Chapter !10 - Proteomics Code : KSI0019

<Index of Chapter 10>

 

Introduction

- The proteome is the complete set of proteins associated with a sample of living matter. Proteomics deals with the proteins that form the structures of living things, are active in living things, or are produced by living things. or are produced by living things. This includeds their nature, distrivution , activities , interactions , and evloution. Many fields contribute to proteomics.

 

Protein nature and types

Proteins are where the action is

1.  Proteins have a great variety of functions .

2. The amino acid sequences of proteins dictate their 3D structures and their folding pathways. (Folding pathway) 

3. Advances in protein science have spawned the biotechnology industry. 

 

Protein structure

 Backbone or mainchain / sidechain /

> Hydrogen bonding  / Hydrophobic interactions/ Disulphide bridges  ~ 

 

Different types of residues make different types of interactions , including hydrogen bonds, hydrophobic interactions, and disulphide bridges. Formation of the native structure allows optimal formation of favorable inter-residue and residue- solvent interactions 

 

> A protein is a message writeen in a 20 letter alphabets. 

Helices and sheets

> Helices and sheets are recurrent structures, stablized by mainchain hydrogen bonding, that appear in many protein structures. 

-The chemical structure of proteins 

-Conformation of the polypeptide chain

>Diheadral angles. 

-Protein folding patterns

> Folding patterns. 

1. Primary structure 

2. Sencondary structure.

3. Tertiary struture

4. quanteanry structure .

We describe protein folding patterns according to a hierachy of primarym secondary, tertiary and quaternaty sturctures

Domains  / Modular proteins / Polypeptide chain / mainchain / sidechains / primary structure / hydrogen bod / secondary structure / alpha helix / beta sheet / folding pattern / tertiary structure / quantenary structure / native state / denaturant / denatured state / post-translational modification / disulphide bridge  

 

Post-translational modifications

-Why is there a common genetic code with 20 canorical amino acids?

Level of transcription 

Formation of different splice variants 

mRNA editing 

the nature and binding sites of ligands integral to the final sturcture 

post translational modifications 

 

Seperation and anylsis of proteins 

All methods of separating molecules require two things

1. A difference in some physical property, between the molecules to be separated

2. A mechanism taking advantage of that property, to set the molecules in motion, the speed differing according to the value of the property selected. This moves apart molecules with different properties. 

 

To measure an inventory of the proteins in a sample, the proteins must be 1. separated 2. identified. 3. counted 

-Polyacrylamide gel electrophoresis (PAGE)

> SDS (Sodium Dodecyl Sulphate) - PAGE

-Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

> Isoelectric point / isoelectric focusing 

-Mass spectrometry

> Rapid identification of the components of a complex mixture of proteins 

> sequencing of proteins and nucleic acids

> Anaylsis of post-translational modifications or substitutions relative to an expected sequence

> Measuring extents of hydrogen deuterium exchange to reavel the solvent exposure of individual sites 

> Mass spectrometry is oftern used to characerize  proteins isolated from mixtures. The peptide mass fingerprint is usually sufficiennt to identify a protein 

 

Classification of protein structures

-SCOP

> Strucutural Classification of Proteins) offers facilities for searching on ketwords to identify structures, navigation up and down the hierarchy, generation of pictures, access to the annotaion records in the PDB entries and links to related databases. 

 

-Changes in folding patterns in protein evolution

 

Many proteins change conformation as part of the mechanism of their function

Many proteins are microscopic machines, with internal parts moving in precise ways to support their function 

By their nature, transition states are reactive and difficult to trap long enough for structure determination. Possible solutions include enzymes binding transition -state analogues or inhibitors, or lowering the temperature to slow down the reaction 

 

-Conformational change during enzymatic catalysis

-Motor proteins

Myosins /Kinesins / Dyneins /Linear motors / rotary motor.  / sarcomere

-Allosteric regulation of protein function

> Allosteric proteins deviate from the michaelis -menten curve in ligand binding or in the cases of allosteric enzymes, in reaction velocity as a function of substrate concentration. The cooperativitiy is achieved by ligation - induced conformational change. 

 

-Conformational states of serine protease inhibitors (serpins)

The differnce in colour between arterial and venous blood reveals the different state iron in ligated and unligated hemoglobin.

 

Protein structure prediction and modelling

-Homology modelling

> is one of the most useful techniques for protein structure prediction - when it is applicable 

Attempts to predict secondary structure 

Fold recognition

Prediction of novel folds 

 

-Available protocols for protein structure prediction

-Structural genomics

Critical Assessment of Structure Prediction (CASP) 

CASP categories change as the field progresses. Secondary structure prediction and fold recognition have been discontinued. Prediction of residue- residue contacts of disordered regions and the ability to refine models have been added. 

 

 

Directed evolution and protein design

-Directed evolution of subtilisin E

Artificial selection and evolution / natural selection

-Enzyme design

 

<The procedure of directed evolution comprises these steps>

1. Create variant genes by mutagenesis or genetic recombination

2. Create a library of variants by transfecting the genes into individual bacterial cells.

3. Grow colonies from the cells and screen for desiable properties.

4. Isolate the genes from the selected colonies and use the as input to step 1 of the next cycle.