tivamifu's version from 2016-02-17 12:57

Fragmenting reagents


Question Answer
Cyanogen Bromidecleaves on the C-side of Met
Submaxillarus proteasecleaves on the C-side of Arg
Endoproteinasecleaves on the C-side of Lys
Trypsincleaves on the C-side of Arg or Lys
Chymotrypsincleaves on the C-side of Aromatics (Phe, Trp, or Tyr)
Staphylococcal proteasecleaves on the C-side of Asp or Glu
Asp-N-proteasecleaves on the N-side of Asp or Glu
Pepsincleaves on the N-side of Aromatics (Phe, Trp, Tyr)
Edman reagent (phenyl-N=C=S)combines with the N-terminal of an amino acid
Fluoro-2,4-dinitrobenzene (FDNB)combines with the N-terminal of an amino acid. The N-terminal amino acid will combine with a phenyl group

Protein Purification Techniques


Question Answer
SDS-PAGE-sodium dodecylsulfate Polyacrylamide Gel Electrophoresis
-SDS denatures proteins which allows the protein to unfold
-SDS has a negative charge which overwhelms any intrinsic charge the protein may have (all molecules will ultimately have same charge)
-electrophoreses is then used on the gel strip to separate molecules based on molecular weight
-molecules with higher charge/mass ratios (smaller MW) will travel father in the gel
isoelectric focusing-A solution of ampholytes (compound that can be a base or acid) is electrophoresed through a gel in a small tube which creates a pH gradient
-A protein mixture is applied to gel and electrophoresis is continued
-A molecule will move in the gel until it reaches its pI, where the positive and negative electric forces balance each other out
ammonium sulfate precipitation-The solubility of proteins varies according to the salt concentration
-the solubility of the protein increases with increasing salt concentration until it reaches a saturation point where protein solubility decreases
-at high salt concentrations the protein will precipitate out
-salt ions attract water molecules away from the protein which causes proteins to interact with themselves and aggregate
ion-exchange-Solutes in a liquid phase are poured over a porous column.
-The column contains charged resin particles that attract charged solutes in the solution that is poured over the column.
-To separate the amino acids a salt solution is poured over the column which washes out the amino acids.
-A dilute salt solution is used first to wash out weakly bonded molecules and the salt solution is increased to wash out stronger bonded molecules
size exclusion (gel filtration chromatography)-Fine, porous beads are packed into a chromatography column.
-A solution of molecules is pored over the column where the smaller molecules enter the porous beads and the larger molecules elute out.
-As more solvent is added to the column eventually the smaller molecules will elute out
hydrophobic interaction-Proteins bind to a hydrophobic ligand on the surface of a support resin under high salt concentration conditions
-The salt promotes the hydrophobic interaction between the protein and the solid support
-to desorb the protein, the salt concentration is lowered via a decreasing salt gradient which diminishes the hydrophobic interaction
specific interaction-Proteins bind to a specific ligand on the surface of a support resin.
-The interaction is very specific and only proteins that can bind the ligand will bind while the rest washes down the column.
-To desorb the protein, buffer is added that contains the same ligand free in solution
absorbance spectroscopy-Proteins have a characteristic absorption between 275-280 nm due to the presence of tyrosine and tryptophan residues
-calculating the extinction coefficient allows one to estimate the amount of protein present

Protein Characterization and structure


Question Answer
What is the sequencing procedure for a protein?1) Obtain amino acid composition
2) Determine which amino acid is present at the N-terminus
3) Break disulfide bonds if present
4) First fragmenting agent and sequencing
5) 2nd fragmenting agent and sequencing
6) Fit all fragmented pieces together

if protein still can't be sequenced
7) Use another fragmenting agent and fit pieces together
What is the basis for the ribbon model?-peptide bonds
-Each peptide bond has some double-bond character and cannot rotate
alpha helix-carbonyl of residue i hydrogen bonds to the backbone imino group of residue i + 4
-R-groups positioned on the outside
-often contains amino groups MALEK
-Tyr, Ser, Gly and Pro do not tend to be in the α-helix
-bends usually contain proline
beta sheet-The backbones are laterally connected by the hydrogen bonds between carbonyl group from one chain to the peptide-bonded imino group from the adjacent chain
-R-groups stick out above and below sheet
-can be parallel (N terminals are on same side of adjacent chains) or anti-parallel ((N terminals are on opposite side of adjacent chains)
-turns are 180 degrees and usually contain pro or gly
random coilThere is no repetitive structure that can be recognized.
info gained from NMR vs crystal structure-both methods will give info about the structure of a molecule
-crystal structure only works on molecules able to crystalize
-NMR works for smaller molecules and molecules that can't crystallize


Question Answer
What are the main groups of amino acids?1. common or coded AA modified AA
3. AA with different side groups than common AA
4. D-amino acids
Describe Common or coded amino acidsThese are the 22 different amino acids found in proteins and peptides. They are coded for by the 3-nucleotide codons on DNA/RNA and have their own tRNA that is used during peptide synthesis on ribosomes.
Describe Amino acids that are due to post-translational modificationmodifications of the 22 coded amino acids are methylation, phosphorylation, carboxylation and hydroxylation.
Describe AA with different side groups than the coded AAThese can be intermediates in metabolic pathways or are found in small peptides made by bacteria.
Describe D-amino acids Also found in small peptides made by bacteria that can have antibiotic properties.


Question Answer
Describe membrane proteins-α–helices and beta-barrels located in the Transmembrane
-Tyr and Trp located at water-lipid interface
Describe globular proteins-outside of protein is polar
-inside of protein is hydrophobic
Describe fibrous proteins-usually simple, linear structure that serves structural role in cells
-it is a single type of secondary structure that is repeated many times
What are the 3 types of membrane proteins?1. Peripheral proteins - can move around and attach to integral proteins and polar head of lipids
2. Integral proteins - embedded inside a membrane (lipid, bilayer)
3. Covalently attached membranes - covalently attach themselves to membrane lipids


Question Answer
Hydropathy plot-plot of hydropathy index vs residue number
-(+) values are hydrophobic and (-) values are hydrophilic.
-membrane proteins have at least 20 residues
Describe the ribbon modelatoms that make up the peptide bond are found in the same plane, there is a small flat area that contains these 6 atoms. The “flow” of the peptide bonds through the protein structure can be followed by connecting all these flat areas by a ribbon
Describe the worms modelThe amino acid backbone is indicated by a tube like structure. Sheets are indicated by flat ribbon arrow. The helices are indicated by solid tubes or cylinders. This is to emphasize that these structures are not hollow.
Describe surface contoursshow what surface is available to other molecules and proteins to interact with. The Ribbon or Worms models might give the impression that these structures have a lot of empty space which is not the case.
What is a purification table?-lists purification steps and the specific activity after each step
-tells how many steps are needed to purify protein and if a particular step is useful or not
How to calculate specific activity and what is it?Specific Activity = Total Activity/Total Protein

You want the specific activity to increase each step in a purification table. If the SA stays the same from one step to the next, sample may be pure. If the SA goes down need to get rid of step and find new on


Question Answer
conserved amino acids-when looking at the sequence of a protein, some positions can have different types of amino acids yet maintain the same protein function.
-positions where the amino acid can't change are conserved amino acids
-if an amino acid is highly conserved it play an important role in the function of the protein
Sequence alignmentway of aligning multiple sequences of amino acids in rows so that each column in a row has similar or identical amino acids
scoring matrix-used to compare sequence alignments, the larger the value the more similar the sequence
-amino acids at particular positions are swapped for another AA and given a score
-score can be based on size, charge, polarity, and whether the protein still functions the same
evolutionary relationships-come from mutations in gene duplication
-some mutated proteins have completely new functions which may create new species
protein/gene family-group of evolutionarily related proteins
-similar three-dimensional structures, functions, and significant sequence similarity
protein superfamilysimilar three-dimensional structures but generally differ in function and sequence.


Question Answer
subunitis a separate amino acid chain (individual polypeptide chain in a protein)
domainpart of a subunit that if separated from the rest of the subunit, still forms a structurally stable unit.
fold (motif)combination of 2nd degree structures (α/β barrel, β barrel, etc)
primary structurethe linear amino acid sequence of the polypeptide chain
secondary structure-due to H-bonds
-contains helices, beta structures, and turns/bends
tertiary structurethree-dimensional arrangement of all the atoms in a polypeptide chain
quaternary structurearrangement of the separate polypeptide chains (subunits) into the multi-subunit functional protein
Protomerrepeating structural unit in a multimeric protein. Can be a single subunit or a group of subunits.
tetramer of α protomers
dimer of dimers/dimer of αβ protomers
Dimer of trimers/dimer of αβγ protomers