Lecture 3 - Protein Folding Flashcards
How to water-soluble proteins fold
compact structures with nonpolar cores
What does myoglobin do
- protein responsible for carrying oxygen in muscle tissue, containing a Heme group
How many amino acids are in myoglobin
- polypeptide chain consists of 153 amino acids
How is myoglobin organised
- 70 % of the main chain is folded into eight ⍺-helices.
- Porforin Ring, and Iron ion in Heme group
What is protein folding driven by
tendency of hydrophobic residue is to be excluded from water giving a hydrophobic effect
This means that secondary structures are amphipathic (different Hydro faces), and NH and CO groups are paired in hydrophobic core (through H bonding).
What are covalent bonds
– 2 atoms share electrons to fill a covalent shell – strongest
What is ionic bonding
donation of an electron to form electrostatic attractive ions
What are Van Der Waals forces
Temporary Dipole-Dipole interactions
How is tertiary structure dictated
by side-chain reactions –
Positive and negative salt bridges between amino acid
What do ribonuclease do
Degrade RNA
Why are disulphide bonds rarely found in the cell
High glutathione concentrations
What is a chaotropic agent
Breaks up hydrogen bonding
What is a reducing agent
Causes reduction reactions
What are native disulphide parings
contribute to the stabilization of the thermodynamically preferred structure
How does Urea (chaotropic agent) convert denatured ribonuclease into an active rn
Urea prevents side chain interactions, and preventing correct interactions between amino acids to form proper shape, only disulfide bonds present
How do reducing agent help turn a denatured ribonuclease into an active rn
When Reducing agent is added, weak disulfide bridges and incorrect bonds are broken, so only the strongest bonds remain
- This occurs as there is a decrease in free energy as the scrambled ribonucleases are converted to the stable, native conformation
What is a glycocelated protein
Protein covered in sugars
What is alternative splicing
exons from the same gene are joined in different combinations, leading to different, but related, mRNA transcripts
What is proteome
- The entire complement of proteins expressed in a cell
- The set of proteins expressed under a specific condition
What are some post-translational modifications a protein undergoes
- Addition of small chemical groups
- Amino Acid Processing
- Addition of Complex Molecules
- Addition of Small proteins
How does Protein production occur to create a desired drug eg. insulin
- Plasmid contains gene of interest
- E. Coli cells are heat shocked at 42 degrees for 30 seconds, making outside membrane perforated
- Plasmid enters cell and transforms, proteins are translated and produced.
- This can then be purified and used eg. insulin
How is a desired protein purified from the bacteria that created it
- Transformation of cell (plasmid with gene of interest) (antibiotics will kill any cell that hasn’t taken up plasmid which is encoded with antibiotic resistance)
- Selection
- Cell Growth and Protein Production
- Cell Lysis (Homogenization Sonication)
- Chromatography – pull out desired protein
- Dialysis – swap protein dissolved fluid for desired medium
- SDS-PAGE – to check purity
- Protein Assay – ensures protein is fit for purpose
What is column chromatography
Used to separate soluble proteins based on differences in molecular characteristics
What are some methods of purifying a product
- Gel Filtration – Separation depends on molecular size
- Ion Exchange – Separation depends on molecular charge
- Affinity Chromatography – Separation depends on specific binding interaction
Gel Filtration (Size exclusion chromatography – SEC) – separates proteins based on molecular size
Smaller proteins get trapped in polymer bead by entering aqueous spaces
Large molecules cannot enter beads do fall to the bottom
Ion exchange – Separates proteins based on differences between molecular charge
Negatively charged protein repulsed by resin, pushed to the bottom
Reverse for positive
Affinity Chromatography – Separates proteins based on specific binding interactions
What is a SDS-PAGE
the migration of a protein only depends on size
What are protein assays used for
Check activity of the protein/viability of product
