SBT 2233 PROTEIN BIOTECHNOLOGY

by Dr Tengku Haziyamin Abd Hamid

References Texts
R.M. Twyman (2004) Principle of Proteomic. Advanced Text series.
Pub Bios scientific (Taylor and Francis)
Micheal M Cox and Gorge N Philips Jr.(2007) Handbook of Protein
Structure function and method Vol 1 and 2 ELS (Wiley)
Brandon and Tooze (1999) Introduction to Protein Structure (2nd Ed)
Garland
Gregory Petsko and D. Ringe Protein Structure and Function.
Blackwell Pub.
Other relevant reading!!! :
Any Textbooks on Biochemistry , Enzymology , Protein Structure,
Protein purification or Protein Methods (available in IIUM Library)
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The need for proteomic and protein biotechnology

• Function of protein on its structure/interaction cannot be

predicted from DNA sequences
• Mutation are coarse tool in structure function studies
• Abundance of trancriptomes do not reflect relative
abundance of protein
• Protein diversity is generated post-trancriptionally
• Protein activity depends post-translational modification
• Function of protein often depend on its localization
• Some samples contain no nucleic acid
• Protein are the most therapeutically relevant molecules

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Protein structure
for
biotechnology

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LECTURE COMPONENTS
• Protein Structure
• Protein purification Techniques
• Proteomics in Biotechnology

REFERENCES
• Principle texts
• Extended Reading

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Primary Structure:

20 amino acids –
• chemical structure & properties:
chirality
• different types of side chain
• relevance to mutation
• size R
• aliphatic/aromatic
• polarity
• charge H3N C COOH
• hydrophobicity;
• disulphide bonds
• molecular models H

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Peptide bond

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 Peptide bond

General structure of an amino acid. Amino

acids are joined by forming peptide bonds.

H3N C COOH

resonance all peptide bonds

in protein structures are
found to be almost planar, i.e.
atoms C (i), C(i), O(i), N(i+1)
and Cα(i+1)
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 Dihedral Angles

• torsion angles:
main chain phi/psi - helices,
sheets, turns;
side chain rotamers
• Ramachandran plots
• molecular surfaces; molecular graphics

• Since bond length and angles are fairly invariant in the known protein structures,
the key to protein folding lies in the torsion angles of the backbone.
• Repeating phi,psi angles always lead to some type of regular structure providing
that it is sterictly allowed.
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Ramachandran Plot

• (after G. N. Ramachandran) or conformational map.

• Repeating values of phi and psi along the chain result in regular
structure.

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For example, repeating values of phi ~-57o and psi ~-47o give a
right-handed helical fold (the alpha-helix). The structure of
cytochrome C-256 shows many segments of helix and the
Ramachandran plot shows a tight grouping of phi-psi angles near
to -50,-50.

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Similarly, repetitive values in the region of phi = -110 to -140 and psi =
+110 to +135 give extended chains with conformations that allow
interactions between closely folded parallel segments (beta sheet
structures). The structure of plastocyanin is composed mostly of beta
sheets and the Ramachandran plot shows a broad range of values in the
-110,+130 region.

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FOUR LEVEL STRUTURE

• Protein structure can be

discussed in terms of four
levels of complexity
defined as follows:

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Secondary structure
α-helices

• α-helix and β-sheet have different

ways of H-bonding arrangement

• In α-helix H-bonds are formed

from one residue to the next few
residues in the same chain
forming a helix of sizes 3.6
residues per turn.

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In β-sheet, H-bond form between amino acids
located on one chain and the one on neighboring
chains.

Antiparallel
Parallel

Parallel

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WHAT DO WE KNOW ABOUT PROTEIN FOLDING?

• around 4000 known structures from X-ray crystallography and 2-D NMR
studies
• structure data base widely available for analysis, the Protein Data Bank
• water soluble proteins are "globular," tight packed, water excluded from
interior, folded up.
• bond lengths and bond angles don't vary much from equilibrium positions.
• structures are stable and relatively rigid, folding possibilities are limited, both
along the backbone chain and within the side chain groups.
• folding motifs are used repetitively.
• proteins with similar function typically have similar structure.
• with similar proteins (say from different organisms) structure tends to be
more conserved than the exact sequence of amino acids.
• although sequence must determine structure, it is not yet possible to predict
the entire structure from sequence accurately. 17
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Tertiary structure

• The tertiary structure results from assembling of the

secondary structure elements such as motifs to form
domains.
• Stabilized by mainly hydrophobic forces but ionic
interaction and H-bonds may play some roles.
• Commonly found motifs in protein include helix-loop-
helix or HLH motif, Rossman fold motif, β-meander
motif and β-α-β motif. Several simple motifs combine to
form a more complex motif such as ‘Greek Key’ and
‘Swiss’ or ‘Jelly roll’.

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Domain Folds

• All-Alpha
The Lone Helix • Alpha/Beta
Helix-loop-helix Rossman Fold
The Four-helix Bundle alpha/beta horseshoe
DNA-binding domains alpha/beta barrels
Globins • Alpha+Beta
• All-Beta
Up-and-down Anti-parallel Beta • Small disulphide rich proteins
Sheets
Greek Key
Jellyroll
Beta Propellors
Beta Trefoils
Beta Helix
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Examples on commonly found secondary structures motifs:
(a) β-meander, (b) Greek key and (c) Jelly rolls

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 Protein structure classification

Protein structure classified into three main groups based on

secondary structure motifs (Levitt and Cothia, 1976)
• α-structure,
• β-structure
• α/β structure

These groupings are based on two consideration;

(1) either presence or absence of α-helices and β-sheets
(2) β strand in either parallel or antiparallel.

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CATH hierarchy
structure
classification

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Example on proteins from each class
(a) α structure, (b) β structure and (c) α/β structure.
(Adapted from Branden and Tooze, 1991).

a) Human growth b) Human plasma retinol binding c) FMN binding redox

hormone: protein (RBP) protein flavodoxin. An
α structure with four β structure with antiparallel β example of α/β
helices bundles. sheets structure.

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Example on varieties in α/β structures (a) closed barrel in triose
phosphate isomerase and (b) open barrel ribonucease inhibitor.
(Adapted from Branden and Tooze, 1991).

b) Ribonuclease inhibitor
in open barrel that shape
like a horseshoe

a) TIM barrel of triose

phosphate isomerase as α/β
structure. Look for the parallel β
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sheets that form closed barrel.
Quaternary structure

• The highest level in protein structure hierarchy

• association of two or more polypeptides, each already
fold in their tertiary structure to form multi subunits
protein.
• Even though not all protein forms quaternary structure, it
is a common feature for protein with complex functions
such electron transport or gene expression.
• Some quaternary structures are also held together by
disulphide-bridges between different polypeptides chain,
but many multimeric protein comprise looser association
of subunits stabilized by hydrogen bonding, hydrophobic
effects or electrostatic interactions.
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