Ch. 5 Flashcards
Fibrous Protein
Simple, linear structure
Insoluble in water
Ex. of Fibrous Protein
Collagen, Keratin
Globular Protein
Spherical structure
nonpolar inside and polar outside
soluble in water
Ex. of Globular Protein
Enzymes
Membrane Protein
Embedded in cell membrane
polar inside and nonpolar outside
not soluble in water
Crude Isolation/Purification Techniques
Centrifugation, Salting out, and Dialysis
Centrifugation
Separates based on density
Salting out
Increase salt concentration until proteins precipitate out
Dialysis
Separate Proteins from small molecules/ions by diffusion across semipermeable membrane
Chromatography Techniques
Ion Exchange, Size Exclusion, Hydrophobic Interaction, Affinity Chrom., and HPLC
Ion Exchange
Separates based on charge
charged matrix
like charges move faster through matrix
Size Exclusion
Matrix made of porous beads
Larger proteins move faster bc small proteins get trapped in the beads
Hydrophobic Interaction
Matrix of covalently linked hydrophobic groups
Hydrophobic sticks, hydrophilic pass through quickly
Affinity Chromatography
Proteins bind certain functional groups or ligand
Matrix has ligand or functional group attached
Binding interaction traps proteins
HPLC (High-performance liquid chromatography)
Limited to small scale due to tightly packed matrix and high pressure
Works well with other techniques
Electrophoresis Techniques
SDS-PAGE, Isoelectric Focusing, and 2D Electrophoresis
SDS-PAGE (Sodium dodecylsulfate)
Disrupts tertiary structure and gives everything a (-) charge
Proteins separated based on MW (Smaller=Faster)
Makes protein soluble
Isoelectric Focusing
Based on relative amts. of acidic and basic AA
Matrix is a pH gradient from low to high, protein stops at pI
Isoelectric Point
pH at which protein is neutral
Low pI = more acidic groups
High pI = more basic groups
2D Electrophoresis
Mixture separated by pI then SDS-PAGE
How to break a protein into individual AA’s
6M HCl and 110C
Structure Determination Steps
- Separate and purify polypeptide chains
- Cleave S-S bonds
- Determine N-terminal and C-terminal AA
- Cleave polypeptide chain into shorter fragments
- Repeat step 4. with diff cleavage method
- Reconstruct AA sequence through overlaps
- Separate and purify polypeptide chains
Disrupt noncovalent forces by
changing pH, high salt conc., treat with urea or guanidinium chloride (breaks up noncovalent interactions)
- Cleave S-S bonds
Oxidation-performic acid
Reduction- 2-mercaptoethanol/dithiothreitol
Cap thiols to prevent disulfide bond from refroming
- Determine N-terminal and C-termial
N-terminal - Edmans reagent
C-terminal - carboxypeptidase Y
- Cleave Polypeptide Chain
Enzymatic and Chemical Cleavage
Enzymatic Cleavage Methods
Trypsin, Chymotrypsin, Staphylcoccal
Trypsin
Cuts peptide bond on C-terminal side of Lysine (Lys) and Arginine (Agn)
Chymotrypsin
Cuts peptide bond on C-terminal side of Phenylalanine (Phe), Tyrosine (Tyr), and Tryptophan (Trp)
Staphylcoccal
Cuts peptide bond on C-terminal side of Aspartic Acid (Asp) and Glutamic Acid (Glu)
Chemical Cleavage Methods
Cyanagen bromide (CNBr)
Cuts peptide bond on C-terminal side of Methionine
Hydrophobic Proteins are most commonly found where?
Globular Proteins
Sequence Similarity implies evolutionary relatedness (Name?)
Homologous Proteins
Conjugated Protein
Protein containing a non-protein part
NH2 Protecting Groups
FMOC and BOC
Reagent used to make OH a better leaving group in protein synthesis
DIPCDI
Purification and Protection in protein synthesis
Attach C-terminal to insoluble resin
(Solid phase peptide synthesis)
Purification by filter
Direction of synthesis in nature and chemically
Nature: Starts at N-Terminal
Chemically: Starts at C-Terminal
Examples of Ligand Bound Proteins
Transport proteins, enzymes, regulatory proteins, and scaffold proteins
