Abiotic Stress Adaptation: Protein Folding Stability and Dynamics - Abiotic Stress: Plant Responses and Applications in Agriculture - page 9

function of osmolytes in preventing heat induced activity loss. To get further insights into
folding stability and dynamics of proteins under stress conditions, more detailed analysis
and extended methods are needed.
3.3. Global conformational stability of proteins under stress
Circular dichroism (CD) spectroscopy has been introduced as a quick and valuable techni‐
que for examining the structure and stability of proteins in solution. CD is used for deter‐
mining whether a protein is folded and for characterizing its secondary structure (alpha-
helices, beta-strands) and some aspects of the tertiary structure (aromatic amino acids,
disulfide bonds). Conformational changes during the acquisition of the native structure are
measured in the near-UV (250-350) and far-UV (190-250). This technique has been used
widely to determine the folding stability of proteins dependent on temperature, pH and un‐
der denaturant conditions [36, 37]. CD is a convenient tool to characterize the interactions
between co-solvents and proteins and to find co-solvent conditions that increase the melting
temperature or fully refold proteins after thermal unfolding. If the melting is fully reversi‐
ble, the melting temperature is directly related to conformational stability, and the thermo‐
dynamics of protein folding can be extracted from the data [38].
CD studies have been employed to investigate how osmolytes such as glycerol, trehalose
and myo -Inositol affect the global folding of native proteins and its thermal unfolding proc‐
ess. CD signals arising from protein chromophors reflect an average of the protein popula‐
tion. The resulting spectrum is a sum of individual spectra arising from secondary structure
elements present in the protein sample (Figure 4).
Figure 4. Circular dichroism spectra of Malate Dehydrogenase (insert) in 20mM NaP-buffer at pH 7.0. The far-UV spec‐
trum recorded from 260 to 190nm at 20 o C displays a typical α-helical protein with two negative maxima at 208 and
222nm. Addition of 0.4M glycerol (-), myo -Inositol (--) or trehalose (- -) did not change protein secondary structure
and did not show self-absorbance in this spectral region [39]
Thermally induced protein unfolding was monitored in the far-UV region by gradually in‐
creasing the temperature in the protein sample. Thermal denaturation curves were moni‐
tored at a fixed wavelength of 222nm (Figure 5) and acquired data were fitted to a simple
thermodynamic unfolding model. The melting temperature, T m (midpoint transition temper‐