Stability of uniformly labeled (13C and 15N) cytochrome c and its L94G mutant

Abstract

Cytochrome c (cyt c) is widely used as a model protein to study (i) folding and stability aspects of the protein folding problem and (ii) structure–function relationship from the evolutionary point of view. Databases of cyts c now contain 285 cyt c sequences from different organisms. A sequence alignment of all these proteins with respect to horse cyt c led to several important conclusions. One of them is that Leu94 is always conserved in all 30 mammalian cyts c. It is known that mutation L94G of the wild type (WT) horse cyt c is destabilizing and mutant exists as molten globule under the native condition (buffer pH 6 and 25 °C). We have expressed and purified uniformly labeled (13C and 15N) and unlabeled WT horse cyt c and its L94G mutant. We report that labeling does not affect the thermodynamic stability of proteins. To support this conclusion, the secondary and tertiary structure of each protein in labeled and unlabeled forms was determined by conventional techniques (UV–Vis absorption and circular dichroism spectroscopy).

Effect of pH on Diclofenac–Lysozyme Interaction: Structural and Functional Aspect

“… With the values of T m , Δ H m , Δ C p , and Δ G D ( T ), the values of Δ G D were estimated at any temperature using the Gibbs–Helmholtz equation ( Eq. 3 ) ( Singh et al, 2005 ; Khan et al, 2013 ; Dragan et al, 2019 ; Naiyer et al, 2021 ). The far-UV CD of lysozyme was measured in a Jasco spectropolarimeter (Model: J-810) having a temperature controller (peltier model-Jasco PTC-424S).…”

“…With the values of T m , ΔH m , ΔC p , and ΔG D (T), the values of ΔG D were estimated at any temperature using the Gibbs-Helmholtz equation (Eq. 3) (Singh et al, 2005;Khan et al, 2013;Dragan et al, 2019;Naiyer et al, 2021).…”