Abiotic Stress Adaptation: Protein Folding Stability and Dynamics - Abiotic Stress: Plant Responses and Applications in Agriculture - page 10

ature) can be extracted from thermal denaturation curves, reflecting the global stability of
the folded versus the unfolded protein. 0.4M glycerol, myo -Inositol and trehalose increased
the melting temperature of malate dehydrogenase by 3 to 5 o C as compared to proteins alone
[39].

Figure 5. Thermally induced unfolding of Malate Dehydrogenase (•) in the presence of 0.4M glycerol (□), trehalose
(▵), glucose (▪) and myo -Inositol (▴). The horizontal line indicates the midpoint transition temperature (T m ). Osmolytes
and proteins were mixed to protein solution and equilibrated at room temperature prior to heating. Temperature pro‐
files at 222nm were recorded for 1 °C increments in the temperature range 20-90 °C at a heating rate of 1 °C min −1
[39]. Thermal unfolding measurements were set up in quartz cuvettes, placed into a Peltier controlled sample holder
unit connected to a temperature probe to provide an accurate temperature record. Thermal unfolding curves were
analyzed using a sigmoidal curve function according to (Equation 1) [40]:

é ù

( m T b ´ - ) (

1 ( / ) T

- m T b ´ -

D D N N )

q = ê + m T b ´ - ú

(1)

T N N

m

ê + T T ú

ë û

m

where θ T is the ellipticity at temperature T, m T is the slope of the curve within the transition
region, and the inflection point of the curve the melting temperature T m . At each tempera‐
ture b N and b D can be extrapolated from the pre- and post-transition baselines, (m N × T − b N )
and (m D × T − b D ), respectively. The fraction of unfolded protein can be calculated by sub‐
tracting these baselines (Equation 2):

q q - q - ( m T b ´ - )

T N T N N

f = =

(2) v

q q - ( m T b ´ - ) ( - m T b ´ - )

v N D D N N

The stabilization of protein global folds through naturally occurring osmolytes seems to be a
general mechanism. Other studies also reported increases in the midpoint transition temper‐
ature (ΔT m ) of 2 to 18 o C upon the addition of 0.1-2M glycerol, trehalose and sucrose meas‐
ured on various proteins [41-43]. Additionally, all proteins studied in the presence of
osmolytes showed a remarkably retention of secondary structure at T m relative to proteins