Amino Acids and Protein Primary Structure Flashcards
pKa of amino group of amino acids
9.5
pka of carboxyl group of amino acids
2
Zwitterion
Ionized molecule with net charge of 0
Form of amino acids most commonly found in nature
L-amino acids
Aliphatic amino acids
Glycine, alanine, valine, leucine, isoleucine, proline
Aromatic amino acids
Phenylalanine, tyrosine, tryptophan
Sulfur-containing amino acids
Methionine, cysteine
Alcoholic amino acids
Serine, threonine
Basic amino acids
Histidine, lysine, arginine
Guanidinium group
Functional group of arginine
High stabilization through resonance in protonated form- deprotonated form is very basic
Acidic amino acids
Aspartate, glutamate, asparagine, glutamine
Making of biosynthetic amino acid derivatives
Decarboxylation and deamination enzymes combine and modify amino acids
Isoelectric point (pI)
pH where a molecule is electronically neutral
pI values of amino acids with basic sidechains and pI values of amino acids with acidic sidechains
Basic amino acids have basic pI values
Acidic amino acids have acidic pI values
pKa of cysteine’s sidechain
8.4
pKa of tyrosine’s sidechain
10.5
pKa of aspartic acid’s sidechain
3.9
pKa of glutamic acid’s sidechain
4.1
pKa of lysine’s sidechain
10.5
pKa of arginine’s sidechain
12.5
pKa of histidine’s sidechain
6.0
Primary structure of protein
Linear sequence of amino acids in a polypeptide chain
Polypeptide nomenclature
Amino acid residues in a polypeptide chain change their “-ine” or “-ate” to “-yl”
Named from N-terminus to C-terminus
Drawing an L-amino acid
Alpha-amino acid is going up in chain: wedge
Alpha-amino acid is going down in chain: dash
Crystallization
Protein purification technique
Good for separating peptides from other types of molecules
Difficult to separate mixtures of proteins
Ion-exchange chromatography
Protein purification technique
Matrix is charged, proteins are eluted by increasing salt concentration
Gel-filtration chromatography
Protein purification technique
Matrix is porous, separation based on molecular size
Affinity chromatography
Protein purification technique
Matrix contains covalently-bound small molecule, separation based on degree of interaction with immobilized small molecule
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)
Separation of protein based on their differential migration in an electric field
SDS- detergent that imparts negative charge to proteins
Top of gel: high molecular weight
Bottom of gel: low molecular weight
Electrospray ionization (ESI) and matrix-assisted desorption ionization (MALDI)
Charged proteins are dispersed in gas phase
Fragmentation patterns can be deciphered to determine amino acid sequence and post-translational modifications
Percent composition
Acid hydrolysis followed by phenyl ITC treatment and detection
Phenyl ITC labels amino acid -> can detect absorbance that corresponds to label
Only tells which amino acid, not order
Edman degradation
Method of determine amino acid sequence
- Protein is treated with phenyl ITC
- PTC- peptide is treated with anhydrous acid: N-terminal peptide bond is cleaved
- Anilinothiazolinone product is extracted and treated with aqueous acid to cause rearrangement to phenylthiohydantoin derivative
- Amino acid is identified through chromatography
- Repeat sequentially with remaining peptide chain
Maximum limit of Edman degradation
30 amino acids
Technique becomes less accurate over time: extra amino acid residues are picked up
Cyanogen bromide (BrCN)
Protein sequencing technique
Cleaves polypeptide chains on the C-terminal side of methionine residues
Trypsin
Protease
Cleaves on C-terminal side of basic residues Lys and Arg
Chymotrypsin
Protease
Cleaves on C-terminal side of aromatic hydrophobic sidechains (Phe, Trp, Tyr)
Polypeptide sequencing steps
- Treat polypeptide with hydrolytic enzymes
- Analyze fragments via Edman degradation
- Deduce structure from overlapping evidence
