PROTEINS BIT 230 Biochemistry Purification Characterization.

PROTEINS

BIT 230BiochemistryPurification

Characterization

Review (familiar material)

Review of Central DogmaReview of TranslationClasses of proteinsAmino Acid CharacteristicsProtein StructuresSequencing

Fig 13.3 The four levels of organization in proteins.© 2003 John Wiley and Sons Publishers

Fig 13.1 Structures of the 20 amino acids commonly found in proteins.© 2003 John Wiley and Sons Publishers

20 amino acids (residues)R groups

1. Hydrophobic, nonpolar aliphatic (side chain only has C H)met is non reactive-often a part of this grouppro R group covalently bond to amino grouptend to interact non-covalentlyfound in membranes or inside the 3D structure

2. Hydrophilic, polar, non-charged, aromaticcysteine -sulfur group MOST reactive

disulfide bonds (intra and inter)Aspargine and Glutamine can deaminate to Aspartic acid and glutamic

acid

3. Negatively charged

4. Positively charged

Phe, Tyr Trp are aromatic - absorb UV light (280 nm)

•Bulky R groups

p. 304 – All the amino acids except proline contain a free amino group and a free carboxyl group.© 2003 John Wiley and Sons Publishers

Fig 13.2 The formation of a peptide bond between two amino acids by the removal of water.© 2003 John Wiley and Sons Publishers

Fig 13.4 Secondary structure in proteins.© 2003 John Wiley and Sons Publishers

Fig 13.5 The five types of molecular interactions that determine the tertiary structure.© 2003 John Wiley and Sons Publishers

Features•Denaturation•Structure and Function

•Classes •Fibrous

•simple, elongated, structural, protective

•Globular complex, spherical

Transmembrane proteins

Signal Sequence (N terminal region)

Simple vs Conjugated

•Conjugated•prosthetic groups (non-peptide)

•Glycoproteins•phosphoproteins•flavoproteins•metalloproteins

Sequencing Amino Acids

•Edman Degradation•http://www.cartage.org.lb/en/themes/Reference/dictionary/Biologie/E/11.html

•Mass Spectrometry•http://chipo.chem.uic.edu/web1/ocol/spec/MS1.htm

•Swiss Prot Database

Secondary Structures•Alpha Helix

•Ala, Leu, Met, Glutamate•why would proline and trp not be found here?

•Beta Sheets•zigzag

•Loops•outside of protein (hydrophilic)•active sites of enzymes•antigen binding region of Ab•susceptible to proteolytic cleavage• B turn Glycine at bend (small side) and proline (induces kink)

Tertiary Structures

•Domains•structural subunits within a single polypeptide

•intracellular region•TMR•extracellular domain

Post Translational Modifications

•FUNCTION

•Proteolytic Processing•Pro form (zymogen, inactive)•cleave some amino acids-•ACTIVE clotting factors, digestive enzymes

•Glycosylation•p 30•O linked serine threonine• N linked asparagine

PTM Continued

•Phosphorylation•activate or inactivate•donor often ATP•kinase adds PO3•phosphorylase removes PO3

•Acetylation add acetyl group

•Acylation add fatty acid

Inhibition of protein synthesis•Antibiotics

•Cycloheximide -inhibits tranlocation•Erythromycin- binds ribosome inhibits tranlocation•Streptomycin - inhibits initiation of translation•Tetracycline - blocks aminoacyl tRNA from binding to ribosome

•Antimicrobials•Triclosan disrupts membrane of bacteria

•non-specific? No tolerance developed?•Inhibits a reductase (synthesis of fats)