Thermostability

Question 1

What are the most abundant replacements moving from mesophilic to thermophilic organisms?

Answer 1

Lys –> Arg
Ser –> Ala
Gly –> Ala/Pro
Asp –> Glu

Question 2

What is a B factors and how does it compare from mess to thermophiles?

Answer 2

Measure of thermal vibration of atom about a mean position.
Cold adapted have high B factors at RT (more flexible)
Thermostable have lower.

Question 3

What is Tm and how is it linked to enzyme stability?

Answer 3

T at which 50% of enzyme inactivated during reversible heat denaturation.

Question 4

How do we know core packing is so important?

Answer 4

Thermozymes tend to show increased internal hydrophobicity.

Engineering cavities into Barnase protein core destabilises WT enzyme by ~1kcal/mol

Question 5

Aside from lysozyme, what other enzyme has undergone disulphide bond engineering?

Answer 5

Subtilisin
6 S-S bonds engineered in 1989 - NONE stable
G61C-S98C added in 1990 - Tm increase of 4.5 degrees.

Question 6

What causes inactivation at high thermal temperatures?

Answer 6

Deamidation of Asn, Gln –> Asp, Glu
PP chain hydrolysis at Asp
B-elimination at Cys.
(lysozyme loses activity at 100C.

Question 7

What structure has showed additional salt bridges in a themophilic form?

Answer 7

RNase from thermophilus - pH has strong effect so must have additional SS which stabilise B strand and not found in E. Coli form.