Protein Folding and Techniques Flashcards
What are the three classifications of proteins?
- Globular (cytosolic)
- Fibrous
- Membrane
What are the properties of globular proteins?
- Soluble
- Hydrophobic residues are buried (interior)
- Hydrophilic residues are exposes (exterior)
What are the properties of fibrous proteins?
- Have regularly repeating elements
- Protective and tough
ex. keratin, collagen
What are the properties of membrane proteins?
- Very hydrophobic
- Hydrophobic residues outside
- Hydrophilic residues insides
ex. transmembrane receptors, channels, pores
What are the dynamic properties of proteins?
- Equilibrium between folded and unfolded proteins (tends to favors native fold/conformation)
- Flexible structures
- Constantly moving, fluctuating, “breathing”
- Interact with solvent
What are the 2 side chains that move the most?
Lys and Arg (long and floppy)
What are the properties of intrinsically disorded proteins?
- Do not adopt folds
- Rich in low complexity sequence (repetitive sequence)
- Polar/charged amino acids (cannot fold to accommodate hydrophobic residues)
- Lack bulky hydrophobic amino acids
What are proteins sensitive to?
- pH
- Temperature
- Detergents (disrupt hydrophobic interactions, exposes them)
- Chaotropic agents (disrupt hydrogen bonding) (uear and guanidium)
- Reducing agents (betamercaptoethanol (BME) and dithiotheritol (DTT))
What are the major takeaways from Anfisen’s classical experiement on RNase A?
- Denatured proteins can (often) be refolded
- Protein folding is a reversible process
- Protein folding (tertiary structure) is determined by primary structure/amino acid sequence
What is Anfinsen’s experiment?
- Protein is denatured with urea and disulfide bonds are cleaved by mercaptoethanol
- Only mercap is removed causing incorrect disulfide bonds to from
- Subsequent removal of urea leaves an inactive protein due to messed up primary structure
- Mercap added back in absence of O2 to cleave disulfide bonds and allow correct disulfide bonds to form
- Urea and mercap are removed and active protein is reformed
How do we know proteins do not fold randomly?
- Levinthal’s paradox (takes too long for protein to randomly fold correctly)
- Proteins fold in seconds
What are the stages of the protein folding pathway?
- Hydrophobic interactions bury nonpolar side chains (hydrophobic collapse)
- Secondary structures form (alpha helix and beta sheets)
- Supersecondary structure forms
- Molten globule forms (intermediate between secondary and tertiary strucure)
- Molten globule stabilized to get an ensemble of native folds
How do chaperone proteins aid in protein folding?
-Helps protein navigate rugged terrain of many kinetic barriers
-Pushes protein down energy pathway to native state rather than aggregation (much lower energy state than native fold but inactive protein)
What do protein disulfide isomerases do? (accessory protein/chaperone)
Mediate disulfide bridge formation
What are molecular chaperones and what are their properties>
- Proteins that protect folding proteins (interact with or stabilize protein as it folds)
- Not present in final protein structures
- Recognize and bind hydrophobic surfaces to block aggregation
- require ATP for energy
- Work cooperatively with each other
example: Heat shock proteins (HSP) in prokaryotes and eukaryotes,
GroEL/GroES chaperonin in E.coli, HSP60/HSP10 in eukaryotes
How does the GroES/GroEl chaperonin work?
- GroEL binds ADP and GroES. Hydrophobic regions in GroEL interact eith hydrophobic regions of folding protein.
- Binding of 7 ATP promotes binding of new GroES on cis side and dissociation of GroES on trans side.
- ATP hydrolysis causes conformational change to expose hydrophillic patches to help protein fold
- GroES dissociates and native protein is releases
How does the Sanger method help us sequence proteins?
Got his first Nobel Prize in 1958 for this methods
Can only be used in short proteins (40-200 amino acids)
1. Reducing agent separates protein chains
2. Each chain is broken down with a protease and process is repeated with a different protease on different set of chains
3. PITC (Edman’s reagent) removes 1 amino acid at the N terminus
4. TFA cleaves the amino acid off to create a new N terminus
5. Sequence is determined by lining up fragments created by different proteases (look for overlapping regions)
6. Repeat without reduction to identify disulfide bonds
How do we determine protein sequence?
Use mRNA nucleotide sequence
What is conserved in proteins across species?
Structural/functional residues==> determine the fold
How are proteins obtained for study?
- Extracted from a mixture (cells, tissues, whole organism)
- Expressed in E. coli or other organisms
- Isolated from cell debris and contaminates (centrifugation or column chromatography)
How are proteins expressed in E.coli?
Used as factories to mass produce protein
1. DNA sequence (to be inserted) amplified by PCR
2. DNA and circular plasmid treated with specific restriction enzymes (digestion)
3. Digested DNA placed in plasmid (ligation)
4. Plasmid introduced into bacteria by heat shock (transformation)
5. Transformed bacteria grow to certain density
6. Add small molecule to turn expression of gene on
How can proteins be purified with centrifugation?
- Lyse cells with high pressure instrument or sonicator
- Separate soluble proteins from cell debris
2a. Differential centrifugation: low g force separates soluble protein from junk (early step) and high g force separates soluble protein from aggregates/precipitated protein (later step)
2b. Density gradient centrifugation: Prepare a solution with different densities in test tube and protein separates to layer with similar density
What is Svedburg (S)?
unit used to express protein sedimentation coefficient
What is column chromatography?
- Separates particles based on chemical and physical properties
- Uses a solid matrix (natural or synthetic polymer) with specific chemical properties (stationary phase(
- Pass a solution of protein and other components (mobile phase) through column
- Collect fractions and determine which contains protein (running assay)
What are the types of chromatography used in protein purification?
- Affinity: tags or antibodies are attached to protein and matrix selects for them
ex. 6xHis tag binds to Ni-NT matrix and can be flushed with imidazole - Gel filtration (size exclusion): separated by size (large diffuses faster than small) and shape (rod diffuses faster than spherical)
Start with low salt buffer and increase to high salt buffer to release polar molecules - Ion exchange: separate based on electrostatics (cations and anions)
DEAE (anion exchanger, + charge) and CM (cation exchanger, - charge) - Hydrophobic separate by solubility (use nonpolar side chains)
Applied using high salt buffer to promote binding of hydrophobic groups (polar groups will be very dissolved, exclude hydrophobic groups)
Elute hydrophobic groups by decreasing salt, adding detergent, or changing pH
How are proteins identified?
- SDS-Page (mass)
Proteins carry a overall net charge in presence of SDS and travel towards anode in gel electrophoresis. The distance traveled in set time period is associated with a certain mass. Proteins are visualized using a Coomassie stain. Proteins are denatured by heat and SDS. - Western blotting (shows protein is present)
Done after running a gel electrophoresis, transferred onto a membrane using an electrical current, use specific antibodies to probe for protein on interest - Absorbance spectroscopy (concentration)
Proteins absorb strongly in UV range due to aromatic amino acids (Trp, Tyr, Phe). Absorbance is measured at wavelength-280nm. Use A=ecl. - Circular dichroism (secondary structure)
Type of absorbance spectroscopy that uses circularly polarized light. Different secondary structures have characteristic spectra (alpha helix, beta sheet, random coils)
