-

helix face outward.

Pitch of α-helix: length of one complete turn

along helix axis and is equal to product of rise

(1.5 Å) and number of aa per turn (3.6), or 5.4

Å.

Fig.4.4 b: Ball-and-stick model of a right handed α helix. Lehninger Principles

of Biochemistry

Amino terminus

Cont--

Largely α-helical protein: Ferritin

~75% of residues in ferritin, a protein that

helps store iron, are in α-helices.

~25% of all soluble proteins are composed

of α-helices connected by loops and turns

of polypeptide chain.

Many proteins that span biological

membranes also contain α-helices.

Fig.2.28: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Type of constraints affect stability of α-helix

Intrinsic susceptibility of aa residue to form α-helix

Interaction bet R groups (spaced 3 or 4 residues apart)

Bulkiness of adjacent R groups

Occurrence of Pro and Gly residues

Interaction bet aa residues at end of helical segment and electric dipole

inherent to α-helix

β-Sheets Stabilized by Hydrogen Bonding Between Polypeptide Strands

Backbone of polypeptide chain

extended into zigzag

Arrangement of many segments side

by side in β-conformation is called β-

sheets

Hydrogen bonds form bet adjacent

segments of polypeptide chain within

sheet

Side chains are above and below

plane of strands.

Fig.2.30: Biochemistry 7th edition by Berg, Tymoczko and Stryer

NO

CCα

H

Parallel β-sheet

In parallel arrangement, for each aa,

NH group is hydrogen bonded to CO

group of one aa on adjacent strand

1:2 H-bond pattern: 1 a.a bonds with 2

other a.a in an opposing stand.

Fig.2.32: Biochemistry 7th edition by Berg, Tymoczko and Stryer

N

HO

C

Cα

R

O

C

Antiparallel β-sheet

Fig.2.31: Biochemistry 7th edition by Berg, Tymoczko and Stryer

N

HO

C

Cα

Adjacent chains in a β sheet can run

in opposite directions (antiparallel β

sheet) or in same direction (parallel β

sheet).

Follow 1:1 H-bond pattern

In antiparallel arrangement, NH group

and CO group of each aa are

respectively hydrogen bonded to CO

group and NH group of a partner on

adjacent chain.

N

H

C

o

Reverse Turns

In globular proteins, has compact folded

structure, some aa residues are in turns or

loops where polypeptide chain reverses

direction. Turns connect helical twists and

sheets.

Structure is a 180° turn involving four aa

residues, with CO of residue i or 1st residue

forming a hydrogen bond with NH of residue

i+3 or 4th residue

Fig.3.42: Biochemistry 7th edition by Berg, Tymoczko and

Stryer

C O

NH

Cont--

• Gly and Pro residues occurs in β-turn, Gly because its small and flexible

but Pro because peptide bond involves imino nitrogen assume cis conf,

a form a tight turn

• β-turn found near surface of protein where peptide groups of central two

aa residues in turn can hydrogen bond with water.

Cont--

Loops are responsible for chain reversals

and overall shape.

Turns and loops invariably lie on surfaces

of proteins and participate in recognition

role of proteins, such as recognition of

specific antigens by antibodies.

Ex. part of antibody molecule has surface

loops (shown in red) that mediate

interactions with other molecules.Fig.3.43: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Tertiary Structure

Overall 3-D arrangement of all atoms in protein is

referred as protein’s tertiary structure

Myoglobin, oxygen carrier in muscle. It functions

both to store oxygen and to facilitate oxygen

diffusion in rapidly contracting muscle tissue.

Capacity of myoglobin to bind oxygen depends on

presence of heme, a non-polypeptide prosthetic

group consisting of protoporphyrin IX and a central

iron atom.Fig.3.44 b: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Cont--

Interior consists of nonpolar residues such as leucine, valine,

methionine, and phenylalanine

Only polar residues inside are two histidine residues, play critical roles

in binding iron and oxygen.

Outside of myoglobin, consists of both polar and nonpolar residues.

Higher level of structure

Useful to designate two major groups into fibrous and globular proteins:

Fibrous Proteins with polypeptide chains arranged in long stands or

sheets

• Consist largely of a single type of 2⁰ structure and their 3⁰ structure is

simple

• Its structure provide support, shape and external protection

• Tertiary structure of the fibrous proteins: α-keratin, collagen and silk

fibroin

1. α-keratin helix is a right handed α-helix,

arranged as a coiled coil

• These combine in higher-order structures

called protofilaments and protofibrils.

• Four protofibrils—32 strands of α-keratin

altogether—combine to form an

intermediate filament

• 2 strands of α-keratin, oriented in parallel

wrapped each other to form super-twisted

coiled coilFig.4.11: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Cont--

• Coiled coil of this type are common structural elements in filamentous

proteins and in the muscle protein myosin

• Contribute to cell cytoskeleton (internal scaffolding in a cell)

Cont--

2. Collagen is main fibrous component of skin, bone,

tendon, cartilage, and teeth.

• Super helical twisting is right handed, opposite in sense

to left handed helix of α-chains.

• Three aa residue per turn. Glycine appears at every third

residue in a sequence.

• Hydrogen bonds within a strand are absent.

Fig.2.40: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Fig.4.12 (a). Lehninger Principles of Biochemistry, 6th Ed.

Cont--

• Helix is stabilized by steric repulsion of pyrrolidine rings of proline andhydroxyproline residues

• Seq of aa in collagen is repeating tripeptide unit, Gly-X(pro)-Y(4-Hyp)

• Only Gly residues accommodated at very tight junction bet individual α-chains

• Pro and 4-Hyp residues permit sharp twisting of collagen helix

• Tight wrapping of α-chains in collagen triple helix provides tensilestrength

Cont--

Osteogenesis imperfecta: Abnormal bone formation in babies, at least

eight variants, with different degrees of severity.

• Results from substitution of an aa residue with larger R group (Cys or

Ser) for single Gly residue in α-chain in one or another collagen protein

• They disrupt Gly-X(pro)-Y(4-Hyp) repeat gives collagen its unique

helical structure

Cont--

Globular Proteins: Different segments of polypeptide chain fold backon each other, generates more compact shape

• Consists of variety of tertiary structure

• 3-D structure of typical globular protein consider assemblage ofpolypeptide segments in α-helical and β conformations linked byconnecting segments

• Most of enzymes, transport proteins, motor proteins, regulatoryproteins, Ig are globular proteins

Quaternary Structure

Some proteins contain two or more separate polypeptide chains orsubunits, may be identical or different.

Arrangement of these protein subunits in 3-D complexes constitutesquaternary structure

Many multisubunit proteins have regulatory roles. Ex. each ribosome,incorporates dozens of protein subunits along with a number of RNAmolecules

Quaternary structure implies non-covalent interaction that stabilise foldedpolypeptides leads to multisubunit proteins.

Cont--

Multisubunit proteins/multimer have a number of identical (homomeric)

subunits (in symmetric arrangements and simplest) or non-identical

(heteromeric) subunits (asymmetric and complicated)

Repeating structural unit in multimeric protein whether a single or

groups of subunit is called protomer

Multimer with just a few subunits is called oligomer. First oligomeric

protein to have its 3D structure determined was Hemoglobin (Hb).

Cont--

• Ex. Hb (oxygen carrying protein) in

blood contains four polypeptide chains

and four heme prosthetic groups, in

which iron atoms are in ferrous (Fe2+)

state.

• Protein portion, called globin, consists

of two α-chains (141 residues) and two

β-chains (146 residues)

Fig.3.49: Biochemistry 7th edition by Berg, Tymoczko and Stryer

Cont--

Subunits of Hb are arranged in symmetric pairs, each pair having one

α and one β subunit.

Hb exists as an α2β2 tetramer or dimer of αβ protomers.

Subtle changes in arrangement of subunits within Hb molecule allow

it to carry oxygen from lungs to tissues with great efficiency.

Summary

Most important element of primary structure is sequence of aminoacid residues.

Nature of covalent bonds in polypeptide backbone placesconstraints on structure.

Peptide bond has a partial double bond character that keeps entiresix-atom peptide group in a rigid planar configuration.

N-Cα and Cα-C bonds can rotate to assume bond angles of φ and ψ,respectively.

Cont--

Gene-encoded primary structure of a polypeptide is sequence of its

amino acids.

Secondary structure refers to stable arrangements of amino acid

residues giving rise to recurring structural patterns.

Folding of polypeptides into hydrogen-bonded motifs such as the α helix,

β-pleated sheet, β bends, and loops.

Cont--

Tertiary structure is complete three-dimensional structure of a

polypeptide chain.

When a protein has two or more polypeptide subunits, their

arrangement in space is referred to as quaternary structure

Reference Books

1) Harper’s Illustrated Biochemistry-30th edition

2) Biochemistry. 4th edition. Donald Voet and Judith G. Voet.

3) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer

4) Lehninger Principles of Biochemistry, 6th Ed by David L. Nelson, Michael M. Cox

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