Amino Acids and Proteins

1. Structure

   Nota:

   • Favorable interactions: Hydrophobic effect Hydrogen bonds London dispersion forces Electrostatic interactions
   1. Primary Structure

      Nota:

      • All the information required for polypeptides to form structure is encoded in primary structure
      1. Peptide bond

         Nota:

         • Rigid and almost planar, caused by resonance structures, less reactive than ester linkages, each bond has small dipole moment
         • Bonds go C-alpha, C, N starting at N terminus
         1. Usually trans conformation
         2. Shorter than a single bond but longer than a typical double bond

            Nota:

            • About 0.133 nm long
         3. Planar
         4. Phi is N-Calpha bond, Psi is Calpha-C bond

            Nota:

            • Unfavorable when both are 0 degrees or one is 180 and one is 0
            1. Ramachandran plot shows interactions in a backbone
   2. Native Fold

      Nota:

      • Creates biological function, made up of favorable interactions.
      1. Chaperones and Chaperonins

         Nota:

         • Chaperones: proteins that interact with partially folded or improperly folded proteins to help them fold right. Chaperonins: elaborate protein complexes required for some cellular proteins that don't fold spontaneously
   3. Secondary Structure
      1. Alpha helices

         Nota:

         • First proposed by Pauling and Corey, identified by Perutz
         1. Favorable H-bonds between peptide carbonyl and peptide NH group four residues up the chain
         2. Angles allowed (phi -57, psi -47)
         3. 3.6 residues per turn, 5.41 angstrom pitch
         4. R groups are perpendicular to the helix axis and stabilize favorable interactions 3-4 residues apart
         5. Destabilized by stretches of similarly charged residues
         6. Has a large dipole moment since all peptide bonds have the same orientation
         7. Factors determining stability
            1. Interactions of R groups 3-4 helices apart
            2. Occurence of Gly and Pro residues
            3. Interaction between AA residues at ends of helical segment
            4. Electrostatic interatctions between successive AA w/R groups
      2. Beta pleated sheets

         Nota:

         • Also first postulated by Corey and Pauling 
         1. Strands can be parallel or antiparallel
         2. Rise/residue is 3.47 A for antiparallel and 3.25 A for parallel
         3. H bonds between neighboring chains, alpha carbons in folds, side chains protrude up and down
         4. Not flexible
         5. Beta Turn
            1. Allows peptide chain to reverse direction
            2. Stabilized by H bond from carbonyl to amide proton 3 residues down
            3. Often contains Pro or Gly
   4. Tertiary Structure
      1. Fibrous proteins

         Nota:

         • Typically insoluble, made from a single secondary structure
         1. Alpha keratin (alpha helices), silk fibrin (beta pleated sheets), collagen (triple helix)
      2. Globular proteins

         Nota:

         • Water soluble globular proteins, lipid soluble membrane proteins
         1. Hydrophobic residues tend to face inside, polar residues tend to face out
         2. Domains are sections of the protein that can fold independently
      3. Secondary structures form wherever possible
      4. Helices and sheets pack together
   5. Quaternary Structure
      1. Typical Kd for two subunits is 10^-8-10^-16 M
      2. Entropy loss due to association but entropy gain due to burying of hydrophobic groups makes up for it
      3. Provides stability, genomic efficiency, brings catalytic sites together, provides cooperativity

1. Display acid-base properties
2. Able to polymerize into proteins
3. All except Gly are chiral
4. Metabolites, essential to human nutrition, hormones
5. Nonpolar AAs
   1. Glycine, Gly, G
   2. Alanine, Ala, A
   3. Proline, Pro, P
   4. Valine, Val, V
   5. Leucine, Leu, L
   6. Isoleucine, Ile, I
   7. Methionine, Met, M
6. Aromatic AAs
   1. Phenylalanine, Phe, F
   2. Tyrosine, Tyr, Y
   3. Tryptophan, Trp, W
7. Polar AAs
   1. Serine, Ser, S
   2. Threonine, Thr, T
   3. Cysteine, Cys, C
   4. Asparagine, Asn, N
   5. Glutamine, Gln, Q
8. Positive AAs
   1. Lysine, Lys, K
   2. Arginine, Arg, R
   3. Histidine, His, H
9. Negative AAs
   1. Aspartate, Asp, D
   2. Glutamate, Glu, E

Nota:

• General Method: 1. Obtain/grow cells 2. Lyse cells 3. Fractionation based on protein solubility differences 4. Dialysis 5. Chromatography

1. Types of Chromatography
   1. Column chromatography
   2. Ion exchange chromatography

      Nota:

      • Cation exchange elutes negatively charged proteins first, anion exchange elutes positively charged proteins first.
   3. Gel filtration chromatography

      Nota:

      • Separate by size, smaller particles go through smaller channels and take longer to elute.
   4. Affinity chromatography

      Nota:

      • Binds protein to a ligand, protein of interest elutes last.
2. Specific Activity

   Nota:

   • One unit of enzyme activity = the amount of enzyme that can transform one um of substrate to product/min at 25C
   • Total activity or total units is the total number of enzyme units in a sample
   • Specific activity is the number of enzyme units per mg of protein
   • Yield is the total number of units at each step divided by the initial total in the crude extract x100
3. Purification Techniques
   1. SDS-Page

      Nota:

      • PAGE = polyacrylamide gel electrophoresis, SDS = sodium dodecyl sulfate
      • SDS binds and unfolds proteins and gives a uniformly negative charge, native shape doesn't matter so they will elute by size (smaller faster)
   2. Isoelectric Focusing

      Nota:

      • Protein is applied to gel with immobilized pH gradient, protein will move along strip based on pI values (left side is more alkaline, right side is more acidic)
   3. Running SDS-PAGE and isoelectric focusing at the same time can give indication of weight and pI of proteins at same time
4. Primary Sequence Determination
   1. Small Proteins (<~50 residues)

      Nota:

      • Steps: 1. Reduce disulfide bonds and alkylate Cys residues 2. Determine total AA composition via hydrolysis with 6M HCl 3. Determine identity of N-terminal residue by: a. Treating with Sanger's reagent, FDNB, then acid workup (peptide destroyed) or b. Do first step of Edman degradation (liberates one AA at a time, peptide intact) 4. Sequence by Edman degradation
   2. Large Proteins (>~50 residues)

      Nota:

      • Complete steps 1-4 from small protein 5. Digest protein into smaller peptides suitable for Edman degradation via proteases a. Trypsin cleaves on C side of Arg and Lys (+) b. Chymotrypsin cleaves on C side of Phe, Trp, and Tyr (aromatic) c. Cyanogen bromide cleaves on C side of Met 6. Sequence by Edman degradation on each fragment (deduce AA sequence from overlap)
   3. Reagent used for N-terminal determination is in pink, rest of molecule is AA

1. Ligand

   Nota:

   • Molecule that binds reversibly to a protein (can be another protein)
2. Binding site

   Nota:

   • Site on the protein where the ligand binds, complimentary to the ligand in size, shape, charge, and hydrophilic/phobic character
3. Induced fit

   Nota:

   • Binding of ligand to protein results in conformational change that results in increased binding
4. Myoglobin
   1. Oxygen storage protein
      1. Ineffective for transport b/c it has a very high affinity for O2

         Nota:

         • Has a hyperbolic curve
         1. Good transporters must vary affinity with pO2
   2. Contains a heme group so Fe2+ can be oxidized to Fe3+ without creating free radicals
   3. Structural Features

      Nota:

      • -First protein structure determined by x-ray crystallography -Peptide bond in trans config -All alpha domain -3/4 of Pro were at bends -All hydrophobic residues interior -All but 2 polar R groups exterior -Dense, only room for 4 H20  -8 alpha helices (A-H) -Heme group is in hydrophobic pocket between E and F -Fe(II) at center of heme has *4 porphyrin N atoms *His F8 on proximal side *O2 on distal side (E7 binds to this) -Val E11 and Phe CD1 on O2 side of heme helps keep it in place
      1. Porphyrin and Heme
   4. Affinity for CO
      1. Similar size and shape to O2 but has lone e- pair that can be donated
      2. CO binds over 20k times better than O2
5. Hemoglobin
   1. Structure
      1. Tetramic, contains 4 alpha2beta2 subunits
         1. Each subunit has 1 heme and binds 1 O2 (Mr=16000)
         2. Most interactions between alpha and beta subunits (not alpha-alpha or beta-beta)
   2. Very sensitive to changes in [O2], [CO2], [H+], and [BPG]
   3. Uses cooperativity to have affinity changes
      1. Positive cooperativity: first binding event increases affinity at remaining sites
      2. Negative cooperitivity: first binding event decreases affinity at remaining sites
      3. Generally has sigmoidal binding curves
   4. Conformations
      1. Oxyhemoglobin (HbO2): relaxed (R) state, greater affinity for O2
      2. Deoxyhemoglobin (Hb): tense (T) state, lower affinity for O2, stabilized by more ion pairs than R state
      3. Movement from T-->R state triggered by movement of heme iron

         Nota:

         • When O2 binds Fe is pulled into the heme plane pulling the His F8 and F helix with it. This binding changes Fe's spin state from high to low so it can fit in the porphyrin ring
         1. Solvent filled channel narrows as ab dimers slide 15 degrees past each other, switch from T-->R is simultaneous
   5. Allosteric Protein

      Nota:

      • All or none model: two conformations are in equilibrium and the ligand can bind to either conformation. Molecular symmetry is conserved
      • Sequential model: ligand binding to a subunit causes a conformational change that may cause changes in adjacent subunits. The more ligand is bound the more likely more conformational changes will occur
      • T---&gt;R conversion more closely follows concerted model
   6. Bohr Effect
      1. Affinity of Hb for O2 decreases with decreasing pH (increasing [H+])
      2. Hb releases more O2 at more acidic pHs because increased [H+] favors ion pair formation driving R-->T transition, releasing O2
   7. Effect of CO2
      1. CO2 produces HCO3- and H+ in water, H+ then decreases Hb affinity
         1. In tissues this drives R-->T releasing O2
         2. In lungs O2 binds driving T-->R which releases H+ and creates CO2 from the HCO3- present
   8. BPG
      1. Binds to deoxyhemoglobin (T)
      2. Helps stabilize T state by binding in the channel in the middle
   9. Fetal hemoglobin has a higher affinity for O2 than adult Hb
   10. In sickle cell anemia, the Hb interacts and crystallizes