Protein analysis & structure Flashcards
2.7, 2.9, 2.10 (23 cards)
what is a βαβ structure (labeled diagram)
helix connecting 2 parallel sheets
what is a β hairpin (labeled diagram)
antiparallel strands
what is an α α motif (labeled diagram)
2 successive helices antiparallel
what is the greek-key motif
a pattern similar to an ornamental design from ancient greece
α β hairpin folded over to form a 4-stranded antiparallel β sheet
what is the structure of all α
helices spaced by short connection links
what is the structure of all β
sandwich of 3 or 4 stranded antiparallel β
sheets
what is the topology of the protein
how motifs are connected
what classifies a protein as a domain
> 200 amino acids folded into 2 or more globular clusters
during evolution is sequence or structure conserved
structure
why have TIM barrel and Rossman fold been retained
because they are stable, tolerate mutation and can support biological structure
what are the 4 classifications proteins (CATH)
class- secondary structure composition
Architecture - shape and orientations of secondary structure
Topology - connectivity of secondary structure
Homologous superfamily - proteins with high sequence identity or structural identity
give 2 superfold structures
TIM barrel
Rossmann fold
what is net charge of an amino acid when pH=Pi
0
if pH > Pi what will the net charge of the molecule be
negative
if Pi > pH what will the net charge of the molecule be
positive
how does charge effect elution
protein contains mans R groups & charges, the overall charge on a protein is affected by the pH when overall charge becomes 0 at the isoelectric point protein is eluted but some proteins may denature at the pH of their isoelectric point
what leaves first during size exclusion chromatography
big things leave then small things
what’s affinity chromatography
separates molecules according to their ability to form specific interaction
why are buffers used in analysis of protein structure
to keep a protein at a constant pH to prevent unfolding
give the 4 key steps in protein purification
- break up cell (chemically & mechanically)
- remove contaminating material via differential centrifugation
- isolate the protein by column chromatography
- check purify via electrophoresis
draw a general elution profile
what is the purification factor
the number of times by which the specific activity has increased
what are the main signs of purification
- total protein decreases as contaminants removed
- reduction in activity (some protein of interest lost)
- increase in specific activity
