Chapter 4: Part 2

Protein 3-D structure: 3o and 4o structure and protein folding.

3o Structure
• Third level of protein organization • Results from the folding of a polypeptide into a closely packed three-dimensional structure by interacting of 2o structures . • Amino acids that are far apart in the primary structure are brought together to have side chain interactions. • Tertiary structure is stabilized primarily by noncovalent interactions, mostly hydrophobic effects. • Disulfide bridges also contribute to tertiary structure. • formation of motifs and domains.

Motifs (supersecondary structures)
• Combinations of α-helices, ß-strands, and loops. Often have a particular function, such as a protein binding site.

in calcium-binding proteins

2amphepathic hleces -hydrophobic interaction

Several α-H

• Composed of several independently folded compact units in a 3o structure of a protein formed from combination of motifs. • 25 - 300 amino acid residues. • Each domain contains various elements of secondary structure. • Domains are usually connected by loops, but bound to each other through R-group interactions (hydrophobic interaction). • Individual domains have specific function

Protein domain classification

1- All-α

2- All-β

3- α + ß

4- α/ß

Protein family members share common domain structures
lactate dehydrogenase malate dehydrogenase

4 Structure

• Quaternary structure describes the organization of subunits in a protein with multiple subunits (oligomeric protein) • Each subunit is a separate polypeptide chain. • Can have homo-multimers or hetero-multimers • The hydrophobic interaction is the main force involved in the holding of the subunits to each others. (electrostatic forces may contribute also). • Determine molecular weight of native protein by gel permeation chromatography • Determine molecular weight of individual subunits by SDSPAGE • Can use the information to determine subunit composition • If Native protein – 160,000 daltons and α-Subunit – 50,000 daltons ß -Subunit – 30,000daltonsThen Protein can have 2 α and 2ß structure.

4o Structure




Importance of 4 Structure

• Oligomeric protein is more stable than disassociated subunits • Active site often made up of AA residues from different subunits • 4o and 3o structure is often affected by ligand (substrate or inhibitor) binding. Important in the regulation of the biological activity of certain oligomeric protein.

Protein denaturation
• Denaturation – disruption of native conformation • Heat commonly used to denature proteins • Tm = temperature where 50% folded/50% unfolded. • Tm affected by pH and ionic strength • Typical Tm = 40-60oC • Tm for thermophiles >100oC polymerase) (Taq DNA

Heat denaturation of riboneclease


20 mM KCL pH 2.1

Chemical denaturants
•Chaotropic agents weakly dissolved ions that enhance the solubility of non polar compounds such as Urea and guanidinium salts. Disrupting the hydrophobic interaction Detergents (SDS) : the hydrophobic tail penetrate the protein interior

Denature protein

*Renature protein: reformation of the native protein conformation after removing of denature chemical or thermal agents.