Proteins

• What proteins do:

  1. Binding (ex. hemoglobin)
  2. Catalysis (ex. DNA polymerase)
  3. Switching on/off (ex. rhodopsin)
  4. Structure (ex. microtubules)
• A symptom of sickle cell anemia is respiratory failure
• Sickle cell anemia is caused by low levels of O2
• Electrophoresis showed that HbS has 2-3 fewer negative charges in comparison to HbA
• Ingram divided the HbA and HbS beta chains into multiple fragments instead of trying to sequence the whole thing
• Sickle cell mutation at position 6 on the beta strand:

  HbA is glutamic acid (Glu,E)

  HbS is now valine (Val,V)
• The hydrophobic βVal6 on the surface of HbS drives polymerization as a consequence of the hydrophobic effect
• βVal6 from HbS tetramer 1 fits into a hydrophobic pocket on HbS tetramer 2, pocket formed by βPhe85 and βLeu88
• By fluke of nature, each end of an HbS alpha/beta dimer interacts with another dimer, leading to long polymers

  
• In HbS, the hydrophobic effect drives βVal6 to interact with βPhe85/βLeu88, leading to polymerization

  
• In HbA, the interactions between βGlu6 and βPhe85/

  βleu88 are NOT favourable for dimerization/polymerization to occur

  
• High O2 concentration induces conformational change in hemoglobin, eliminating the hydrophobic binding pocket, therefore the mutated valine cannot interact with the pocket
• Anfinsen's dogma / the thermodynamic hypothesis:

  The native structure of a protein is determined by the protein's amino acid sequence (i.e. primary structure)
• Almost all naturally occurring amino acids are “ alpha amino acids”, because the “amino” group is connected to the alpha carbon.
• At pH values << 6, there is a high [H+] that favours the protonated state. If we titrate by adding OH-, we raise the pH and lower the [H+] so that we now favour the deprotonated or neutral form

  -> The pKa is the pH at which the side chain is 50% protonated and 50% deprotonated

  
• • The pKa is a measure of the relative ease with which the side chain gives up its proton
  • His with a moderately low pKa (pKa≈6) deprotonates much more easily than Ser (pKa≈13.6). In other words, a higher pH, or a lower [H+] is required for Ser to deprotonate
  • The given pKa values are for side chains in aqueous solution

  
• • The pKa of HisA will be higher than in aqueous solution; The negatively charged Asp stabilizes the protonated form of HisA and shifts its pKa to a higher pH. The HisA holds on to its proton stronger than in aqueous solution
  • The pKa of HisB will be lower than in aqueous solution; The neutral Val stabilizes the neutral form of HisB and shifts its pKa to a lower pH value. In other words, HisB gives up its proton more easily than in aqueous solution

  
• • In Conformation A, His is close to a Val and will prefer to be deprotonated thus shifting the pKa lower (i.e. the His will likely be deprotonated at neutral pH=7)
  • In Conformation B, His is close to an Asp and will prefer to be protonated thus shifting the pKa higher (i.e. the His will likely be protonated at neutral pH=7)

  
• We can intuitively estimate polarity from the structures of side chains. For example, the aliphatic chain of Isoleucine with four carbons (carbon is an atom with weak electro-negativity) is non polar; Serine with one carbon, but a polar hydrogen bonding OH group is

  moderately polar, while the charged Aspartate is very polar

  
• Determining polarity: Isoleucine has a greater affinity for the organic phase, while Serine has moderate affinity for both the organic and the aqueous phase, and Aspartate has a greater affinity for the aqueous phase. By measuring the partition coefficients, we can establish a relative polarity scale

  
• Side chain polarity dictates protein structure, which in turn dictates protein function;

  • Non-polar residues are found in the interior of globular proteins, and at the interfaces between different subunits
  • Hydrophobic residues (red) are clustered in the interior of a protein away from the aqueous environment. Polar hydrophilic residues (green) are typically found on the surface of the protein

  A) what

  
• Side chain polarity can also be used as a predictive tool

  
• womp womp

  A) H20

  B) peptide bond

  
• Properties of the peptide bond:

  • resonance leads to partial double bond character and restricted rotation
  • C=O and N-H groups are all coplanar
  • peptide bonds have a dipole moment;
  • peptide C=O is an H-bond acceptor (red arrow)
  • peptide N-H is an H-bond donor (blue arrow)

  
• Rotation around N-Cα and Cα-C is not really “free” due to steric blocking, as bulky R-groups of AA side chains effect favourability of the orientation
• Free rotation around N-Cα = phi (Φ)

  Free rotation around Cα-C = psi (Ψ)

  A) psi

  B) phi

  
• phi and psi are always between +180° and -180°

  • clockwise = + ; counter-clockwise = -
• Most regions of the Ramachandran plot are inaccessible to proteins because of steric clash
• To determine structure we can't only use phi and psi, we also need the omega bond angle
• The vast majority of peptide bonds are in the trans configuration

  • Refers to the orientation of the alpha carbons adjacent to the peptide bond
  • The peptide bond angle (“ω”)(omega) is 180 ° in this configuration
• The energy difference between the cis and trans configuration is not as dramatic in proline

  
• Summary of the interactions that stabilize folded proteins

  A) Covalent

  B) Disulfide

  C) Salt

  D) hydrogen

  E) electrostatic

  F) Van der Waals

  G) 1.5

  H) 2.2

  I) 2.8

  J) 3

  K) 3.5

  
• The inside the cells is a reducing environment, thus in the

  cytoplasm most proteins are reduced so disulfide bonds are rare

  
• Hydrogen bonds:

  • 2 - 6 kJ/mole (if donor/acceptor is charged, and can be stronger in low dielectric field)
  • Shorter distance between donor and acceptor = STRONGER, however optimal at 3.0 Å.
  • Longer distance (> 3.0 Å) between donor and acceptor = WEAKER
  • If donor-H-acceptor well aligned = STRONGER

  
• Electrostatic interactions

  -> more negative = more favourable

  A) hydrogen

  B) Salt

  
• Van der Waals interactions

  A) electronic

  B) london

  
• Lipid bilayer = many van der Waals interactions add up!

  Hydrophobic core of proteins = many van der Waals interactions add up!

  A) head

  B) acyl

  
• Secondary Structure:

  Segments of the main chain in nearly all proteins adopt conformations in which the phi (Φ) and psi (Ψ) torsion angles of the backbone repeat in a regular pattern
• Types of Helices:

  A) Alpha

  B) 3 10

  C) Pi

  D) Polyproline I

  E) Polyproline II

  
• α helix (most prevalent helical form in proteins!)

  • 1.5 Å per residue “rise” along helix axis
  • 3.6 residues per helix “turn” (360˚)
  • peptide dipoles align, summing to a substantial “helix dipole”
  • almost exclusively right -handed (because all are L amino acids)
  • peptide N-H and C=O are similarly aligned along the helix axis leading to a repeating hydrogen bond pattern
  • side chains all project away from the helix