Williams – Proteins1
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chemical bonds (in order of decreasing strength)
o 1. Covalent vs. ionic bonds (covalent are the strongest biochemical bond; i.e. the
most energy is involved in breaking and forming these bonds)
 covalent bonds are required to complete the outer valence shell
o 2. electrostatic interactions
 reversible
 ionic
o 3. H bonds – weak electrostatic
 between H and N, O
o 4. Van der Waals interactions
 distance between atoms
 nonsymmetrical charge distribution around each atom (i.e. the
hydrophobic interaction); usually take place in the center of a protein
 Van der Waals contact distance – point of greatest attraction between
two atoms
Water does not interfere with covalent interactions, though it strongly affects noncovalent interactions (due to its high dielectric constant)
o the water molecule itself has no net charge, but it may interact with charged
particles
o the hydrophobic effect (which releases free energy) is responsible for proper
protein folding
Acid-Base Chemistry
o pKa is the pH at which [conjugate acid] = [conjugate base]
o Henderson-Hasselbalch Equation – to predict pH when [HA] and [A-] is known
 pH = pKa + log([A-]/[HA])
o H+ + HCO3-  H2CO3  H2O + CO2
o Respiratory acidosis
 caused by hypoventilation  buildup of CO2
o Respiratory alkalosis
 caused by hyperventilation  decreased CO2
AAs
o studying tips:
 first, get a good idea of each AA’s name and structure
 then, study them as they are presented in Dr. Williams first lecture (as
organized by their side chains)
 I do remember there being a question concerning the essential amino
acids
Williams – Proteins2
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Peptide bond backbone: amine + high energy acyl derivative = amide
o planar, due to double bond-like characteristics (from its resonance structures)
o uncharged and rigid; the N and O do not rotate around one another
o much H-bonding potential
o stable
Polypeptide chains
o Phi Psi, N O
o Be able to identify individual AA residues if given a polypeptide chain
Disulfide bonds
o S-S = one cystine (from oxidation of two cysteine residues)
1 AA = ~110 daltons (0.11kD)
Secondary structure
o alpha helices
 H bonding between N-H and C=O groups (4 residues apart)
o Beta-pleated sheet
 more relaxed structure
Reverse (Beta, hairpin) turn
o H bonding between C=O of i with N-H of i + 3
Omega loops for chain reversals
Reverse turns
o on surfaces of proteins
o e.g. myoglobin
Prions
o insoluble Beta-pleaded sheets (instead of alpha helices)  cause for protein
aggregates
Three dimensional protein structures – responsible for their functions
o concerns tertiary and quaternary structures, and post-translational mods
 interior of protein = nonpolar, hydrophobic regions
 exterior of protein = polar and nonpolar residues
 tertiary structure – spatial arrangement (think thermo. stability and
various electrostatic bonds), and disulfide bonds
 coiled coil – two amphipathic helices wound (polar residues face exterior)
 protein domains – within a single protein
 quaternary structure – more than one polypeptide
Williams – Proteins3
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Primary vs. secondary vs. tertiary vs. quaternary structures of proteins
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o AA residues – information that contains the correct catalytically active structure
 can use AA sequence to predict 2 and 3 structures
o alpha helices, beta-pleated sheets
o interactions within polypeptide chain (e.g. disulfide bonds)
o interactions between protein subunits (e.g. dimers, trimers, tetramers)
Polarity
o importance: formation of porins within the plasma membrane
Use of BME (strong reducing agent)
o –S-S-  -SH + SH- (cystines to cysteines)
Use of 8M urea
o disruption of non-covalent bonds
o original conformation returns upon removal of agent
Summary of BME and 8M urea
o remove BME  protein still denatured; randomly formed disulfide bridges
 remove urea  protein renatures, but incorrect bridges formed
 add small amount of BME  correct bridges form (active protein)
Protein folding
o not random
o cooperative transition – proteins are either in an active or inactive form (an “all
or none” process)
o cumulative selection – relation to entropy
Post-translational mods
o most common: phosphorylation – various cellular functions
o hydroxylation: collagen hydroxylation process (proline)
o acetylation: for resistance of ubiquitinization
collagen cont’d
o >33% glycine (at every 3rd position)
o ~20% proline, hydroxyproline
o vitamin C (ascorbate) as cofactor for prolyl hydroxylase (addition of hydroxyl
group to proline residue)
marginal stability of proteins
o contributes to their ability to have multiple roles
Protein purification
o AAs, evolution, function, structure
o 1. isolation
o 2. purification based on solubility, size, charge, binding affinity
 dialysis – unwanted material diffuses out of bag
 gel filtration – based on protein size
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affinity chromatography – use of Ab/Ag, DNA binding, receptor binding
 discard FT then collect solutions containing ligand
 generally very successful in terms of specific activity, yield, and
therefore fold purification
 ion exchange – separation based on pH
 high pressure liquid chromatography
 isoelectric focusing gel – use pH gradient to separate based on isoelectric
point (pI) of the protein
 two-dimensional electrophoresis: isoelectric focusing and SDS-PAGE
 followed by MALDI-TOF
o generation and acceleration of protein ions via electric
field (separation based on TOF (time of flight))
o 3. check yield and specific activity
 gel electrophoresis (e.g. SDS PAGE) to determine yield (how dark is the
band?) and specific activity (is this the correct protein of interest?)
 SDS – denatures and provides for uniform charge on proteins
o large (-) charge on SDS
 LDH assay to measure protein specific activity
 measure NADH production
Determine AA sequence
o 1. first determine AA composition
 harsh reagents, ion-exchange chromatography, reaction w/ ninhydrin to
measure absorbance
o 2. Edman degradation to determine AA sequence
 begin at N-terminal
 label: phenyl isothiocyanate (ID with chromatography)