Analysis of rigidity, stability, and activity in Thermotoga neapolitana adenylate kinase.

摘要

Proteins from hyperthermophiles are adapted to be stable and active at temperatures ≥80°C, conditions in which mesophilic proteins denature. Because the sequence and structural similarity between hyperthermophilic proteins and their mesophilic homologues is very high, it is not readily apparent as to what causes the enhanced stability of hyperthermophilic proteins. Understanding the mechanisms of protein thermostability has been one of the most intensely studied problems in the last three decades. The activity-temperature profiles of hyperthermophilic and mesophilic proteins show that the profile is right-shifted for hyperthermophiles. In other words, they attain similar activity as their mesophilic homologues at a much higher temperature and they are inactive at low temperatures. These facts have lead to the proposal of the 'rigidity' hypothesis regarding hyperthermophilic proteins. Hyperthermophilic proteins achieve high thermostability through increased structural rigidity. These proteins are highly rigid in their native structure that allows them to maintain their structural integrity at high temperatures. The increased rigidity of hyperthermophilic proteins freezes out fluctuations required for activity at room temperature making them inactive at low temperatures.; The studies described here use adenylate kinase from the hyperthermophilic bacteria Thermotoga neapolitana (TNAK) as a model system in which to test the rigidity hypothesis. What makes TNAK a really interesting model system is the fact that it is highly active at 30°C, an unusual property for a hyperthermophilic protein. Special attention was paid to the techniques used to investigate motions in TNAK. NMR and MD simulations, techniques that can access a wide range of timescales in atomic detail, were used to compare dynamics in TNAK with its mesophilic homologue from Escherichia coli (ECAK).; Results from 15N NMR relaxation data shows that TNAK is uniformly more rigid than ECAK in the ps-ns timescales as well as mus timescale. Although, overall, TNAK has higher rigidity than ECAK, several residues in the AMP-binding and lid domains exhibit high flexibility in the ps-ns timescale. Residues in the hinge regions between the lid and core domains of TNAK exhibit flexibility in the ps-ns timescales as well mus timescales. H-D exchange data, which provide information on timescales greater than seconds, show that TNAK's lid and AMP-binding domains are more stabilized compared to ECAK. Again, in spite of this increased rigidity, several residues in these domains of TNAK show considerable local fluctuations. (Abstract shortened by UMI.)