Amino Acids and Proteins

Amino Acids and Proteins

1. Proteins

   • Structure
     • Favorable interactions: Hydrophobic effect Hydrogen bonds London dispersion forces Electrostatic interactions
     • Primary Structure
       • All the information required for polypeptides to form structure is encoded in primary structure
       • Peptide bond
         • 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
         • Usually trans conformation
         • Shorter than a single bond but longer than a typical double bond
           • About 0.133 nm long
         • Planar
         • Phi is N-Calpha bond, Psi is Calpha-C bond
           • Unfavorable when both are 0 degrees or one is 180 and one is 0
           • Ramachandran plot shows interactions in a backbone
     • Native Fold
       • Creates biological function, made up of favorable interactions.
       • Chaperones and Chaperonins
         • 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
     • Secondary Structure
       • Alpha helices
         • First proposed by Pauling and Corey, identified by Perutz
         • Favorable H-bonds between peptide carbonyl and peptide NH group four residues up the chain
         • Angles allowed (phi -57, psi -47)
         • 3.6 residues per turn, 5.41 angstrom pitch
         • R groups are perpendicular to the helix axis and stabilize favorable interactions 3-4 residues apart
         • Destabilized by stretches of similarly charged residues
         • Has a large dipole moment since all peptide bonds have the same orientation
         • Factors determining stability
           • Interactions of R groups 3-4 helices apart
           • Occurence of Gly and Pro residues
           • Interaction between AA residues at ends of helical segment
           • Electrostatic interatctions between successive AA w/R groups
       • Beta pleated sheets
         • Also first postulated by Corey and Pauling 
         • Strands can be parallel or antiparallel
         • Rise/residue is 3.47 A for antiparallel and 3.25 A for parallel
         • H bonds between neighboring chains, alpha carbons in folds, side chains protrude up and down
         • Not flexible
         • Beta Turn
           • Allows peptide chain to reverse direction
           • Stabilized by H bond from carbonyl to amide proton 3 residues down
           • Often contains Pro or Gly
     • Tertiary Structure
       • Fibrous proteins
         • Typically insoluble, made from a single secondary structure
         • Alpha keratin (alpha helices), silk fibrin (beta pleated sheets), collagen (triple helix)
       • Globular proteins
         • Water soluble globular proteins, lipid soluble membrane proteins
         • Hydrophobic residues tend to face inside, polar residues tend to face out
         • Domains are sections of the protein that can fold independently
       • Secondary structures form wherever possible
       • Helices and sheets pack together
     • Quaternary Structure
       • Typical Kd for two subunits is 10^-8-10^-16 M
       • Entropy loss due to association but entropy gain due to burying of hydrophobic groups makes up for it
       • Provides stability, genomic efficiency, brings catalytic sites together, provides cooperativity

2. Amino Acids

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

3. Protein Purification & Primary Sequence

   • General Method: 1. Obtain/grow cells 2. Lyse cells 3. Fractionation based on protein solubility differences 4. Dialysis 5. Chromatography
   • Types of Chromatography
     • Column chromatography
     • Ion exchange chromatography
       • Cation exchange elutes negatively charged proteins first, anion exchange elutes positively charged proteins first.
     • Gel filtration chromatography
       • Separate by size, smaller particles go through smaller channels and take longer to elute.
     • Affinity chromatography
       • Binds protein to a ligand, protein of interest elutes last.
   • Specific Activity
     • 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
   • Purification Techniques
     • SDS-Page
       • 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)
     • Isoelectric Focusing
       • 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)
     • Running SDS-PAGE and isoelectric focusing at the same time can give indication of weight and pI of proteins at same time
   • Primary Sequence Determination
     • Small Proteins (<~50 residues)
       • 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
     • Large Proteins (>~50 residues)
       • 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)
     • Reagent used for N-terminal determination is in pink, rest of molecule is AA

4. Protein Function

   • Ligand
     • Molecule that binds reversibly to a protein (can be another protein)
   • Binding site
     • Site on the protein where the ligand binds, complimentary to the ligand in size, shape, charge, and hydrophilic/phobic character
   • Induced fit
     • Binding of ligand to protein results in conformational change that results in increased binding
   • Myoglobin
     • Oxygen storage protein
       • Ineffective for transport b/c it has a very high affinity for O2
         • Has a hyperbolic curve
         • Good transporters must vary affinity with pO2
     • Contains a heme group so Fe2+ can be oxidized to Fe3+ without creating free radicals
     • Structural Features
       • -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&nbsp; -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
       • Porphyrin and Heme
     • Affinity for CO
       • Similar size and shape to O2 but has lone e- pair that can be donated
       • CO binds over 20k times better than O2
   • Hemoglobin
     • Structure
       • Tetramic, contains 4 alpha2beta2 subunits
         • Each subunit has 1 heme and binds 1 O2 (Mr=16000)
         • Most interactions between alpha and beta subunits (not alpha-alpha or beta-beta)
     • Very sensitive to changes in [O2], [CO2], [H+], and [BPG]
     • Uses cooperativity to have affinity changes
       • Positive cooperativity: first binding event increases affinity at remaining sites
       • Negative cooperitivity: first binding event decreases affinity at remaining sites
       • Generally has sigmoidal binding curves
     • Conformations
       • Oxyhemoglobin (HbO2): relaxed (R) state, greater affinity for O2
       • Deoxyhemoglobin (Hb): tense (T) state, lower affinity for O2, stabilized by more ion pairs than R state
       • Movement from T-->R state triggered by movement of heme iron
         • 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
         • Solvent filled channel narrows as ab dimers slide 15 degrees past each other, switch from T-->R is simultaneous
     • Allosteric Protein
       • 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
     • Bohr Effect
       • Affinity of Hb for O2 decreases with decreasing pH (increasing [H+])
       • Hb releases more O2 at more acidic pHs because increased [H+] favors ion pair formation driving R-->T transition, releasing O2
     • Effect of CO2
       • CO2 produces HCO3- and H+ in water, H+ then decreases Hb affinity
         • In tissues this drives R-->T releasing O2
         • In lungs O2 binds driving T-->R which releases H+ and creates CO2 from the HCO3- present
     • BPG
       • Binds to deoxyhemoglobin (T)
       • Helps stabilize T state by binding in the channel in the middle
     • Fetal hemoglobin has a higher affinity for O2 than adult Hb
     • In sickle cell anemia, the Hb interacts and crystallizes

Images and files in the map