L3

Protein structure

Dr. Sujin Bao, SJSM MD 2 Biochemistry 2013

Primary Structure
A peptide bond is formed from a condensation reaction
(dehydration) involving two amino acids.
A molecule of H2O is eliminated.
Dipeptide formation
H
H H O
O H N C C
H3N C C O
H CH
O
CH3
H3C CH3

alanine valine

H2O
peptide bond
H O H
O
amino H3N C C N C C carboxyl
terminus H O terminus
 CH3  CH
( amino group)
H3C CH3
 

alaninylvaline
Characteristics of the peptide bond

H O H
O
H3N C C N C C
H O
R1 R2
 Characteristics of the peptide bond
H O H
O
1. partial double-bond character H3N C C N C C
H O
R1 R2
• due to resonance

O R2 O R2
H H
C C O C C O
C C C
N C N
H 3N H H3N H
H O H O
R1 R1


O R2
H
C  C O
C C
N
H3N H
H O
R1
Characteristics of the peptide bond

2. rigid and planar

• rotation occurs around single bonds but not around

double bonds

no rotation around
peptide bond
H O H
O
H3N C C N C C
H O
R1 R2
 O
O H
C
Rotation around single bonds H
C C
O

C N R2
H3N
H
R1

Because no rotation is possible O R2

around double bonds, the H
O
C C
stereochemistry of the peptide C N
C
bond is fixed. H3N H
H O
R1

O R2
R1
C C O
H
C N C
H
H O
NH3
 sigma () bond only = “single bond”  rotation possible

pi () bond = “double bond”  no rotation possible

O
R2
H
C C O
C C
N
H3N H
O
R1 H
Characteristics of the peptide bond

3. Trans configuration

• minimizes steric hindrance

Steric hindrance in cis
conformation reduces stability
Characteristics of the peptide bond

4. Uncharged but polar

• dipole moment exists due to separation of charge


O R2
H
C  C O
C C
N
H3N H
H O
R1
Characteristics of the peptide bond - summary
• partial double bond character
• rigid and planar
• trans configuration
• uncharged but polar
amide
trans plane
config 
O R2
H
C  C O
C C
N
H3N H
H O
R1
 Determination of amino acid composition
1. Acid hydrolysis
• cleaves peptide bonds
• destroys (acidifies) Gln and Asn
• Trp mostly destroyed

2. Chromatography
• separates amino acids based on charge and pH

Two types of chromatography:

Anion exchange – separates positively-charged aa’s

Cation exchange – separates negatively-charged aa’s

Cation exchange chromatography

• separates anionic aa’s

• resin is negatively-charged

• anionic amino acids move through

column faster because they are repelled
by the negative charge of the resin

• charge is controlled by pH

• at low pH, all aa’s are cations!

• anion exchange chromatography is the

opposite of cation exchange
Quantitative analysis
• As amino acids are separated based
on charge, the relative number of
each one must be determined.

• Ninhydrin reacts with amino acids

to give purple complexes (yellow
with Pro)

• Color intensity of amino acids is

measured using spectrophotometry.

• If MW is known, relative number of

amino acids may be determined, but
not primary structure.
Peptide sequencing from N-terminal
end
• Phenylisothiocyanate – Edman’s reagent – reacts with N-
terminal residue to form phenylthiohydantoin derivative (PTH)
• The peptide bond of the PTH derivative is easily cleaved as a
thiolzolinone derivative, so the N-terminal amino acid may be
removed and identified without breaking the other peptide bonds.
• The thiazolinone amino acid is then selectively extracted into an
organic solvent and treated with acid to form the more stable
phenylthiohydantoin (PTH)- amino acid derivative that can be
identified by using chromatography or electrophoresis.
• only useful for peptides that contain 100 amino acids or less.
 O
H2N CH C Lys His Phe Leu Arg COOH
CH3
N C S
N-terminal 1. Labeling
Phenylisothiocyanate
alanine
(Edman’s reagent)
H
O
HN CH C Lys His Phe Leu Arg COOH
S C CH3 labeled peptide
H N 2. Acid
cyclization and expulsion of
hydrolysis
shortened peptide chain

O S
C
H2N CH C His Phe Leu Arg COOH + N NH
(CH2)4 C CH
O
NH2 CH3

N-terminal PTH-alanine
lysine
Cleavage of peptide into smaller fragments

• occurs before Edman degradation

• necessary if peptide is > 100 amino acids in length

• need to use more than one cleaving agent in order to

determine amino acid sequence

• different enzyme/chemical specificity

• overlap peptide fragments in order to determine original

sequence
Determining of a protein’s primary structure by
DNA sequencing
Secondary Structure
• Secondary structures result from local folding
of adjacent amino acids into only a few 3
dimensional forms.

• H-bonds are key to stabilizing these structures.

Secondary structures include:

• Helical Structures
• Beta Structure (maximally extended primary
sequence)
• Random chain (nonrepetitive)
Helix

Left-hand Right-hand
 helix  helix
Intrachain Hydrogen Bonding is important in maintaining
secondary protein structure. Here (in the α helix) the carbonyl
oxygen from one amino acid is H-bonded to an alpha nitrogen of
the 4th distant amino acid in the polymer.

Hydrogen
bond
• 3.6 residues per turn

• R groups extend outward

helix is disrupted by:

1) Pro and Gly

2) large numbers of charged

aa’s

3) aa’s with bulky R groups

Myoglobin
Sheet
• “pleated”

• all peptide bond components involved in H-bonding

• strands visualized as broad arrows

N terminal
C terminal

• may be parallel or antiparallel

Sheet
-Sheet in fibrous (Amyloid) protein

• Amyloid protein deposited in brains of

Alzheimer’s disease patients –
twisted -pleated sheet fibrils with 3-D
structure virtually identical to silk fibrils
 -turn

• function to reverse the direction of polypeptide

chain

• often include charged residues

• stabilized by ionic and/or H-bonds

• usually composed of 4 amino acids including Pro

and Gly
 Secondary structure
-turn

Structural feature

A hydrogen bond forms in

between amino acids i and
i +1
i+3 i +2

i i +3
 Secondary structure
loop

Structural feature

- A loop links regular

secondary structures
Supersecondary structure (motif)
• result from local folding of secondary structures
into small, discrete, commonly-observed aggregates
of secondary structures:
• loop

•  corner
• extended super secondary structures are known as
domains:

• barrel

• twisted  sheet
porin –  barrel
Prion
Tertiary Structure
• Tertiary structure is the 3 dimensional form of a
molecule resulting from distant protein-protein
interactions within the same polypeptide chain
(caused by folding of secondary structures):

Globular proteins are characterized as generally

having:

• a variety of different kinds of secondary structure

• spherical shape

• good water solubility

• a catalytic/regulatory/transport role i.e. a dynamic

metabolic function
Fibrous proteins are characterized as generally
having:

• one dominating kind of secondary structure

(i.e. collagen helix in collagen)

• a long narrow rod-like structure

• low water solubility

• a role in determining tissue/cellular structure and

function (e.g. collagen, keratin)
Forces involved in maintaining tertiary structure
Hydrogen bonds -N-H• • •N-
-N-H• • •O-
-O-H• • •O-
-O-H• • •N-

Disulfide bonds 2 cysteine (-SH)  cystine (-S-S)-

Hydrophobic interactions
(Van der Waals forces)

Electrostatic interactions (ionic

and/or dipole-dipole)
Most proteins do not revert to their original tertiary
structures after denaturation.

Ribonuclease enzyme is an exception.

Role of chaperones in protein folding
Chaperone proteins (polypeptide chain-binding proteins
(PCB proteins) increase rates of final stages of folding
process
Quaternary Structure
Quaternary structure consists of the association
of multimeric proteins (identical or nonidentical)
held together by one or more of the following
noncovalent interactions:

Hydrogen bonds

Hydrophobic interactions
(Van der Waals forces)

Electrostatic interactions (ionic

and/or dipole-dipole)
Hemoglobin

Hemoglobin is a
heme-containing
protein comprised of
four subunits: 2β2