6 research outputs found
Expression and molecular characterization of the Mycobacterium tuberculosis PII protein
The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with Kd in the range 1.93–6.44 μM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the γ-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins
Classification of Chemical Chaperones Based on Their Effect on Protein Folding Landscapes
Various small molecules present in
biological systems can assist
protein folding <i>in vitro</i> and are known as chemical
chaperones. <i>De novo</i> design of chemical chaperones
with higher activity than currently known examples is desirable to
ameliorate protein misfolding and aggregation in multiple contexts.
However, this development has been hindered by limited knowledge of
their activities. It is thought that chemical chaperones are typically
poor solvents for a protein backbone and hence facilitate native structure
formation. However, it is unknown if different chemical chaperones
can act differently to modulate folding energy landscapes. Using a
model slow folding protein, double-mutant Maltose-binding protein
(DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide
(TMAO), accelerates refolding by decreasing the flexibility of the
refolding intermediate (RI). Among a number of small molecules that
chaperone DM-MBP folding, proline and serine stabilize the transition
state (TS) enthalpically, while trehalose behaves like TMAO and increases
the rate of barrier crossing through nonenthalpic processes. We propose
a two-group classification of chemical chaperones based upon their
thermodynamic effect on RI and TS, which is also supported by single
molecule Förster resonance energy transfer (smFRET) studies.
Interestingly, for a different test protein, the molecular mechanisms
of the two groups of chaperones are not conserved. This provides a
glimpse into the complexity of chemical chaperoning activity of osmolytes.
Future work would allow us to engineer synergism between the two classes
to design more efficient chemical chaperones to ameliorate protein
misfolding and aggregation problems