Lecture 4: About Amino Acidd Flashcards
Catalysis
•Enolase (in the glycolytic pathway)
•DNA polymerase (in DNA replication)
Transport
•Hemoglobin
•Lactose permease (transports lactose across the cell membrane)
Structure
•Collagen (connective tissue)
•Keratin (hair, nails, feathers, horns)
Motion
•myosin (muscle tissue)
•actin (muscle tissue, cell motility)
Properties of Amino Acids
•Capacity to polymerize
•Useful acid-base properties
•Varied physical properties
•Varied Chemical functionality
Each amino acid consists of
•A central carbon atom
•An amino group
•A carboxyl group
•a R side chain
Most a-amino acids are
•Chiral
What does the “R” Group do?
•determines its identity
•determines its physiochemical properties
•20 different amino acids in proteins
The R Group is identified
•name
•three-letter code
•One-letter symbol
What are Amino acids classified as?
•Nonpolar, aliphatic (7)
•Aromatic (3)
•Polar, uncharged (5)
•Positively charged (3)
•Negatively charged (2)
Cofactor
Many proteins (especially enzymes) require associated chemical constituents (or cofactors) for function
Conjugated protein
If the cofactor is attached covalently, the result is a conjugated protein, and the attached group is called a prosthetic group
Why is protein purification important?
•if you want to determine a proteins properties (if it’s an enzyme and you want to study its activity)
•structural studies (X-ray crystallography, NMR)
•sequence analysis
•pharmaceuticals (insulin)
What is the goal with protein purification?
•Maximize specific activity while losing as little of your protein as possible
Purification=
•specific activity/initial specific activity
Ion-exchange chromatography
•Negatively Charged Analyte: Anion (attracted to positive surface)
•Positively charged Analyte: Cation (Attracted to negative surface)
Size-exclusion chromatography
•protein mixture added to column containing cross-linked polymer
•Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions
Affinity chromatography
•Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest
•Solution of ligand is added to column
•Unwanted proteins are washed through column
•Protein of interest is eluted by ligand solution
Electrophoresis
•Method to separate proteins
•Polyacrylamide matrix acts as sieve
•PAGE (polyacrylamide gel electrophoresis)
•Migration (m) depends on charge (Z) and frictional coefficient (f)
•f is a function of shape/size
M=
Z/f
SDS-PAGE
•SDS is a detergent (soap)
•it binds protein rather uniformly (1 SDS every 2 amino acids)
•it negates differences in mass/charge ratios between different proteins
•allows separation based on size (lengths)
•migration is inversely proportional to log(Mr)
To sequence a protein…
•Break it up into smaller pieces
•Sequence the small pieces
•Assemble the resulting sequences to get full length
