CH339K. Proteins: Primary Structure, Purification, and Sequencing. a -Amino Acid. a. All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules. I prefer this layout, personally…. 2 Amides.
Proteins: Primary Structure, Purification, and Sequencing
Virus infects bacterium
Infected bacxterium produces toxin
Toxin binds receptor on cell
Receptor-toxin complex is endocytosed
Endocytic vessel becomes acidic
Receptor releases toxin
Toxin escapes endocytic vessel into cytoplasm
Bad things happen
DG0’ = +10-15 kJ/mol
Polymerization of activated a.a.:
DGo’ = -15-20 kJ/mol
How to Purify and Sequence a
How do I measure the amount of protein I have?
c = concentration
l = path length
e = extinction coefficient
An Absorbance = 2 means that only 1% of the incident beam is getting through.
Absorbance vs. Concentration
Transmittance vs. Concentration
How can I spot my protein in the great mass of different proteins?
The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so:
i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein
Sodium Dodecyl (Lauryl) Sulfate
SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein
Below the pI, a protein has a positive charge and migrates toward the cathode
Above the pI, a protein has a negative charge and migrates toward the anode
1 unit = amount of enzyme that catalyzes conversion of 1 mmol of substrate to product in 1 minute
Ricin B chain
(the attachment bit)
endocytosis by coated pits and vesicles or,
endocytosis by smooth pits and vesicles. The vesicles fuse with an endosome.
Many ricin molecules are returned to the cell surface by exocytosis, or
the vesicles may fuse to lysosomes where the ricin would be destroyed.
If the ricin-containing vesicles fuse to the Trans Golgi Network, (TGN), there ís still a chance they may
return to the cell surface.
Toxic action will occur when RTA, aided by RTB, penetrates the TGN membrane and is liberated into the cytosol.
The last time I was qualified to know for sure, there were no effective antidotes.
The solubility of haemoglobin in different electrolytes as a function of ionic strength.
Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47
Solubility reaches minimum at pI
Salting out: there’s simply less water available to solubilize the protein.
Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+>Al3+> guanidinium / urea
Anions: SO42−> HPO42−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3−> ClO4−> SCN−
You have a stationary phase
You have a mobile phase
Your material partitions out between the phases.
Agarose is a polymer of agarobiose, which in turn consists of one unit each of galactose and 3,6-anhydro-a-L-galactose.
Ricin sticks to galactose, so store-bought agarose acts as an affinity column right out of the bottle, with ricin binding the beads while other proteins wash through.
Vm = matrix volume
Vo = void volume
Vp = pore volume
Vt = total volume
Ve = elution volume
(1a) Vt = Vo + Vp or
(1b) Vp = Vt - Vo
(2) Ve = Vo + Kav*Vp
Combining 1b with 2
Fig. 3. Measurement of molecular weight of native NAGase enzyme of green crab by gel filtration on Sephadex G-200: standard proteins (empty circles); green crab NAGase (filled circle).
From Zhang, J.P., Chen, Q.X., Wang, Q., and Xie, J.J. (2006) Biochemistry (Moscow)71(Supp. 1) 855-859.
21 residue A chain
31 residue B chain
Connected by disulfides
In order to sequence the protein, the chains have to be separated
From Himeno et al (1996) J. Biol. Chem. 271: 15769-15775.
Time of Divergence
Our green genes are evolutionarily superior!
Phylogenetic trees built from the amino acid sequences of type 1 RIP or A chains (A) and B chains (B) of type 2 RIP (ricin-A, ricin-B, and lectin RCA-A and RCA-B from castor bean; abrin-A, abrina/b-B, and agglutinin APA-A and APA-B from A. precatorius; SNAI-A and SNAI-B, SNAV-A and SNAV-B, SNAI'-A and SNAI'-B, LRPSN1-A and LRPSN1-B, LRPSN2-A and LRPSN2-B, and SNA-IV from S. nigra; sieboldinb-A, sieboldinb-B, SSAI-A, and SSAI-B from S. sieboldiana; momordin and momorcharin from Momordica charantia; MIRJA from Mirabilis jalapa; PMRIPm-A and PMRIPm-B, PMRIPt-A and PMRIPt-B from Polygonatum multiflorum; RIPIriHol.A1, RIPIriHol.A2, and RIPIriHol.A3 from iris hybrid; IRAr-A and IRAr-B, IRAb-A and IRAb-B from iris hybrid; SAPOF from S. officinalis; luffin-A and luffin-B from Luffa cylindrica; and karasurin and trichosanthin from Trichosanthes kirilowii)
Hao Q. et.al. Plant Physiol. 2010:125:866-876
Phylogenetic tree of Opisthokonts, based on nuclear protein sequencesIñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz Lang (2008) Molecular Biology and Evolution, Jan 9