Proteins -II

Protein Folding
  • Information for correct folding is in the primary sequence of proteins.
  • Driven by thermodynamics; proteins fold to maximize weak non-covalent interactions, reducing free energy.
  • Protein folding is cooperative.
  • Some proteins require chaperones (e.g., Heat-shock proteins, Peptidyl-prolyl cis-trans isomerases, Protein-disulfide isomerases) to assist folding, prevent aggregation, and stabilize unfolded chains.
Denaturation of Proteins
  • Loss of protein structure and function.
  • Caused by: pH changes, temperature (T), chaotropic agents (urea, guanidine hydrochloride), detergents (SDS).
Post-Translational Modification (PTM) of Proteins
  • Introducing chemical groups onto polypeptide chain after translation.
  • Glycosylation:
    • O-linked glycoproteins: Golgi, ether linkage to -OH of Ser and Thr.
    • N-linked glycoproteins: ER, amide linkage to N of Asn within consensus sequence (Asn-X-Ser; Asn-X-Thr).
  • Lipid modification (Lipoprotein):
    • Acetylation: a- and ϵ\epsilon-NH2_{2} groups (e.g., histones).
    • Myristoylation: N-terminal Gly (C14_{14}), amide linkage, involved in protein-protein/protein-lipid interactions, signal transduction.
    • Palmitoylation: Cys residues near C-terminus (C16_{16}), thioester linkage, protein-protein interactions, membrane association.
    • Polyisoprenylation: Cys residues near C-terminus (farnesyl PP [C:1515], geranylgeranyl PP [C:2020]), protein-protein interactions, signal transduction.
    • GPI (glycosylphosphatidylinositol) anchor: At C-terminus of some membrane proteins.
  • Phosphorylation:
    • Dynamic, regulatory modification by protein kinases and phosphatases.
    • Protein kinases: Transfer of γ\gamma-phosphate from ATP onto -OH group of Ser, Thr, or Tyr.
    • Types: Ser/Thr kinases, Tyr protein kinases.
  • Other modifications: Hydroxylation (e.g., HO-proline in collagen), Sulfation (Tyr), Ubiquitination (e.g., histones), Sumoylation (e.g., histones), Carboxylation.
Pathophysiology of Misfolding Proteins
  • Misfolding leads to denaturation (loss of function) or partial function loss, causing diseases.
  • Alzheimer's Disease (AD): Neurological degenerative disease marked by memory loss, confusion, and behavioral changes.
    • Results from dense plaques of fibrillar β\beta-amyloid proteins with well-ordered β\beta-sheet secondary structure.
    • A protein misfolding disease, possibly initiated by oxidative stress.
  • Cystic Fibrosis (CF): Chronic disease affecting lungs and pancreas due to thick, sticky mucus.
    • Caused by misfolding of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein.
    • Approximately 70%70\% of cases involve a deletion of phenylalanine at position 508508 (Phe508508) in CFTR.
Protein Purification
  • Separation of a protein of interest from a complex mixture.
  • Procedure: Cell breakage/homogenization, protein extraction, purification (precipitation, chromatography).
  • Requires an assay method for protein abundance (activity or antibody).
  • Principles based on differences in:
    • Molecular weight (gel filtration / size-exclusion chromatography).
    • Charge properties (ion-exchange chromatography).
    • Hydrophobicity (hydrophobic interaction chromatography).
    • Affinity for substrates/ligands (affinity interaction chromatography).
  • Gel filtration: Separates proteins by size; larger molecules elute first.
  • Ion-exchange chromatography: Separates by net charge.
    • Anion exchanger (positively charged stationary phase) binds negatively charged analytes.
    • Cation exchanger (negatively charged stationary phase) binds positively charged analytes.
    • Elution occurs via pH or salt gradient.
Protein Analysis
  • Electrophoresis: Follows protein purification progress.
    • SDS-PAGE: Migration is determined only by polypeptide size; SDS (anionic detergent) binds to proteins (1 SDS per 2 amino acid residues), dissociates them, and overwhelms native charge.
    • Native PAGE: Migration is determined by both size and charge.
  • 2D Gel Electrophoresis: Identifies proteins by two steps:
    • 1. Isoelectric focusing (IEF): Separates proteins based on their isoelectric point (pI, charge).
    • 2. SDS-PAGE: Separates proteins based on size.
  • Proteins are often identified by mass spectrometry (MS) or Western blot after 2D gel.
  • Western blot: SDS-PAGE followed by transfer of proteins onto a membrane and visualization with a specific antibody (Y).