Background:
The dipeptide composition-based Instability Index (II) is one of the protein
primary structure-dependent methods available for in vivo protein stability predictions. As per this
method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles
guided the original development of the II method. However, the use of the II method for in vitro
protein stability predictions raises questions about the validity of applying the II method under
experimental conditions that are different from the in vivo setting.
Objective:
The aim of this study is to experimentally test the validity of the use of II as an in vitro
protein stability predictor.
Methods:
A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that
rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent
degradation properties of CCM by generating CCM mutants to represent stable and unstable II
values. A comparative degradation analysis was carried out under in vitro conditions using wildtype
CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1-
casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions.
The effect of temperature and a protein stabilizing agent on CCM degradation was also tested.
Results:
Data support the dipeptide composition-dependent protein stability/instability in wt-CCM
and mutants as predicted by the II method under in vitro conditions. However, the II failed to
accurately represent the stability of other tested proteins. Data indicate the influence of protein
environmental factors on the autoproteolysis of proteins.
Conclusion:
Broader application of the II method for the prediction of protein stability under in
vitro conditions is questionable as the stability of the protein may be dependent not only on the
intrinsic nature of the protein but also on the conditions of the protein milieu.
