Protein structure

Protein structure

1. Primary

   • amino acids are covalently linked together
   • proteins with similar AA seqs and conformations have similar functions (i.e. DNA-binding proteins)
   • point mutation in T-form hemoglobin-->sickle cell anemia

2. Secondary

   • Alpha helix
     • hydrogen bonds, which form between the carbonyl O of one amino acid and the amino H of another.
       • Free rotation around these bonds
     • Helical conformation allows for linear alignment of hydrogen bonds
       • Maximum bond strength
       • Stable conformation
       • Coil of helix is clockwise or right-handed
       • 3.6 residues per turn
       • C═O≡H—N H bonds parallel to helical axis
         • every backbone N−H group donates H bond to the backbone C=O group of AA located three or four residues earlier along the protein sequence
       • R groups point outward
     • Each peptide bond is trans and planar
     • Disruption
       • Bending of polypeptide backbone due to Pro
         • Restricts rotation due to cyclic structure
         • Results in absence of N—H for H-bonding in α-amino group
       • electrostatic repulsion caused by proximity of like charges (i.e. Lys and Arg or Glu and Asp)
       • Steric repulsion caused by proximity of bulky side chains (i.e. Val, Ile, and Thr)
     • Irregularities
       • Found in shorter stretches than standard α-helix
       • Sometimes break up regular nature of α-helix
         • 310 helix: 3 residues per turn and 10 atoms in ring
   • Know the Ramachandran Angles!
     • phi (Φ): bond between the α-carbon and amino nitrogen
     • psi (Ψ): bond between α-carbon and carbonyl carbon
   • Hydrogen-bonded arrangement of polypeptide chain (backbone only)
   • Each amino acid residue has two bonds with free rotation
   • Beta pleated sheets
     • Backbone mostly extended
     • Hydrogen bonds between peptide chains can be interchain or intrachain
     • Polypeptide chains lie adjacent to one another
       • If chains run in the same direction, the sheet will be parallel
         • less stable because individual AA geometry forces H bonds to occur at an angle, making them longer and thus weaker
       • If chains run in opposite direction, sheet will be antiparallel
     • H bonds perpendicular to protein chain (not parallel)
     • R groups alternate above and below the plane
     • Each peptide bond is trans and planar
     • β-bulge
       • Common nonrepetitive irregular secondary motif in antiparallel β-sheets
       • Between 2 normal β-structure H bonds
       • Involves 2 residues on one strand and 1 on the other
     • Reverse turns
       • Contain glycine
       • Formed due to (steric) reasons
       • Proline also encountered in reverse turns because of cyclic structure

3. Supersecondary

   • βαβ unit (hairpin) - Two parallel β-sheets connected by α-helix
   • αα unit (helix turn helix) - two antiparallel α-helices
   • Motifs: Repetitive supersecondary structures
     • Provide information about protein folding
     • Don’t predict biological function of protein

4. Tertiary

   • Fibrous Proteins
     • Backbone of protein does not fold back on itself
     • long, narrow rod
     • Chains organized in parallel along single axis
       • Consist of long fibers or large sheets
       • mechanically strong
     • H2O insoluble
     • Tropocollagen
       • coiled coil interaction
       • triple helix
         • Strands held together by H bonds involving hydroxyproline and hydroxylysine
         • With age, collagen helices become cross-linked by covalent bonds formed by reactions of Lys and His residues
           • Leads to tough meat in animals
       • single collagen molecule
       • X—Pro—Gly or X—Hydroxyproline—Gly
   • Globular
     • nonpolar inside, polar outside (Ion–dipole interactions with H2O environment)
     • compact
     • substantial sections of α-helix and β-sheet
     • soluble in water and salt solutions
     • Noncovalent interactions
       • Backbone H bonding between polar side chains
       • Hydrophobic interactions between nonpolar side chains
       • Electrostatic attraction between side chains
     • covalent interactions
       • disulfide bridges restrict folding patterns
       • Side chain metal ion coordination
   • Experimental determination
     • X-Ray crystallography
       • Perfect crystal: all molecules have same 3-D structure and orientation
       • atoms scatter X-Rays
       • Fourier series analysis
     • 2D NMR (aqueous solution)

5. Quaternary

   • disulfides
   • Allostery: Conformational change in one subunit induces change in another subunit
   • van der Waals
   • ionic

6. Denaturation

   • Sodium dodecyl sulfate (SDS): disrupts hydrophobic interactions
   • β-mercaptoethanol reduces disulfide bridges
   • Guanidine HCl breaks H bonds
   • Urea
     • breaks H bonds
     • Increase accessibility of RNApol disulfides to reducing agent

7. Hemoglobin and Myoglobin

   • Myoglobin
     • Structure
       • single polypeptide chain of 153 amino acid residues + heme prosthetic group
       • 8 α-helices,0 beta strands
     • O2 binding
       • Two His residues interact with heme group and O2
       • Hyperbolic
         • Linear at low pO2
         • Curve flattens at high pO2
         • All molecules bound to O2
       • Function - Oxygen storage
         • Requirement - Bind O2 strongly at very low pressures
         • Saturation - 50% at 1 torr
   • Heme
     • protoporphyrin IX
       • Four 5-membered rings based on the pyrrole structure
       • Linked by methine (—CH═) groups
     • In hydrophobic pocket
     • Fe(II) coordination
       • Four N atoms of four pyrrole-type rings
       • 5th coordination one of N atoms of His F8
       • O2 bound at 6th coordination site
     • Affinity of free heme for carbon monoxide (CO) 25,000 X >O2
       • His E7 forces angled (weakened) binding
         • E6V mutation in T form of Hb-->sickle cell anemia
   • Hemoglobin
     • Structure
       • 2 alpha chains 141 residues long
       • 2beta chains 146 residues long
     • O2 binding
       • up to 4 molecules
       • Function - Oxygen transport
         • Requirement - Bind strongly to O2 and release O2 easily (need 2 conformations
         • p50 ≅ 26 torr (pO2 in tissues)
         • 100% saturated at pO2 = 100 torr (pO2 in lungs)
         • sigmoidal graph
     • fetal version has gamma instead of beta chains, binds BPG less well and O2 better