VL 2 - Protein Structure

• Primary - Amino Acid sequence: covalent bonds

• Secondary - local 3D patterns: backbone H-bonds, avoiding steric clashes

• Tertiary - overall 3D fold of protein chain: Disulfide bridges (S-S), H-bonds, Aromatic Interactions, Ionic Interactions, Van Der Waals Interactions

• Quaternary - complex of proteins (two or more chains)

• peptide bond: 

  • partly double bond character → strong bond & highly stable

  • is hydrolyzed in acidic or basic conditions

  • planar, no free rotation → cis/trans (trans preferred, cis sometimes with proline)

• properties: rigid (peptide bond), variability (sidechains), polar atoms at defined distances

• Alpha helix: 5-40 AAs (avg: 14), 3.6 AAs per turn, 31% of residues are in alpha helix

• Beta strand: 

  • different polypeptide chains run alongside each other, linked by hydrogen bonds, 5-10 AAs per strand, alternating pattern of hydrophobic and polar amino acids, parallel or antiparallel

  • “Twist”: most beta sheets found in proteins are “twisted” (more in parallel) 

• Loops: unstructured areas connecting SS domains, various lengths and shape → structured and unstructured regions

  • Drug targets: disordered proteins → great uncertainty

• Turn: connects secondary structure elements, non-regular SS that causes reversal of direction, 2-6 AAs (very short loop)

Glycine: smallest AA, only one hydrogen atom as sidechain

Proline: distinctive sidechain with cyclic structure, high rigidity, bends the protein

Disulfide bridges

• usually from the coupling of 2 thiol (R-S-H group)

• Cysteine

Ion interactions (salt bridges)

• electrostatic interactions

Hydrogen Bonds (between side chains)

Aromatic interactions (pi-stacking or pi-pi-stacking)

• non-covalent interactions between aromatic rings

• aromatic molecules are cyclic and planar with resonance between ring bonds

Hydrophobic effect: observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules

Globular Protein: hydrophobic AAs bounded inward & hydrophilic AAs outward → hydrophobic core → protein stays soluble in water

Protein folding is the physical process by which a protein chain acquires its native three-dimensional structure, a conformation that is usually biologically functional, in an expeditious and reproducible manner. 

Anfinsen´s Dogma: “Protein structure is determined only by amino acid sequence” (thermodynamic hypothesis)

For small globular proteins: at environmental conditions at which folding occurs, the native structure is a unique, stable, and kinetically accessible minimum of the free energy.

Experiment:

• reduce S-S bonds from Ribonuclease and denature with Urea

• remove Urea -> SH oxidizes back to S-S

Result: sequence determines structure - proteins indeed can adopt their native conformation spontaneously

a) Levinthal´s Paradox

Difference between theoretical calculation and observed data: “Levinthal's Paradox”

Theoretical calculation: Consider a small protein of 100 AAs. If each AA can assume 2 conformations, then the protein can adopt 2100 ~ 1030 conformations. Assuming the conformation shift in a constant time of 10-10 s.

→ time to sample all conformations: 1012 years (age of the universe = 1.2*1010)

But: proteins fold spontaneously (large ones in minutes)

b) Main result:

⇒ Proteins do not randomly sample conformations, there must be pathways!

a)  Similar sequences suggest similar structure.

b) Workflow

1. Template selection: sequence identity  > 30 %, pairwise or multiple sequence alignment

2. Target-template sequence alignment: structure-based seq. alignment (gaps into unstructured regions), find regions of similarity (may be the consequence of functional, structural, or evolutionary relationships)

• pairwise alignment: global (Needleman-Wunsch) vs. local (Smith-Waterman), BLAST, dynamic Programming, Heuristic Methods

• MSA: progressive alignment (ClustalW2, Cobalt), iterative methods (ClustalO, MAFFT, MUSCLE), consensus method (MergeAlign)

1. Model construction: 

   a) backbone

   b) loops (variable): < 10 AAs and refine

   c) sidechains

2. Assessment: check model quality