Protein Analysis Flashcards
(34 cards)
What is the primary structure of a protein?
The linear sequence of amino acids, encoded by single-letter or three-letter codes. It determines all higher levels of protein structure.
What are the main types of secondary protein structures?
Alpha helices and beta-pleated sheets, formed via hydrogen bonding.
What is the tertiary structure of a protein?
The complete 3D folding of a single polypeptide chain; includes loops and complex folds.
What is quaternary structure in proteins?
The assembly of multiple polypeptides into a functional complex, stabilized by non-covalent interactions (e.g. hydrogen bonds, Van der Waals).
Give an example of a quaternary protein.
Homo-tetramer (4 identical subunits) or hetero-oligomer (different subunits).
What causes protein denaturation?
Heat, pH changes, and chemicals. Denaturation disrupts 3D structure → loss of function.
Real-life example of protein denaturation?
Cooking an egg—egg white proteins denature and solidify; another is fever affecting enzyme function.
Where does GFP come from?
Originally from jellyfish Aequorea victoria and sea anemones.
What is unique about GFP structure?
Beta-barrel structure with an internal autocatalytic chromophore that fluoresces.
What happens when blue light hits GFP?
It emits green light—this is its fluorescence.
Why is GFP useful in molecular biology?
It allows live imaging and tracking of proteins, organelles, and cell structures.
What is a GFP fusion protein?
A recombinant protein formed by linking GFP to another protein without a stop codon in between—produced as a single polypeptide.
What does GFP-tagged beta-actin show?
Localizes to cytoskeleton fibers, allowing visualization of cytoskeletal structures.
What did mitochondrial GFP imaging reveal?
Mitochondria form dynamic, tubular networks, not just round blobs.
Why were sea anemone proteins studied?
They provide GFP and red fluorescent proteins, useful for multi-color imaging.
What is a His-tag?
A sequence of 6 histidine residues added to a protein to aid purification via metal affinity.
What metals are commonly used in IMAC?
Nickel (Ni²⁺) and cobalt (Co²⁺).
How does IMAC purify proteins?
His-tag binds to metal ions on beads in the column. Non-tagged proteins wash out.
What are two elution methods in IMAC?
1) Imidazole competition; 2) Acidification with protons.
Why is imidazole effective in elution?
Its structure mimics histidine’s side chain, displacing the His-tag from the metal.
What does gel filtration chromatography separate proteins by?
Molecular size using porous beads.
Why do larger proteins elute faster in gel filtration?
They bypass the pores and take a shorter path through the column.
Ideal primer length and Tm range?
18–30 nucleotides, with Tm values within 2–3°C of each other.
Key features of good primers?
40–60% GC content, no strong secondary structures, specificity to target.
