BIOPHYSICAL AND BIOCHEMICAL

ANALYSIS OF
RECOMBINANT PROTEINS
Protein Structure
&
Protein Folding
Outline

 Primary Structure
 Secondary Structure (alpha helix, beta sheet, loops and turns)
 Tertiary structures
 Forces (Hydrophobic Interactions, Electrostatic Interactions, van der
Waals Interactions)
 Protein Folding

Pharmaceutical Biotechnology - Crommelin

Protein Functions

 Three examples of protein functions

Alcohol
dehydrogenase
 Catalysis: oxidizes alcohols
Almost all chemical reactions in a living to aldehydes or
cell are catalyzed by protein enzymes. ketones

 Transport:
Some proteins transports various
substances, such as oxygen, ions, and so Haemoglobin
carries oxygen
on.

 Information transfer:
For example, hormones. Insulin controls
the amount of
sugar in the
blood
 Amino acid: Basic unit of protein

R Different side chains, R,

NH3 + C COO properties of 20 amino
- determine the
Amino group Carboxylic
acid group acids.
H
An amino acid
 20 Amino acids

Glycine (G) Alanine (A) Valine (V) Isoleucine (I) Leucine (L)

Proline (P) Methionine (M) Phenylalanine (F) Tryptophan (W) Asparagine (N)

Glutamine (Q) Serine (S) Threonine (T) Tyrosine (Y) Cysteine (C)

Asparatic acid (D) Glutamic acid (E) Lysine (K) Arginine (R) Histidine (H)

White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

Each protein has a unique structure!

Amino acid sequence

NLKTEWPELVGKSVEEAK
KVILQDKPEAQIIVLPVGTI
VTMEYRIDRVRLFVDKLD

Folding!
 Protein Structure

Primary Assembly
STRUCTURE

PROCESS
Secondary Folding

Tertiary Packing

Quaternary Interaction
 Protein Assembly

• occurs at the ribosome

• involves polymerization of amino acids
attached to tRNA
• yields primary structure
 Primary Structure
primary structure of human insulin
CHAIN 1: GIVEQ CCTSI CSLYQ LENYC N
CHAIN 2: FVNQH LCGSH LVEAL YLVCG ERGFF YTPKT
• linear
• ordered
• 1 dimensional
• sequence of amino acid polymer
• by convention, written from amino
end to carboxyl end
• a perfectly linear amino acid
polymer is neither functional nor
energetically favorable  folding!
 Protein Folding

• occurs in the cytosol

• involves localized spatial interaction among • yields secondary structure
primary structure elements, i.e. the amino
acids
Secondary Structure

 non-linear
 3 dimensional
 localized to regions of an
amino acid chain
 formed and stabilized by
hydrogen bonding,
electrostatic and van der Waals
interactions
Secondary structure

α-helix β-sheet

Secondary structures, α-helix and β-sheet,

have regular hydrogen-bonding patterns.
 Protein Packing

• occurs in the cytosol (~60% bulk water,

~40% water of hydration)
• involves interaction between secondary
structure elements and solvent
• yields tertiary structure
 Tertiary Structure

• non-linear
• 3 dimensional
Components of Tertiary Structure

 Fold – used differently in different contexts – most broadly a

reproducible and recognizable 3 dimensional arrangement
 Domain – a compact and self folding component of the
protein that usually represents a discreet structural and
functional unit
 Motif (aka supersecondary structure) a recognizable
subcomponent of the fold – several motifs usually comprise
a domain
 Protein Interaction
• occurs in the cytosol, in close proximity to other folded and packed proteins
• involves interaction among tertiary structure elements of separate polymer
chains
Quaternary Structure
• non-linear
• 3 dimensional
3D structure of proteins

Tertiary
structure
Quaternary structure
Class/Motif

• class = secondary structure composition,

e.g. all , all , / , +
• motif = small, specific combinations of secondary
structure elements,
e.g. -- loop
• both subset of fold

/
Fold
• fold = architecture = the overall shape and
orientation of the secondary structures,
ignoring connectivity between the structures,
e.g. / barrel, TIM barrel
• subset of fold families/superfamilies
Fold families/Superfamilies

• fold families = categorization that takes into

account topology and previous subsets as well as
empirical/biological properties, e.g. flavodoxin
• superfamilies = in addition to fold families,
includes evolutionary/ancestral properties

CLASS: +
FOLD: sandwich
FOLD FAMILY: flavodoxin
Hierarchical nature of protein structure

Primary structure (Amino acid sequence)

↓
Secondary structure （α-helix, β-sheet）
↓
Tertiary structure （Three-dimensional structure
formed by assembly of secondary structures）
↓
Quaternary structure （Structure formed by more than
one polypeptide chains）