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Compiled notes
Outline
Amino Acids
Stereochemistry
Non-standard amino acids
Amino Acids
carboxylic acid group amino group on the alpha (a) carbon Properties dictate behavior of AAs
R side chain | H2N C COOH | H
Zwitterions
Both the NH2 and the COOH groups in an amino acid undergo ionization in water. At physiological pH (7.4), a zwitterion forms
Classify by structure of R
Nonpolar Polar
Aromatic
Acidic Basic
Disulfide Bonds
Acidic
Basic
Acid-base Properties
Remember H3PO4 (multiple pKas) AAs also have multiple pKas due to multiple ionizable groups
pK1 ~ 2.2
(protonated below 2.2)
pK2 ~ 9.4
(NH3+ below 9.4)
pKR
(when applicable)
Table 3-1
pH and Ionization
Consider glycine:
O O OH O
H3N
CH H
OHH3O
+
OHH3N CH H C O H2N CH H C O
H3O
Titration of Glycine
pK1
[cation] = [zwitterion]
[zwitterion] = [anion]
pK2
Zwitterion Molecule has no net charge pH = pI (Isoelectric point) pI = average of pKas = (pK1 + pK2) pIglycine = (2.34 + 9.60) = 5.97
Animation
pI of Lysine
pK1
H3N CH C OH H3N CH C O
pK2
H2N CH C O
pKR
H2N CH C O
CH2CH2CH2CH2NH3+
CH2CH2CH2CH2NH3+
CH2CH2CH2CH2NH3+
CH2CH2CH2CH2NH2
Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)
Learning Check
Stereochemistry of AAs
Fischer projections:
D and L Configurations
Importance of Stereochemistry
D-AAs are found in bacteria Geometry of proteins affects reactivity (e.g binding of substrates in enzymes)
Thalidomide
AA derivatives
D-AAs
Bacteria
Chain of amino acids = peptide or protein Amino acid residues connected by peptide bonds Residue = AA H2O
Polypeptides
Linear polymers (no branches) AA monomers linked head to tail Terminal residues:
on left on right
pKa values of AAs in polypeptides differ slightly from pKa values of free AAs
Naming Peptides
(GA)
Glx/Z Asx/B
X = undetermined or nonstandard AA
Learning Check
Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.
CH3 CH3 CH CH3 CH3 O H CH O H SH CH2 O H S CH2 CH2 O
H3N CH C N CH C N CH C N CH C O
Learning Check
Outline
Protein size
Usually 100-1000 residues Percent of each AA varies Proteins separated based on differences in size and composition Proteins must be pure to analyze, determine structure/function
Factors to control
pH
Keep pH stable to avoid denaturation or chemical degradation May affect structure (e.g. proteases/peptidase) Control denaturation (0-4C) Control activity of enzymes Reactive Add protecting group to prevent formation of new disulfide bonds Denature or oxidize Store under N2 or Ar Keep concentration high
Presence of enzymes
Temperature
Thiol groups
Break into fine pieces Cells disrupted Soluble contents mix with buffer Centrifuge to separate soluble and insoluble
Solubility
Selectively precipitate protein Manipulate
Concentration of salts
Adding small amount of salt increases [Protein] Salt shields proteins from each other, less precipitation from aggregation
Salting-in
Salting out
Solvent
pH
Temperature
Chromatography
Mobile phase
Stationary phase
Types of Chromatography
Paper
Stationary phase = small uniform particles, large surface area Adapt to separate based on polarity, size, etc.
Hydrophobic Interaction
Types of Chromatography
Ion-exchange
Cation exchangers
Types of Chromatography
Gel-filtration
Size/molecular exclusion chromatography Stationary phase = gels with pores of particular size Molecules separate based on size
Types of Chromatography
Affinity
UV-Vis Spectroscopy
Electrophoresis
Protein Sequencing
Determination of primary structure Need to know to determine 3D structure Gives insight into protein function Approach:
Denature protein Break protein into small segments Determine sequences of segments
Animation
Number of chains/subunits
Identify specific AA Dansyl chloride/dabsyl chloride Sanger method (FDNB) Edman degradation (PITC)
Bovine insulin
Dansyl chloride
N-terminus
+
H2N CH R SO2 Cl
O C
Yields dansylated polypeptides Dansylated polypeptides hydrolyzed to liberate the modified dansyl AA Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence) Useful method when protein fragmented into shorter polypeptides
SO2 HN CH R
O C
SO2 HN CH R
O C OH
N N
O S O Cl
Sanger method
Edman degradation
Phenylisothiocyanate (PITC) Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC) Treat with TFAA (solvent/catalyst) to cleave N-terminal residue Does not hydrolyze other AAs Treatment with dilute acid makes more stable organic compound
Identify using NMR, HPLC, etc. Sequenator (entire process for proteins < 100 residues)
Fragmenting Proteins
Purify fragments
Sequence fragments
Determine order of fragments and disulfide bonds
2-Mercaptoethanol
HSCH2CH2OH
Dithiothreitol (DTT)
HSCH2CH(OH)CH(OH)CH2SH
Hydrolysis
Enzyme Acid Base Identify using chromatography Quantify using absorbance or fluorescence
After cleavage:
Disadvantages
Doesnt give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results
Enzymatic
Chemical
An example
Another example
The same protein is cleaved with chymotrypsin to yield the following sequences: