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SBT 2233 PROTEIN BIOTECHNOLOGYby 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) GarlandGregory 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 • aliphatic/aromatic • polarity • charge • hydrophobicity; • disulphide bonds • molecular models

C COOHH3N

R

H

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Peptide bond

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Peptide bond

C COOHH3N

R

H

General structure of an amino acid. Amino acids are joined by forming peptide bonds.

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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• 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.

Dihedral Angles

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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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• 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.

Secondary structure

α-helices

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

Parallel

AntiparallelParallel

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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.

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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 Helix-loop-helix The Four-helix Bundle DNA-binding domains Globins

• All-Beta Up-and-down Anti-parallel Beta

Sheets Greek Key Jellyroll Beta Propellors Beta Trefoils Beta Helix

• Alpha/Beta Rossman Fold alpha/beta horseshoe alpha/beta barrels

• Alpha+Beta

• Small disulphide rich proteins

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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 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.

Protein structure classification

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CATH hierarchy structure classification

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b) Human plasma retinol binding protein (RBP) β structure with antiparallel β sheets

a) Human growth hormone: α structure with four helices bundles.

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

c) FMN binding redox protein flavodoxin. An example of α/β structure.

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a) TIM barrel of triose phosphate isomerase as α/β structure. Look for the parallel β sheets that form closed barrel.

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

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).

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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.