Novel Design Flashcards
What six methods are used for the design of new proteins?
Modify existing
Framework of known protein
Analogy to known structures
Entirely novel
In vitro random mutagenesis and selection
In silico mutagenesis and energy calculations.
What examples are there in nature of proteins that have folded differently despite having high sequence identity values?
Cro repressor proteins - 40 % sequence identity
Xfaso1 - entirely helical
Pfl6 - some beta sheet present
As functionally related must have diverged structure.
What is an abzyme?
An antibody enzyme.
Can make S analogues - inject into mouse, raise antibodies and check whether have catalytic activities.
What was the aim for the design of betanova?
To design a small, soluble, monomeric, B sheet protein folding in absence of SS or metal cofactors.
Minimal unit = 20 residues, 3 stranded antiparallel B-sheet, 4 residues per strand and 2 residue beta turns.
What modifications were made to BBA1?
Zn ligands replaced by hypho core.
Residues to enhance alpha and beta structure included.
Type II Beta turn
Helix cap residue (His).
Give an example of an artificial protein that is highly stable.
184 residue 4 fold symmetric TIM barrel (no sequence match with any other TIM barrels)
RMSD 1.3A (low)
Tm = 88
BUT change in G of folding was v low = 4.2!
Why are cyclic proteins so stable?
Disulphide bonds constrain unfolded state –> enhanced stability to denaturation and unfolding.
Naturally occurring sunflower cyclic peptides have trypsin inhibitor activity - under development as antiprotease drugs.
What have Chin et al shown?
In vitro evolution of a quadruplet-decoding ribosome.
Possibility of encoding >200aas.
Incorporated 2 novel AAs into calmodulin.
Briefly outline the structure of alpha/beta barrels.
Pocket created by loops connecting C TERMINI of B strands to amino termini of alpha helices.
AS’ at C terminal ends of Beta barrels.
Substrate binding within barrel
Catalytic within loop.
